Preparation of the recombinant rM179 has been described elsewhere (Moradian-Oldak et al., 2000; Lakshminarayanan et al., 2010). The protein was further purified on a Jupiter C4 semipreparative reversed phase column (10 × 250 mm, 5μm) Varian Prostar HPLC system (ProStar/Dynamics 6, version 6.41, Varian, Palo Alto, CA). A linear gradient of 60% acetonitrile in 0.1% trifluoroacetic acid at a flow rate of 2 ml/min was used (**Figure 1B**, lane 2).

Recombinant porcine amelogenin (rP172) was expressed in *Escherichia coli* strain BL21-codon plus (DE3-RP, Agilent Technologies, Inc., Santa Clara, CA), precipitated by 20% ammonium sulfate and purified as previously described (Hu et al., 1996; Ryu et al., 1999). The precipitate was dissolved in 0.1% TFA and protein purification was accomplished on a reverse phase C4 column (10 × 250 mm, 5μm) mounted on a Varian Prostar HPLC system (ProStar/Dynamics 6, version 6.41 Varian, Palo Alto, CA) and fractionated using a linear gradient of 60% acetonitrile at a flow rate of 1.5 mL/min.

Recombinant murine ameloblastin (rMAmbn) both phosphorylated and glycosylated (**Figure 1C**, lane 2) was prepared in a Drosophila melanogaster expression system using Schneider 2 cells (Invitrogen, CA) as previously described (Zeichner-David et al., 2006). The protein was purified by HPLC with a gradient of 20–80% B for 60 min (buffer B was 60% v/v aqueous acetonitrile in 0.1% v/v trifluoroacetic acid (TFA) and buffer A was 0.1% TFA) at a flow rate of 1.0 ml/min.

Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) was carried out in the presence of 0.1% SDS, using a 10% acrylamide gel (Laemmli, 1970) to characterize the proteins (**Figures 1B,C**). The gel was stained with Coomassie brilliant blue.

#### **IMMUNOCHEMICAL ANALYSIS OF PROTEIN FROM ENAMEL EXTRACELLULAR MATRIX AT DAY 9**

For immunochemical analyses, six 9-day-old (postnatal) mice were used. All mouse studies were conducted according to protocols approved by the USC Institutional Animal Care & Use Committee. The upper and lower molars were extracted from the alveolar bone. The enamel surface was then gently wiped to remove remnants of secretory ameloblasts. The sample was dissolved in 0.5 M acetic acid (four molar /100μl) and dissolution is aided by grinding the samples in the acid with pestle and then total extracted protein was lyophilized. Samples were electrophoresed on 10 % polyacrylamide gel containing 0.1% sodium dodecyl sulfate (SDS-PAGE) under non-reducing conditions and then Western blot analysis was performed as described in previous Section using both anti-amelogenin and M300 primary antibody (**Figure 2**, lane 2, 3).

#### **TISSUE PREPARATION**

Mandibular processes of mice from postnatal days 1–8 (here the day of birth was termed postnatal day 0) were dissected and fixed in 4% paraformaldehyde in PBS at pH 7.4 for 24 h. Samples from day 5–8 were demineralized in 0.2% paraformaldehyde, 0.05% glutaraldehyde and 10% EDTA for ∼1–2 weeks (pH 8.0) at 4◦C with rocking. The tissues were then processed for histological analysis and embedded in paraffin. Tissue sections of 7μm thickness were cut from the wax blocks and mounted onto glass slides.

#### **IMMUNOFLUORESCENCE LABELING**

Tissue sections were subjected to an antigen retrieval step by incubation in 10 mM sodium citrate with 0.05% Tween 20 at pH 6.0 in a 60◦C water bath overnight. The sections were allowed to cool in dH2O before being rinsed in TBS and then incubated with 0.3% H2O2 for 15 min. After washing with TBS, sections were blocked with 1% bovine serum albumin (BSA) for 15 min before incubation overnight at room temperature with primary antibody. We have used 1:500 dilutions for anti-ameloblastin M300 and 1:1000 for anti-amelogenin (Section Description of Antibodies). For simultaneous immunofluorescence double-labeling mixture of both primary antibodies with above mentioned dilution were used. After another wash with TBS, sections were incubated with secondary antibody (for ameloblastin goat anti-rabbit-Texas Red and for amelogenin bovine anti-chicken-FITC with 1:100 dilution each) for 3 h at room temperature, before mounting with

Vectashield® Hard Set™ mounting medium with DAPI. Similar to primary antibody we have used mixture of both secondary antibodies for immunofluorescence double-staining. Several controls were performed to ascertain the specificity of the antibodies, which included incubating each primary antibody with both secondary antibodies, as well as each secondary antibody with both primary antibodies. The sections were visualized using a Leica TCS SP5 confocal microscope.

#### **QUANTIFICATION OF CO-LOCALIZATION OF TWO ANTIGENS**

Using the Leica Application Suite Advanced Fluorescence software (version <IP_ADDRESS>39) confocal images were analyzed with a threshold of each channel at 30% and a background correction of 20%, and scatter grams were generated. Mander's colocalization coefficients were calculated to predict the contribution of each particular antigen to the areas with co-localization, using the following equations (Manders et al., 1993):

$$M\_{red} = \frac{\sum\_{i} R\_{i,\text{ color}}}{\sum\_{i} R\_{i}}; M\_{green} = \frac{\sum\_{i} G\_{i,\text{ color}}}{\sum\_{i} G\_{i}}$$

when, R*i, coloc* = *Ri* if G*i >* 0; G*i, coloc* = *Gi* if R*i >* 0, i.e., M*red* is the sum of intensities of red pixels that have a green component divided by the total sum of red intensities.

#### **CIRCULAR DICHROISM**

CD measurements were carried out at 37◦C on a JASCO J-715 spectropolarimeter between 190 and 260 nm in a quartz cell with a path length of 1 mm. Four spectra were accumulated for each sample, and the contribution of the buffer was always subtracted. Data are represented as mean residue ellipticities. Stock solution of rP172 (89% analogues to mouse amelogenin) was prepared in distilled water and rMAmbn was prepared in Tis-HCL pH 8.0. CD experiments were performed with 10μM protein in 10 mM potassium phosphate buffer at pH7.4. CD spectra were normalized to the protein concentration and normalized spectra were deconvoluted using the CDSSTR algorithm and the Reference set 7 at Dichroweb (Lobley et al., 2002; Whitmore and Wallace, 2004). The values for helix1 and helix2 as well as sheet1 and sheet2 were added to obtain total helix and total sheet content (Sreerama et al., 1999).

## **RESULTS**

#### **DEGRADATION AND DISTRIBUTION OF MOUSE AMELOBLASTIN**

We analyzed the degradation of mouse ameloblastin immunochemically using Western blot analysis (**Figure 2**, lane 3) with proteins extracted from 9 day old mouse enamel. In the enamel matrix sample, M300 antibody reacted with protein bands having molecular weights of near 62, 40, 33, and 29 kDa (**Figure 2**, lane 3). The largest 62 kDa band corresponds to the nascent ameloblastin and the others are presumably C-terminal ameloblastin processing products (Uchida et al., 1997, 1998; Brookes et al., 2001).

To observe the distribution of ameloblastion and its proteolytic products in enamel we have incubate tissue sections from day 1 (**Figure 3A**), 5 (**Figure 3B**) and 8 (**Figure 3C**) with antiameloblastin M300 antibody followed by immunofluorescence

labeling with Texas red conjugated anti-rabbit secondary antibody (see Section Description of Antibodies and Immunofluorescence Labeling). Confocal image of the section at postnatal day 1(P1) indicated that M300 antibody stained the distal cytoplasm of ameloblasts, particularly in Tomes' processes and immature enamel near the secretory surfaces of pre-secretoty ameloblasts (**Figure 3A**). During the early maturation stage when the degradation of ameloblstin has already started, at day 5 (P5), they were present in the enamel epithelium and at the enamel surface (**Figure 3B**). At day 8 (P8), we observed that M300 reacted with the proteins which were accumulated in the juxtanuclear cytoplasm, the distal cytoplasm near the enamel surface, at the enamel surface and dentino-enamel junction (**Figure 3C**) (Lee et al., 1996).

The immunofluoresence study at P8 (**Figure 3C**) with M300 antibody gives us a clear picture of localization pattern of ameloblastin and its C-terminal processing products which were identified by immunochemical analysis (**Figure 2**, lane 3). **Figure 3D** shows the localization pattern of amelogenin and its processing products (**Figure 2**, lane 2) at day P8. The antiamelogenin antibody stained evenly across the enamel layer (visualized by green color FITC conjugated secondary antibody).

### **THE CO-LOCALIZATION PATTERN OF Ambn AND Amgn IN MOUSE MANDIBULAR MOLARS**

To visualize the co-localization of ameloblastin and amelogenin and for further confirmation of distribution of these two proteins in enamel extracellular matrix we acquired confocal images of tissue sections (simultaneously immunostained with two fluorophore mixture) at P1 (**Figure 4A**), P5 (**Figure 4B**) and P8 (**Figure 4C**). In **Figure 4** the regions which were yellow or orange in color represent the co-localization of both the protein. Next quantitative co-localization analysis (QCA) was performed using fluorescence software to quantify the percentage of co-localization of Ambn and Amgn at P1 and P8 (**Figures 5**, **6**) using selected region of these images (**Figure 4**, for P5 data not shown) with higher magnification.

QCA was conducted at day 1 using a selected co-localization region from **Figure 4A** (white square), which presented in **Figure 5A**. We have divided this co-localization region (yellow colored) into 14 small regions of interest (ROIs) and the direction of analyzed region of interest in **Figure 5** is indicated by pink arrow along the secretory face of the ameloblast (at the enamel surface) from the cervical loop to tip of the molar. Then Mander's co-localization coefficients were calculated to characterize the degree of overlap between two channels (**Figure 5B**, green for Amgn and **Figure 5C**, red for Ambn) in the image and to predict the contribution of each particular antigen to the

**FIGURE 5 | Quantitative co-localization of ameloblastin and amelogenin in mouse molar enamel at P1. (A)** Confocal image with higher magnification taken from the sample in **Figure 4A** (see square), considered for QCA analysis along the enamel surface (pink arrow). The individual channels detecting **(B)** only Amgn and **(C)** only Ambn signal. **(D)** Represent

the pixels that have only co-localizing signal after threshold and background corrections of image **(A)**. **(E)** Graph of total intensity sum of each fluophore. **(F)** Plot of Mander's co-localization coefficient with respect to region of interest as shown in **(D)**. Each ROI dimension (5 × 5μm). Note that ROI 1 to 14 in **(E,F)** correspond to the pink arrow in **(A)**.

areas with co-localization, which is the number of pixels that have a co-localizing signal (**Figure 5D**, represented by white dots). First the values for signal intensity sum of the green channel (**Figure 5E**, green line), red channel (**Figure 5E**, red line) and the co-localization intensity (data not shown) were generated at each ROI. Then the co-localization intensity was divided by the signal intensity to obtain co-localization coefficients (**Figure 5F**), which

measured the proportion of overlap of each channel with the other. As can be seen from the co-localization coefficient graph, at the initial stage of enamel development (P1), Amgn and Ambn co-localized near the secretory face of ameloblasts with almost the same proportion and high coefficient values (**Figure 5F**).

On day 8, QCA was performed with a section of **Figure 4C** (square or inset), represented in **Figure 6A**. At P8 the enamel was more mature, we have calculated co-localization coefficients in two different configurations, one along the secretory face of the ameloblast from the cervical loop to tip of the molar (at the enamel surface indicated by pink arrow) and two along the thickness of the enamel layer from the enamel surface to dentinoenamel junction (indicated by cyan blue arrows as Enamel1 and Enamel2) (**Figure 6A**). The two individual channels of this confocal image were presented by **Figure 6B** (green channel for Amgn) and **Figure 6C** (red channel for Ambn) and the number of pixels that have a co-localizing signal in same image indicated by white dots in **Figure 6D**. We have calculated the total sum of green (**Figure 6E**, green line) and red (**Figure 6E**, red line) intensities along, (i) the ameloblast (**Figure 6E1**) and (ii) enamel thickness (**Figures 6E2,E3**). We observed that at day 8 the intensity difference between the two channels increased in both configurations (**Figures 6E1–E3**) relative to day 1 (**Figure 5E**). The co-localization coefficient plot at P8 indicated that Ambn colocalized with Amgn along the secretory face of ameloblasts, and their proportions of co-localization differed (**Figure 6F1**). There was a higher percentage of Ambn co-localizing with Amgn than Amgn co-localizing with Ambn. Along the enamel thickness moving award from the ameloblasts, no significant co-localization was observed (**Figures 6F2,F3**).

It appears that at an early stage both antigens (ameloblastin and amelogenin) are distributed in the same region with high spatial overlap, i.e., we can assume that they may be close enough to form co-assemblies, but with maturation ameloblastin has a high co-localization coefficient in comparison to amelogenin.

This trend can be clearly seen when the average of the colocalization coefficients for each age is calculated and plotted (**Figure 7A**). We have calculated the ratio of the total intensity sum of Ambn-red channel (- *i Ri*) to the Amgn-green channel (- *i Gi*) of each region of interest (**Figures 5E**, **6E**) along the enamel surface obtained from confocal images of day 1 and 8 mouse mandibular molars (**Figure 7B**). At an early developmental stage (day 1) we found that the ratio gradually reaches the value 1 when moving from the root to the tips of the tooth (**Figure 7B**, black line), which indicates that the proteins were secreted simultaneously and in similar proportions (calculated from QCA) from ameloblasts (Zalzal et al., 2008). With enamel maturation, at day 8 the intensity ratio decreases (**Figure 7B**, red line), indicating that at the maturation stage, ameloblastin and its proteolytic products (C-terminal large soluble polypeptides) (Lee et al., 1996; Uchida et al., 1997, 1998; Brookes et al., 2001) are gradually lost from the enamel surface.

Our co-localization studies suggested that amelogeninameloblastin complexes may be the functional entities at the early stage of enamel mineralization. In order to examine how amelogenin would affect ameloblastin's structure, we performed *in vitro* experiment, e.g., CD spectroscopy study with recombinant proteins.

#### **CD SPECTROSCOPIC STUDY OF Ambn AND Amgn INTERACTION**

CD spectra analysis of Ambn (10μM) in the presence of equimolar Amgn revealed that Amgn induced changes in the secondary structure of Ambn (**Figure 8**). The CD spectrum of rMAmbn at 37 ◦C, with a negative minima around 208 nm and a small negative shoulder around 227 nm, is characteristic of an unordered structure (βII structure) (**Figure 8**, black solid line). Additionally, the spectra deconvolution shows rMAmbn contain intermixed alpha-helical (24%), beta-sheet (24%) and beta-turn (18%) regions with 34% unordered structure. The human recombinant full-length ameloblastin also exhibited βII like structural property at 37◦C (Wald et al., 2011, 2013) and almost similar deconvolution pattern with less helical content and more beta character. With the addition of equimolar Amgn to Ambn, we observed an isoelliptic point appears at around 211 nm and a distinct change in CD bands. The intensity of the shoulder around 224 nm became more negative and at 192 nm a positive maxima was appeared. The spectral deconvolution exhibited the alpha-helical content increased to 51% and the betasheet, beta-turn and unordered forms decreased to 17, 12, and 14%, respectively. To avoid the interference of Amgn's structural changes on Ambn due to its self-association at high concentrations (*>*62.5μM) (Lakshminarayanan et al., 2007), we used a very low concentration of Amgn (10μM) and also we have

subtracted its contribution from the CD spectra of equimolar mixture of both the proteins. The structural changes in Ambn at this low Amgn concentration thus clearly indicated that the proteins formed hetero-assemblies.

# **DISCUSSION**

Amelogenin and ameloblastin, the major enamel matrix proteins, are important for well-orchestrated enamel biomineralization (Snead et al., 1985; Krebsbach et al., 1996). They have synergistic roles in enamel development, as suggested by recent study showing that Amel X−*/*− /Ambn−*/*− mice have additional enamel defects not observed in either Amel X−*/*− or Ambn−*/*− mice (Hatakeyama et al., 2009). The co-distribution of amelogenin and ameloblastin in the majority of the secretory granules in Tomes' processes during appositional growth of the enamel layer may reflect a form of functional association between these two distinct proteins (Zalzal et al., 2008). There is *in vitro* evidence of interactions between amelogenin (tyrosyl-binding motif) and ameloblastin (Ravindranath et al., 2004). The same group further produced some indications that during early tooth development the murine ameloblastin 37 kDa isoform interacts with amelogenin (Ravindranath et al., 2007). However, there still remains a gap in our knowledge of how these proteins are distributed in the extracellular enamel matrix, what the contribution pattern of each particular protein is to the areas with co-localization during the different stages of tooth development, and also what structural changes occur when amelogenin and ameloblastin co-assemble.

In order to give clear insight into the putative cooperative function of ameloblastin and amelogenin we took advantage of quantitative co-localization techniques and performed a systematic analysis of the localization of ameloblastin with respect to amelogenin in the mouse mandibular molar. We performed an additional *in vitro* study using circular dichroism (CD) spectroscopy to show the structural changes in ameloblastin when it co-assembles with amelogenin. We concentrated on confocal images from mandibular first molars from postnatal mice from one to eight days old (here the day of birth was termed postnatal day 0). Previous research into the expression of ameloblastin and amelogenin by *in situ* hybridization using DNA biotinylated probes (Torres-Quintana et al., 2005) has indicated that mRNA expression of ameloblastin and amelogenin in mouse molars is first observed in pre-ameloblasts at the tip of the cusp at postnatal day 2 for ameloblastin and day 3 for amelogenin (the day of birth was considered as postnatal day 1) and that expression of both proteins was seen until amelogenin expression terminated in maturation-stage ameloblasts at postnatal day 10. In our present study we used samples up to postnatal day 8 to ensure that both amelogenin and ameloblastin will be present (**Figures 3A–D**). The present study showed that secretion of amelogenin and ameloblastin starts at the cuspal slopes of molars, as seen on postnatal day 1 (**Figures 3A**, **4A**, **5A**). We observed that secretion continues until at least postnatal day 8 (**Figures 3C**, **4C**, **6A**). This is in agreement with the study of mRNA expression of ameloblastin (Torres-Quintana et al., 2005) but amelogenin was observed here earlier than the first detection of mRNA expression of amelogenin in the previous study. This difference could be explained by individual difference between mice and their strains.

Quantitative co-localization analysis is potentially a powerful tool for studying extracellular matrix protein interactions, as spatially related proteins have a high probability of interacting. Moreover, significantly more data can be generated while eliminating bias and errors of visual interpretation (Costes et al., 2004). In a recent report (Gallon et al., 2013) we have used confocal microscopy to successfully determine co-localization between amelogenin and enamelin in enamel extracellular matrix.

We utilized the Mander's co-localization coefficients M1 and M2 as these coefficients are not dependent upon the intensities of the signals and can compare signal intensities for different fluorophores when their signal intensities differ (Manders et al., 1993). These coefficients give a measure of the amount of fluorescence of the co-localizing objects of the image relative to the total fluorescence. By plotting the co-localization co-efficients Mgreen and Mred of amelogenin or ameloblastin against the regions of interest, patterns of co-localization were revealed and comparisons were made. The use of an antibody against ameloblastin may allow us to detect the full-length protein as well as its proteolytic products containing that epitopes. Such products may include the equivalent to the reported rat ameloblastin proteolytic products (Uchida et al., 1997, 1998; Brookes et al., 2001). While the pattern of proteolysis of porcine ameloblastin is well documented (Chun et al., 2010), information regarding the rodent proteolytic pattern is limited. Here the 62, 40, 33, and 29 kDa products were detected by Western blot analysis of protein extracted from 9-dayold mice (**Figure 2**, lane 3). We followed the migration pattern of proteolytic products from the ameloblastin C-terminal in the ECM at day 8. It is notable that signals for amelogenin and/or ameloblastin were not so prominent within the ameloblasts. Following their secretion and processing by MMP-20, epitopes on both amelogenin and ameloblastin can be more available for interaction with the antibody in the ECM.

As seen in **Figure 7A**, the proportion of co-localization between ameloblastin and amelogenin varies with the postnatal age of the mouse. At early stages, when enamel is initially developing, the proteins co-localize with each other to a large degree and in the same proportions (**Figure 5F**). Since we observe that the contribution of each antigen to the areas with co-localization is high, i.e., ameloblastin and amelogenin strongly overlap with each other at day 1, we interpret the data to suggest that at initial stages of enamel development they co-assemble. With enamel maturation this pattern changes. For a time, on day 8, the proportion of co-localizing ameloblastin remain greater than the proportion of co-localizing amelogenin, indicating there is now more "free" or non-co-localizing amelogenin than "free" ameloblastin. This also indicates that there is more amelogenin present in the extracellular matrix than ameloblastin at later stages, which is verified by plotting the intensity ratio with age (**Figure 7B**). These findings are in accordance with the published report that the C-terminal polypeptides of ameloblastin are successively cleaved into smaller peptides and lost from the extracellular matrix (Uchida et al., 1997, 1998) at maturation stage. Co-localization of amelogenin proteolytic products and ameloblastin N-terminal is possible and it is a subject of ongoing investigation (Mazumder et al., in preparation).

#### **ACKNOWLEDGMENTS**

The study was funded by NIH-NIDCR grants DE-020099 and DE-013414. We thank Prof. Malcolm Snead for kindly providing us antibody against amelogenin, Prof Margarita Zeichner-David for providing the Schneider 2 cells and Mrs. Lisha Chen for technical assistance.

#### **REFERENCES**

amelogenin and ameloblastin. *Biochem. Biophys. Res. Commun.* 323, 1075–1083. doi: 10.1016/j.bbrc.2004.08.207

ribbon-like supramolecular structures via an N-terminal segment encoded by exon 5. *J. Biol. Chem.* 288, 22333–22345. doi: 10.1074/jbc.M113.456012

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 22 May 2014; paper pending published: 12 June 2014; accepted: 07 July 2014; published online: 25 July 2014.*

*Citation: Mazumder P, Prajapati S, Lokappa SB, Gallon V and Moradian-Oldak J (2014) Analysis of co-assembly and co-localization of ameloblastin and amelogenin. Front. Physiol. 5:274. doi: 10.3389/fphys.2014.00274*

*This article was submitted to Craniofacial Biology, a section of the journal Frontiers in Physiology.*

*Copyright © 2014 Mazumder, Prajapati, Lokappa, Gallon and Moradian-Oldak. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Role of mineralization inhibitors in the regulation of hard tissue biomineralization: relevance to initial enamel formation and maturation

# *Henry C. Margolis 1,2\*, Seo-Young Kwak1,2 and Hajime Yamazaki 1,2*

*1 Department of Applied Oral Sciences, Center for Biomineralization, The Forsyth Institute, Cambridge, MA, USA 2 Department of Developmental Biology, Harvard School of Dental Medicine, Boston, MA, USA*

#### *Edited by:*

*Bernhard Ganss, University of Toronto, Canada*

#### *Reviewed by:*

*Yuji Mishina, University of Michigan, USA Peter Proff, Regensburg University, Germany*

#### *\*Correspondence:*

*Henry C. Margolis, Department of Applied Oral Sciences, Center for Biomineralization, The Forsyth Institute, 245 First Street, Cambridge, MA 02142, USA e-mail<EMAIL_ADDRESS>
Vertebrate mineralized tissues, i.e., enamel, dentin, cementum, and bone, have unique hierarchical structures and chemical compositions. Although these tissues are similarly comprised of a crystalline calcium apatite mineral phase and a protein component, they differ with respect to crystal size and shape, level and distribution of trace mineral ions, the nature of the proteins present, and their relative proportions of mineral and protein components. Despite apparent differences, mineralized tissues are similarly derived by highly concerted extracellular processes involving matrix proteins, proteases, and mineral ion fluxes that collectively regulate the nucleation, growth and organization of forming mineral crystals. *Nature*, however, provides multiple ways to control the onset, rate, location, and organization of mineral deposits in developing mineralized tissues. Although our knowledge is quite limited in some of these areas, recent evidence suggests that hard tissue formation is, in part, controlled through the regulation of specific molecules that inhibit the mineralization process. This paper addresses the role of mineralization inhibitors in the regulation of biological mineralization with emphasis on the relevance of current findings to the process of amelogenesis. Mineralization inhibitors can also serve to maintain driving forces for calcium phosphate precipitation and prevent unwanted mineralization. Recent evidence shows that native phosphorylated amelogenins have the capacity to prevent mineralization through the stabilization of an amorphous calcium phosphate precursor phase, as observed *in vitro* and in developing teeth. Based on present findings, the authors propose that the transformation of initially formed amorphous mineral deposits to enamel crystals is an active process associated with the enzymatic processing of amelogenins. Such processing may serve to control both initial enamel crystal formation and subsequent maturation.

**Keywords: amorphous calcium phosphate, biomineralization, dental enamel, inhibitors, mineral phase transformation**

# **INTRODUCTION**

*Biomineralization* is the process by a wide variety of living organisms, including mollusks, sponges and unicellular diatoms, for example, produce functional mineralized tissues (Mann, 2001). Vertebrate mineralized tissues, like dental enamel, dentin, cementum, and bone, fulfill specialized functions that reflect differences in their hierarchical organization and composition (Weiner, 1986). Although each of these tissues is comprised of a crystalline calcium apatite mineral phase and a protein component, they differ with respect to overall structure, crystal size and shape, level and distribution of trace mineral ions, the nature of the proteins present, and the relative proportions of mineral and protein components. Differences in structural organization and composition give rise to mineralized tissues with different physical and mechanical properties that are well-suited for their intended biological purpose (Birchall, 1989; Currey, 1999).

Although the mechanisms by which these mineralized tissues form are not fully understood, it is apparent that the unique structure of each mineralized tissue, including dental enamel, is the result of highly concerted cell and extracellular processes that regulate the on-set, growth rate, shape, location and arrangement of forming mineral crystals (Weiner, 1986). Evidence also suggests that critical aspects of hard tissue formation are controlled, in part, through the regulation of specific molecules that inhibit mineralization. This paper addresses the role of mineralization inhibitors in the regulation of biological mineralization and the potential relevance of such mechanisms in the process of dental enamel formation (amelogenesis).

# **FUNDAMENTAL ASPECTS OF VERTEBRATE MINERALIZED TISSUE FORMATION**

#### **EXTRACELLULAR PROTEIN MATRIX AND MINERALIZED TISSUE COMPOSITION**

Biominerals are formed utilizing similar fundamental strategies, although there are unique differences that distinguish one tissue from another, especially dental enamel. Enamel, dentin and bone are each derived from specialized cells; ameloblasts, odontoblasts and osteoblasts, respectively. These cells secrete an extracellular protein matrix that is predominantly comprised of a hydrophobic protein and smaller amounts of acidic hydrophilic molecules. In bone and dentin, the matrix is mostly collagen, while the major enamel matrix component (>90%) is amelogenin. It has been proposed that biomineralization is generally regulated through interactions between hydrophobic components, which provide a skeletal or space-filling structure (e.g., collagen in bone and dentin), and hydrophilic (acidic) molecules (e.g., phosphophoryn in dentin Veis et al., 1991; He et al., 2005) that regulate crystal nucleation and growth (Weiner, 1986; Addadi and Weiner, 1992). Considerable evidence shows that a highly-ordered preassembled collagen matrix serves as a template to guide subsequent mineralization, as we have previously discussed (Margolis et al., 2006). The initial collagenous matrix is mineral free and undergoes a series changes in structure and composition prior to mineralization (Weinstock and Leblond, 1973; Septier et al., 1998; Beniash et al., 2000), resulting in the formation of tissues that are 40–50% mineral and ∼35% organic by volume (Nikiforuk, 1985). The protein matrix of forming enamel is similarly comprised of a predominant hydrophobic protein (amelogenin) and two key minor protein components enamelin (hydrophilic and acidic) and ameloblastin (amphiphilic and acidic). The observations that the amelogenin-null mouse (Gibson et al., 2001) exhibits a marked enamel phenotype and that enamel does not form in the absence of enamelin (Hu et al., 2008; Smith et al., 2009) or ameloblastin (Fukumoto et al., 2004; Smith et al., 2009; Wazen et al., 2009) are in agreement with the proposed general requirement for hydrophobic-hydrophilic molecular interactions in biomineral formation.

Despite similarities in the hydrophobic/hydrophilic composition of developing extracellular bone, dentine and enamel matrices that lead to the formation of a similar mineral phase (i.e., a carbonated hydroxyapatite), mature enamel and the mechanism of its formation differ from those of dentine and bone. First, long thin ribbons of enamel mineral begin to form almost immediately after ameloblasts lay down the enamel matrix (Nylen et al., 1963; Arsenault and Robinson, 1989; Smith, 1998), indicating that mineralization does not take place within a pre-assembled enamel matrix template, as in the case of collagen-based tissues. These long thin mineral ribbons extend hundred of microns to the full thickness of the enamel layer that is laid down during the secretory stage of amelogenesis, although the mineral component occupies only 10–20% of the enamel volume, with the remaining volume occupied by the enamel matrix and water (Robinson et al., 1988; Fukae, 2002). During the maturation stage of amelogenesis (Robinson and Kirkham, 1985), the extracellular enamel matrix is almost completely removed by resident proteases and the initially formed mineral ribbons grow in width and thickness to form a dense mineralized tissue that is >95% mineral by weight, with only 1–2 weight % of remaining protein and a small amount of water. Hence, there is an inverse relationship between the protein and mineral content during enamel development (Robinson et al., 1979), as illustrated in **Figure 1**. Unlike that seen in bone and dentin, the enamel matrix is transient and the resulting enamel tissue is almost fully mineralized. The transient nature

**FIGURE 1 | Inverse relationship between protein and mineral content as a function of enamel development.** Amelogenin is the major component (>90%) of the extracellular enamel matrix in the secretory stage. Adapted from a figure previously published by Mann (2001), with permission from Oxford University Press. This OUP material is for view only and cannot be reused.

of the enamel matrix is uncommon in biomineralization (Weiner, 1986).

#### **CALCIUM PHOSPHATE FORMATION** *IN VITRO* **AND** *IN VIVO*

The formation of a calcium phosphate mineral phase *in vitro* and *in vivo* requires a condition of supersaturation with respect to the forming phase. As shown in Equation 1 for hydroxyapatite (HA: Ca5OH(PO4)3), a prototype for enamel, dentin and bone mineral, the degree of supersaturation (DS) can be defined as the ratio of the mean ionic activity product for a given mineral phase in solution to its thermodynamic solubility product constant (e.g., KSP−HA) defined in the same manner, where "()" represent the activity of the enclosed ion [e.g., (Ca2+ *f* )]. The activity of an ion in solution is equal to the product of the concentration of the ion ([xZ i ]) and an ion activity coefficient (ai) that is a function of the ion size, charge, ionic strength and temperature. Such quantities can be calculated using ion-speciation software, as we have previously described (Moreno and Margolis, 1988). Supersaturation with respect to other calcium phosphate phases, such as octacalcium phosphate [OCP: Ca4H(PO4)3] and dicalcium phosphate dihydrate (DCPD: CaHPO4 . 2H2O), can be defined similarly as indicated in Equations (2) and (3), respectively. A minimum requirement for mineral formation *in vitro* or *in vivo* is the condition of supersaturation. As indicated by Equations 1–3, DS is a function of calcium, phosphate and hydrogen ion activities.

$$\mathrm{DS\_{HA}} = \left[ \left( \mathrm{Ca}\_{f}^{2+} \right)^{5} \left( \mathrm{OH}^{-} \right) \left( \mathrm{PO}\_{4}^{3-} \right)^{3} \right]^{1/9} \Bigg/ \left. \mathrm{K}\_{\mathrm{SP}-\mathrm{HA}} \tag{1} \right]$$

$$\mathrm{DS\_{OCP}} = \left[ \left( \mathrm{Ca}\_f^{2+} \right)^4 \left( \mathrm{H}^+ \right) \left( \mathrm{PO}\_4^{3-} \right)^3 \right]^{1/8} \bigg/ \mathrm{K}\_{\mathrm{SP-OCP}} \quad (2)$$

$$\text{DS}\_{\text{DCPD}} = \left[ \left( \text{Ca}\_{\text{f}}^{2+} \right) \left( \text{HPO}\_{4}^{2-} \right) \right]^{1/2} \int \text{K}\_{\text{SP}-\text{DCPD}} \tag{3}$$

where, (X*z i*) = a*i* × [X*z i*] and Ca2+ *f* is *free* calcium.

Regarding the mineralization process, a given solution can be supersaturated with respect to more than one calcium phosphate phase at the same time. Due, in part, to kinetic factors (for review, see Mann, 2001), however, the more soluble phase will form first and, through a series of *phase transformations*, convert to HA, which is the most thermodynamically stable calcium phosphate phase of the group. This phenomenon, called the *Ostwald-Lussac law of stages*, occurs in nature, as it does *in vitro* (Eanes, 2001). For example, it has been shown (Schroeder, 1969) that DCPD and OCP are the first mineral phases seen in forming dental calculus that subsequently convert to HA over time. Consistent with this observation and *Ostwald-Lussac law of stages*, at physiological pH (pH 7) and ionic strength (163 mM), HA has a relatively low solubility (expressed as the concentration of dissolved Ca2+) of 0.11 mM, in comparison to OCP (1.42 mM) and DCPD (2.09 mM), as calculated (Moreno and Margolis, 1988). Such transformations may go through multiple phases before reaching the stable HA phase, depending on the free energy of activation (-G) associated with nucleation (n), growth (g), and phase transformation (t), as previously discussed and illustrated in **Figure 2** (Colfen and Mann, 2003).

Based on the discussion above, the role of *precursor* phases involved in HA and biomineral formation has received considerable attention. The calcium phosphate mineral phases discussed so far are crystalline in nature, as confirmed using spectroscopic (FT-IR, Raman) and diffraction (X-ray and electron diffraction) methods. However, it has been known for many years (for review, see Eanes, 2001) that amorphous calcium phosphate (ACP) is the first mineral phase to form upon mixing sufficiently high concentrations of calcium and phosphate. ACP is more soluble than the noted crystalline calcium phosphates and is the kinetically favored product (Meyer and Eanes, 1978), although it readily undergoes phase transformation to HA, as shown in

**Figure 3**. The presence of ACP in vertebrate mineralized tissues (Posner and Betts, 1975) has been a topic of considerable debate (Termine and Posner, 1966; Boskey, 1997; Eanes, 2001; Weiner, 2006) because of its transient nature. However, recent evidence suggests that the transient nature of an amorphous mineral phase, that is, the formation of an amorphous mineral phase and its subsequent transformation to a crystalline solid, is a key element in biological mineral formation (Beniash et al., 1997; Weiner et al., 2005; Deshpande and Beniash, 2008; Kwak et al., 2009; Margolis and Beniash, 2010; Wiedemann-Bidlack et al., 2011). More recently, the importance of sequential steps involving ACP formation and transformation has been confirmed *in vitro* (Habraken et al., 2013), showing that the existence of pre-nucleation clusters that precede ACP formation lowers the energy barrier to nucleation and makes amorphous phases "accessible" under conditions that would favor the formation of more stable crystalline phases. Hence, it appears calcium phosphate formation *in vivo* and *in vitro* similarly proceed through a sequence of phase transformations, potentially involving: ACP → DCPD → OCP → HA. As will be discussed below, however, matrix molecules can dramatically influence the phase transformation process.

# **ROLE OF MINERALIZATION INHIBITORS IN BIOLOGICAL MINERALIZATION**

#### **STABILIZATION OF SUPERSATURATION** *IN VIVO*

As illustrated in **Figure 3**, under conditions of relatively high supersaturation brought about by the rapid mixing of calcium and phosphate solutions, mineralization takes place spontaneously. This process involves the *homogeneous* nucleation and formation of precursor ACP nanoparticles, which subsequently transform to crystalline HA. In the absence of a buffer, this process causes a sharp decrease in pH due to the precipitation of a *basic* solid phase (**Figure 4**). In the presence of the full-length native amelogenin, P173, the solution pH does not drop and remains relatively constant for long periods of time, as illustrated in **Figure 4**, by preventing the bulk formation of HA crystals, as shown in **Figure 5** (Wiedemann-Bidlack et al., 2011). As can be seen in this TEM micrograph, P173 has the capacity to stabilize initially formed nanoparticles of ACP and prevent transformation to crystalline HA, as seen in the control (**Figure 3**). Of note, truncated native amelogenin, P148 that lacks the hydrophilic Cterminus was similarly found to be a potent stabilizer of ACP (Kwak et al., 2009). Hence, P173 and P148 are inhibitors of spontaneous HA crystal formation. In doing so, the supersaturation status of the experimental solution is also maintained, along with its inherent *stored* capacity to promote mineralization. Notably, native amelogenins like P173 and P148 contain a single phosphorylated site (serine-16) within the N-terminus (Margolis et al., 2006), as further discussed below.

The stabilization of biological fluids through the inhibition of HA crystal formation is well known and appears to fulfill biologically important functions. A prime example is human saliva that has been shown (Hay et al., 1982) to be supersaturated with respect to all known calcium phosphate phases. Evidence suggests that this condition of supersaturation is maintained by a family of salivary phosphoproteins, which have been shown to

**FIGURE 3 | TEM micrographs of calcium phosphate mineral products formed in the absence (control) of protein examined at selected times: 15 min (A), 45 min (B), 1–4 h (C), and 1 d (D).** As shown at 15 min **(A)**, amorphous calcium phosphate (ACP) was initially formed based on the

observed (inset) selected area electron diffraction (SAED) pattern. At 45 min, ACP phase transformation could be seen. After 1–4 h, randomly arranged plate-like apatitic crystals were found and confirmed by SAED analyses (**D**, inset). This image was reproduced from Kwak et al. (2009).

be potent inhibitors of spontaneous calcium phosphate formation and seeded HA crystal growth *in vitro* (e.g., Hay et al., 1979). Under these conditions that persist in human saliva, calcium phosphate formation on hard and soft tissue surfaces in the oral cavity is prevented, as is the dissolution of tooth enamel in saliva. If saliva did not contain calcium and phosphate ions it would be *undersaturated* with respect to tooth enamel (i.e., with DSEN < 1) and potentially promote the dissolution of enamel surfaces, albeit at a very slow rate. In fact, the observed spontaneous reversal of untreated early carious lesions (Koulourides et al., 1965; Dirks, 1966), where incipient lesions (i.e., white spots) take up mineral ions to become sound surfaces, can be attributed to the condition of saliva supersaturation. Hence, the prevention of unwanted mineral deposits in the oral cavity and the capacity of saliva to promote the repair (remineralization) of beginning enamel lesions is due to resident inhibitors of calcium phosphate formation that maintain saliva in its supersaturated state. Similarly, inhibitors of calcium oxalate formation have been

**formed in the presence of native full-length amelogenin, P173, at 2 mg/mL.** Only ACP was observed, even after 1 d, as confirmed by selected area electron diffraction (inset). This image was reproduced from Wiedemann-Bidlack et al. (2011).

identified in urine and are believed to play an important role in preventing the formation of kidney stones (e.g., Nene et al., 2013). Finally, it has been shown that spontaneous calcification of arteries and cartilage takes place in mice that lack the mineral binding matrix GLA protein, a known mineralization inhibitor. These latter observations, lead the authors of that study to conclude that the calcification of the extracellular matrix is a ubiquitous process that must be actively inhibited in soft tissues that do not normally mineralize (Luo et al., 1997).

#### **REGULATION OF BIOMINERAL FORMATION**

Mann (2001) summation that *"The chemical control of crystallization pathways that involve a sequence of kinetic inhibition and* *phase transformation can result in a high degree of selectivity in crystal structure and composition,"* is perhaps best illustrated in biology where numerous inhibitors of HA formation have been identified, including magnesium ion, carbonate, proteoglycans, polyphosphate and other phosphorylated molecules. In particular, inorganic pyrophosphate (PPi) has been long recognized as a potent inhibitor of mineralization and as a potential regulator of biological mineralization. At micro-molar levels, PPi inhibits the growth (Francis, 1969; Meyer and Nancollas, 1973; Meyer, 1984; Moreno et al., 1987) and dissolution (Fleisch et al., 1966; McGaughey and Stowell, 1977; McGaughey, 1983) of HA, as well as HA crystal formation from ACP (Termine et al., 1970; Robertson, 1973; Blumenthal et al., 1975). A study in our laboratory (Moreno et al., 1987) indicated that the inhibition of HA crystal growth results from PPi adsorption to discrete growth sites on HA surfaces, in the same fashion that has been proposed to explain the inhibition of crystal growth by macromolecules (Moreno et al., 1979). Stabilization of ACP may similarly involve PPi binding to ACP nanoparticles (Ofir et al., 2004).

Based on additional *in vitro* findings (Meyer, 1984) that suggest calcification cannot occur *in vivo* in the presence of physiological levels of PPi, it was proposed (Meyer and Reddi, 1985) that pyrophosphatases regulate mineral deposition in bone and cartilage by hydrolyzing PPi (to two molecules of orthophosphate) to eliminate its inhibitory activity. This overall concept has been supported by the elucidation (Ho et al., 2000) of the role of *progressive ankylosis* (*ank*) gene that encodes a transmembrane protein that appears to regulate calcification (and arthritis) by controlling both intracellular and extracellular levels of PPi. Additional evidence has also been obtained showing that tissue non-specific alkaline phosphatase (TNAP) and plasma cell membrane glycoprotein-1 (PC-1) are key enzyme regulators of the extracellular PPi concentrations required for controlled bone mineralization (Hessle et al., 2002). PC-1 is the major producer of the PPi inhibitor, while TNAP can regulate the promotion of the onset of bone mineralization by hydrolyzing PPi. Consistent with this idea, it has been demonstrated *in vitro* that upon the removal of PPi from solution, either through adsorption or by HA surface-induced hydrolysis (Meyer, 1984; Litman and Margolis, unpublished findings), or by enzymatic degradation of PPi, PPi-stabilized calcium phosphate solutions rapidly form calcium phosphates.

With respect to amelogenesis, alkaline phosphatase (AP) has been found in secretory enamel, leading investigators to suggest that "dephosphorylation" of amelogenin (e.g., by AP) may play a role in regulating enamel formation (Brookes et al., 1998). Although this suggestion has not been fully substantiated *in vivo*, a study using cultured embryonic tooth germs showed that nonspecific inhibition of phosphorylation by casein kinase impaired enamel formation (Torres-Quintana et al., 2000), lending support to the idea that phosphorylated enamel matrix proteins may play a role as mineralization inhibitors. More recently, it has been reported that TNAP is expressed by normal ameloblasts in a stage-specific manner, that is, at the point of transition from secretory to maturation stage (Yadav et al., 2012). In addition, AP was found to be highly expressed by the adjacent stratum intermedium of the developing enamel organ. Importantly, this latter study also showed that the TNAP knockout mice not only exhibited a phenotype similar to infantile hypophosphatasia, but also marked enamel defects, including reduced mineralization and disrupted rod and interrod structures. Hence, the reversal of mineralization inhibition, by PPi hydrolysis or dephosphorylation, may represent a critical regulatory step in the formation of both developing bones and teeth.

Upon mineral growth, macromolecules found in bones and teeth can also effectively influence the *morphology* of calcium phosphate minerals through adsorption to specific crystal planes (e.g., Furedi-Milhofer et al., 1994; Flade et al., 2001).  Consequently, under these conditions, such crystals grow preferably along the long c-axis forming ribbon or needlelike structures resembling enamel crystals. As described in the next section, current evidence suggests that native amelogenins have the capacity to stabilize ACP, prevent unwanted crystallization, and ultimately guide the formation and organization of bundles of aligned enamel crystals during the secretory stage of enamel formation. The authors hypothesize, however, that the transformation of ACP to HA and the subsequent growth of HA crystals in thickness and width during maturation are the result of active regulation processes.

### **MECHANISM OF DENTAL ENAMEL FORMATION**

Studies from our laboratory suggest that amelogenin mediates mineralization through a cooperative mechanism where mineralization and protein self-assembly occur simultaneously (Beniash et al., 2005). This conclusion is based on *in vitro* findings that show that the non-phosphorylated full-length recombinant mouse amelogenin, rM179, can guide the formation of well-aligned bundles of HA crystals *in vitro*, under experimental conditions designed to simultaneously induce amelogenin assembly and mineralization. Organized mineral formation was not observed when protein self-assembly was carried out prior to mineralization or when a truncated recombinant mouse amelogenin (rM166, lacking the hydrophilic C-terminus) was used. Similar results were subsequently obtained under cooperative assembly/mineralization conditions using recombinant fulllength porcine amelogenin, rP172, which was also found to guide the formation of bundles of aligned HA crystals (Kwak et al., 2009). These latter experiments were monitored as a function of time to provide insight into the mechanism of amelogeninmediated mineralization. These studies revealed that rP172 transiently stabilizes ACP nanoparticles (**Figure 6**), which were found to be significantly smaller than those seen in the control (**Figure 3**). As the reaction continued in the presence of rP172, an alignment of ACP nanoparticles was observed at ∼45 min. Arrays of needle-like crystals were then observed between 1–4 h. At 24 h, as shown, multiple bundles of well-aligned needle-like apatite crystals were seen that are similar to those found in the presence of rM179 (Beniash et al., 2005) and distinctly different

**FIGURE 6 | TEM micrographs of calcium phosphate mineral products formed in the presence of 2 mg/ml recombinant full-length amelogenin, rP172, examined at selected times: 15 min (A), 45 min (B), 1–4 h (C), and 1 d (D).** ACP particles seen at 15 min (**A**, inset) were found

to align to form need-like particles (arrows) at 45 min **(B)**. After 1 d, based on selected area electron diffraction analyzes (**D**, inset), aligned bundles of needle-like apatitic crystals were formed. This image was reproduced from Kwak et al. (2009).

in comparison to the randomly arranged plate-like crystals found in the control (at 24 h, **Figure 3**). Truncated recombinant (nonphosphorylated) porcine amelogenin rP147 had relatively little effect on the mineralization process (Kwak et al., 2009), again indicating that the full-length amelogenin has a unique capacity to guide ordered mineralization. This unique capacity is attributed to the fact that full-length recombinant mouse (rM179) and porcine (rP172) amelogenin, as well as full-length native amelogenin (P173), undergo self-assembly to form hierarchical nanosphere (Fang et al., 2011, 2013) and chain-like (Wiedemann-Bidlack et al., 2007, 2011) structures, as a function of solution pH. Truncated amelogenins lacking the hydrophilic C-terminus do not form such organized structures. Notably, the single phosphate group in native amelogenins on serine-16 was found to have only a subtle influence on P173 assembly and structure, as determined by both conventional (Wiedemann-Bidlack et al., 2011) and cryo-TEM (Fang et al., 2013). However, small-angle X-ray scattering studies using phosphorylated and non-phosphorylated leucine-rich amelogenin peptides (LRAP, an alternative splice product that is comprised of the first 33 amino acids and the last 23 C-terminal amino acids of full-length (porcine) amelogenin) showed that phosphorylation of serine-16 had a significant effect on the folding of the peptide in solution (Le Norcy et al., 2011a). LRAP(+P) exhibited an unfolded structure, while LRAP(-P) exhibited a globular structure in solution. LRAP(+P), like P173, was also found to effectively stabilize ACP and inhibit HA formation *in vitro* (Le Norcy et al., 2011b). These influences on protein structure and folding may be of considerable functional importance, given the marked effect phosphorylation has on the capacity of native amelogenins to stabilize ACP and inhibit HA formation for extended periods of time, as further discussed below.

In recent years, two prominent theories of crystal growth aptly named the classical and non-classical crystal growth theories, have received considerable attention, in part, through detailed investigations into the growth mechanisms of biominerals (Niederberger and Colfen, 2006). The classical mechanism for crystal growth is mineral ion mediated where mineral growth progresses through the successive accumulation of mineral ions to form a single crystal. The non-classical mechanism for crystal growth is mediated by initially formed nano-particles that subsequently assemble in an organized fashion to form what appear to be single crystals (so-called mesocrystals). Although it is often difficult to distinguish between these two mechanisms, early studies (Robinson et al., 1981; Robinson, 2007) have suggested that, during amelogenesis, enamel crystals form through the assembly of small amorphous mineral particles, although the amorphous nature of these particles was not verified experimentally. Since this initial proposal, however, data have been obtained that support the presence of ACP in the developing enamel matrix (Diekwisch, 1998; Bodier-Houlle et al., 2000). In a more recent study (Beniash et al., 2009), multiple independent techniques were used to carefully characterize the phase of the long thin parallel arrays of mineral deposits (e.g., as seen in **Figure 7**) that form during the secretory stage of amelogenesis that begin near the DEJ and extend to the enamel surface. Newly deposited mouse enamel, that is, in the outer enamel layers closer to the enamel surface and the ameloblast layer was found to be ACP, while older enamel, found in inner layers of enamel closer to DEJ contained apatitic crystals. Of particular importance, the newly deposited amorphous mineral and the older crystalline deposits had the same ribbon-like shape and organization in parallel arrays. These findings suggest that enamel mineral morphology and organization are established by the enamel matrix prior to the transformation of ACP to HA-like enamel crystals. These findings are also consistent with a proposed hypothesis that initial enamel crystals form from the assembly and fusion of amorphous calcium phosphate particles (e.g., Robinson, 2007). Transient amorphous mineral phases (i.e., ACP and amorphous calcium carbonate) have also been observed to be associated with a number of other biomineralization systems (Lowenstam and Weiner, 1985, 1989; Beniash et al., 1997; Wu et al., 1997; Levi-Kalisman et al., 2000; Aizenberg et al., 2002), including bone (Kim et al., 2005; Crane et al., 2006; Mahamid et al., 2008), leading to the proposal that the transformation of amorphous phases to crystalline materials is an important aspect of biological mineralization (Beniash et al., 1997; Weiner et al., 2005). Other molecules believed to be associated with the regulation of biomineralization, like highly phosphorylated osteopontin from milk (Gericke et al., 2005) and the N-terminal domain of dentin matrix protein 1 (Gajjeraman et al., 2007) have been shown to stabilize ACP.

## **POTENTIAL ROLE OF MINERALIZATION INHIBITORS IN DENTAL ENAMEL FORMATION**

Based on current evidence, inhibition of mineralization by native amelogenins and its reversal *in vivo* may play critical roles in enamel formation. This conclusion is supported by data showing (1) an inverse relationship between protein and mineral content (**Figure 1**), and the notably high protein and low mineral content (10–20% by volume) of secretory stage enamel; (2) the capacity of native amelogenins to stabilize ACP and inhibit mineralization *in vitro*; (3) the apparent requirement for both proteases [i.e., matrix metalloproteinase 20 (MMP20) during the secretory stage Caterina et al., 2002 and kallikrein 4 (Klk4) during transition/maturation Hu et al., 2000; Simmer et al., 2009] and phosphatases (i.e., AP, as discussed above) for proper enamel formation; and (4) the rapid onset of mineral growth coinciding with loss of protein (**Figure 1**). These collective data suggest that during the secretory stage of enamel formation, enamel matrix proteins selectively guide ACP nanoparticle organization and transformation to thin ribbons of co-aligned HA crystals, while at the *same time* preventing unwanted bulk mineralization. Support for this scenario was recently provided by *in vitro* findings showing that under the proper kinetic conditions, controlled by variations in protein concentration, significantly lower concentrations of P173 can guide the transformation of initially stabilized ACP nanoparticles to ordered bundles of HA crystals that have similar morphology to initially formed enamel crystals (Kwak et al., 2014). In contrast to that seen with P173, however, P148 lacked the capacity to regulate ordered mineralization at reduced protein concentrations, although mineralization did take place. At higher concentrations, again, both P173 and P148 are potent stabilizers of ACP. Hence, upon its secretion, the parent phosphorylated full-length amelogenin P173 can initially prevent premature mineralization of HA crystals in forming enamel. However, full-length amelogenin undergoes proteolytic processing soon after secretion, resulting in a significant *decrease* in its concentration. In developing porcine enamel, the P173 content of secretory enamel was found to be only 7.4% of total amelogenin, a reduction in P173 by more than 90% (Wen et al., 1999). P173 was also found to be exclusively associated with newly formed enamel mineral (Uchida et al., 1991). In contrast to P173, the P148 content of the enamel matrix *increases* and becomes the predominant amelogenin cleavage product at 49.5% of total amelogenin (Wen et al., 1999). Based on these combined *in vitro* and *in vivo* findings, the authors propose that the onset of enamel crystal formation is regulated by a marked reduction (>10-fold) in P173 concentration to attain a critical lower level that has the potential to organize forming mineral deposits into oriented bundles of elongated HA crystals, as illustrated in a proposed model presented in **Figure 8**. At the same time, substantially higher concentrations of P148 (∼ 6–7 times higher than P173) that accumulate in the developing (porcine) enamel matrix serve an important role by preventing uncontrolled mineralization throughout the secretory stage of amelogenesis where the mineral volume is only 10–20% (Robinson et al., 1988; Fukae, 2002).

The noted proteolytic processing of amelogenin during the secretory stage of amelogenesis has been primarily attributed to MMP20 that is exclusively expressed during this initial stage of enamel formation. MMP20 has also been shown to be essential for proper enamel formation (Caterina et al., 2002). In its absence, the enamel layer is about one-half the normal thickness

P148 is eliminated by Klk4 proteolysis, along with small amounts of adsorbed P173 growth inhibitor, to initiate the lateral growth of initial enamel ribbons **(C)**. As noted in the text, AP may also play an essential role in regulating mineral formation in the secretory stage and mineral growth during the maturation stage by catalyzing amelogenin dephosphorylation.

**mineral.**

Margolis et al. Regulation of enamel mineral formation

and the decussating prism pattern of normal enamel is substantially disrupted. These latter findings verify that proteolytic processing of full-length amelogenin is required for proper enamel formation, consistent with our proposed model, that is based on the capacity of reduced levels of P173 to guide ordered HA crystal formation, while accumulating P148 levels maintain mineralization driving forces by preventing unwanted bulk mineralization. During maturation, Klk4 is predominantly expressed and is believed to be responsible for removing almost all of the remaining enamel matrix to allow the initial enamel crystals to grow in width and thickness (Hu et al., 2000). Hence, as illustrated in the presented model, we propose that the onset of mineralization during the secretory stage is brought about by the marked reduction of P173 content by MMP20, reversing its inhibitory effect. During maturation, inhibition of bulk mineralization provided by P148 is eliminated by Klk4 proteolysis, along with small amounts of adsorbed P173 growth inhibitor, to initiate the lateral growth of the secretory stage enamel ribbons. As noted above, AP may also play an essential role in regulating mineral formation in the secretory stage and mineral growth during the maturation stage by catalyzing amelogenin dephosphorylation and removing the noted mineralization inhibitory activity found to be associated with native amelogenins. The timing of the expression of key regulators of biomineralization during enamel formation is, therefore, of critical importance (e.g., Smith, 1998; Simmer et al., 2009). Additional studies addressing the potential roles of amelogenin proteolysis and de-phosphorylation in regulating initial enamel mineral formation and maturation are currently underway in our laboratory.

## **CONCLUSIONS**

Mineralization inhibitors can play critical roles in regulating mineralized tissue formation, stability, and regeneration *in vivo*. With respect to dental enamel formation, mineralization events triggered by the proteolysis and/or de-phosphorylation of full-length native amelogenin during the secretory stage of amelogenesis may play an essential role in regulating enamel mineral formation and its hierarchical structure, while predominant amelogenin degradation products, like P148, serve to prevent uncontrolled mineral formation. During maturation, residual inhibition is removed via additional matrix processing, allowing initial enamel crystals to expand to form the highly dense enamel mineral layer.

#### **ACKNOWLEDGMENT**

This work is supported by NIDCR grant R01-DE023091 (Henry C. Margolis).

#### **REFERENCES**

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 15 July 2014; paper pending published: 11 August 2014; accepted: 19 August 2014; published online: 10 September 2014.*

*Citation: Margolis HC, Kwak S-Y and Yamazaki H (2014) Role of mineralization inhibitors in the regulation of hard tissue biomineralization: relevance to initial enamel formation and maturation. Front. Physiol. 5:339. doi: 10.3389/fphys.2014.00339*

*This article was submitted to Craniofacial Biology, a section of the journal Frontiers in Physiology.*

*Copyright © 2014 Margolis, Kwak and Yamazaki. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Preferential and selective degradation and removal of amelogenin adsorbed on hydroxyapatites by MMP20 and KLK4 *in vitro*

#### *Li Zhu1, Haichuan Liu2, H. Ewa Witkowska2, Yulei Huang3, Kataro Tanimoto4 and Wu Li 1 \**

*1 Department of Orofacial Sciences, School of Dentistry, University of California, San Francisco, San Francisco, CA, USA*

*2 Department of Obstetrics, Gynecology and Reproductive Sciences, University of California, San Francisco, San Francisco, CA, USA*

*3 Department of Oral Medicine, Guanghua School of Stomatology, Sun Yat-sen University, Guangdong, China*

*4 Departments of Orthodontics and Craniofacial Developmental Biology, Hiroshima University Graduate School of Biomedical Sciences, Hiroshima, Japan*

#### *Edited by:*

*Megan Pugach-Gordon, The Forsyth Insitute, USA*

#### *Reviewed by:*

*Tom Diekwisch, University of Illinois at Chicago, USA John D. Bartlett, The Forsyth Institute, USA*

#### *\*Correspondence:*

*Wu Li, Department of Orofacial Sciences, School of Dentistry, University of California, San Francisco, Parnassus Avenue, San Francisco, CA 94143, USA e-mail<EMAIL_ADDRESS>
The hardest tooth enamel tissue develops from a soft layer of protein-rich matrix, predominated by amelogenin that is secreted by epithelial ameloblasts in the secretory stage of tooth enamel development. During enamel formation, a well-controlled progressive removal of matrix proteins by resident proteases, Matrix metalloproteinase 20 (MMP20), and kallikrein 4 (KLK4), will provide space for the apatite crystals to grow. To better understand the role of amelogenin degradation in enamel biomineralization, the present study was conducted to investigate how the adsorption of amelogenin to hydroxyapatite (HAP) crystals affects its degradation by enamel proteinases, MMP20 and KLK4. Equal quantities of amelogenins confirmed by protein assays before digestions, either adsorbed to HAP or in solution, were incubated with MMP20 or KLK4. The digested samples collected at different time points were analyzed by spectrophotometry, SDS-PAGE, high performance liquid chromatography (HPLC), and LC-MALDI MS/MS. We found that majority of amelogenin adsorbed on HAP was released into the surrounding solution by enzymatic processing (88% for MMP20 and 98% for KLK4). The results show that as compared with amelogenin in solution, the HAP-bound amelogenin was hydrolyzed by both MMP20 and KLK4 at significantly higher rates. Using LC-MALDI MS/MS, more accessible cleavage sites and hydrolytic fragments from MMP20/KLK4 digestion were identified for the amelogenin adsorbed on HAP crystals as compared to the amelogenin in solution. These results suggest that the adsorption of amelogenin to HAP results in their preferential and selective degradation and removal from HAP by MMP20 and KLK4 *in vitro.* Based on these findings, a new degradation model related to enamel crystal growth is proposed.

**Keywords: amelogenin, tooth enamel, MMP20, KLK4, protein interactions, hydroxyapatites, biomineralization, proteolysis**

# **INTRODUCTION**

The superficial layer of a tooth is enamel, the hardest tissue known in vertebrates. The unique morphological structure and outstanding mechanical properties of the enamel make it different from other mineralized tissues in the body, such as bone, dentin, and cementum. Tooth enamel is comprised mainly of numerous hexagonal carbonated hydroxyapatite (HAP) crystals, organized into rod, and interrod structures with distinct mechanical properties (Ichijo et al., 1992; Plate and Hohling, 1994; Pergolizzi et al., 1995).

The enamel crystals are very thin (20–30 nm in diameter) but extremely long. Many investigators believe that these crystals span the entire thickness of tooth enamel, a distance up to 2.5 mm (Leblond and Warshawsky, 1979; Daculsi et al., 1984; Nanci et al., 1998). The unique morphology and organization of enamel crystals determine its excellent mechanical characteristics while also raise a persistent question of how these enamel crystals form in such a special shape. Solving this puzzle will advance our knowledge of the basic principle of amelogenin-mediated mineralization during enamel development, help us to better understand the fundamental mechanism of an enamel disease, amelogenesis imperfecta, and provide useful information for future acellular tooth enamel regeneration.

The hard enamel tissue develops from a soft protein-rich extracellular matrix secreted by epithelial ameloblasts during the secretory stage of enamel development. At later stages of enamel formation, a well-controlled progressive removal of matrix proteins by resident proteases will provide space for the apatite crystals to grow (Bartlett and Simmer, 1999).

The protein matrix of forming enamel is composed predominantly of amelogenins and its cleavage products (Termine et al., 1980; Fincham et al., 1999). Amelogenin protein is hydrophobic in nature except for its highly charged C-terminus. Amelogenins self-assemble into highly packed and tightly associated nanospheres that interact with apatite crystals to provide the supportive framework for the growth of newly formed crystals with extremely thin and long structure (Robinson et al., 1998; Wen et al., 1999). This intercrystalline protein matrix also serves to prevent premature crystal–crystal fusion during the early stages of enamel formation (Moradian-Oldak et al., 2001).

It has been suggested that amelogenins are critical for the organization of the crystal pattern and regulation of enamel thickness. *In vitro* mineralization studies show that amelogenin can control crystal orientation and growth habit as well as facilitate aggregation of pre-formed hydroxyapatite crystals (Iijima et al., 2002). Mutation of amelogenin can cause amelogenesis imperfecta, a disorder of tooth enamel (Hart et al., 2002; Wright et al., 2003). The essential role of amelogenin in tooth enamel development is also confirmed by the amelogenin-null mice that exhibit defected amelogenesis phenotype with much thinner enamel layers (Gibson et al., 2001).

During enamel mineralization, the step-wise increase in mineral content is concomitant with progressive degradation of amelogenin in the enamel matrix. In particular, during the later stages, amelogenin proteins disappear rapidly and completely to facilitate crystal growth (Fukae and Shimizu, 1974). Matrix metalloproteinase 20 (MMP20) and kallikrein 4 (KLK4) are considered to be the two major amelogenin-processing enzymes during the early and late stages of enamel development, respectively (Bartlett et al., 1996; Bartlett and Simmer, 1999; Simmer and Hu, 2002). However, the relationship between apatite surface, enamel proteases, and enamel crystal growth remains yet to be explored. In this study, we focus on how the adsorption of amelogenin to HAP crystals affects its degradation by enamel proteinases, MMP20 and KLK4. We hypothesize that the binding of amelogenin to apatite may induce protein conformational change and subsequently affect the susceptibility of the adsorbed amelogenin to proteinases. The crystal-protein and protein-proteinase interactions are dynamically coordinated to regulate the mineral accretion. In the present study we have determined the effect of the apatite surface on enamel protease activity and amelogenin cleavage as a means to explain enamel crystal growth through proteolytic amelogenin degradation.

#### **MATERIALS AND METHODS**

#### **SAMPLE PREPARATION OF PROTEINS BOUND TO HAP**

Recombinant human amelogenin (rh174) and MMP20 were produced and purified as previously described (Li et al., 1999; Wang et al., 2006). Commercial recombinant human KLK4 was purchased from R&D Systems (Minneapolis, MN, USA). Hydroxyapatite (HAP) was synthesized according to the method reported by Nelson et al. and identified by X-ray diffraction as described in our previous publication (Zhu et al., 2009).

Amelogenin was incubated with HAP crystals at amount over the saturation binding (875 ± 37µg/m2) (Tanimoto et al., 2008a,b; Zhu et al., 2011). After 2 h incubation at room temperature with constant shaking, the protein-HAP complex was centrifuged at 5000 rpm for 5 min. The amount of protein bound was determined by measuring the change in concentration of protein solution before and after binding using Braford assay (BioRad, Hercules, CA).

#### **REMOVAL OF AMELOGENIN FROM HAP BY MMP20 AND KLK4 DIGESTION**

The digestion of HAP-bound amelogenin (0.2 mg) with MMP20 was performed with enzyme/substrate ratio of 1: 50 (W:W) in 100µl of reaction buffer (50 mM Tris-HCl, 150 mM NaCl, 10 mM CaCl2, 10µM ZnCl2, pH 7.5). At different time points after incubation on an orbital shaker at 37◦C, the samples were boiled for 10 min to inactivate MMP20. After centrifugation at 5000 × rpm for 5 min, the supernatant was carefully and completely removed from the HAP sediment. The HAP was dissolved in 10µl of 0.1% TFA and then reaction buffer was added to bring the volume up to final 100µl and the pH to neutral. The amount of protein released from HAP during incubation and the amount retained on the mineral were quantitatively determined by UV detection at wavelength 220 nm. Control samples, including HAP-bound amelogenin only and HAP with addition of MMP20 only, were treated in the same manner. MMP20 was replenished at same amounts after 24 h of incubation. The experiments were conducted in triplicate, and the average OD values and standard deviations were calculated. The removal of adsorbed amelogenin by KLK4 was carried out in a buffer with 50 mM Tris-HCl at pH 7.5, 250 mM NaCl and 50 mM CaCl2 under the similar conditions and analyzed by an identical methodology as described above.

#### **COMPARATIVE DIGESTION OF HAP-BOUND AND HAP-FREE AMELOGENIN IN SOLUTION**

Same amounts of HAP-bound and unbound amelogenin were digested with MMP20 and KLK4 at enzyme/substrate ratio of 1:50 in equal volumes of their corresponding reaction buffers in a time-course manner. The digestions were carried out at 37◦C under continuous shaking. At each time point, 50µl sample was withdrawn and the hydrolysis was stopped by boiling in a water bath for 10 min. The digested samples were then subjected to SDS-PAGE, reversed-phase high performance liquid chromatography (HPLC), and liquid chromatography matrix-assisted laser desorption ionization tandem mass spectrometry (LC-MALDI MS-MS).

In SDS-PAGE experiments, the digested samples (15 ul for MMP20 or 25 ul for KLK4) were mixed with SDS sample buffer, and resolved by using a 15% SDS-PAGE gel. Prior to HPLC separation, equal volume of 0.1% TFA was added to all the samples to dissolve HAP and release the retained proteins. The HPLC analyses were performed using a C4 column (Varian, Walnut Creek, CA, USA). A linear gradient from 0 to 95% acetonitrile (ACN) in 0.1% TFA was run over a period of 45 min at a flow-rate of 1 ml/min. Elution was monitored via absorbance at 214 nm. The peak areas (mAU/ml) of remaining substrate after hydrolysis were measured and compared.

After 36 h digestion, the samples were centrifuged at 5000 rpm for 5 min. The HAP pellets were dissolved in 50 µl of 0.1% TFA. Both proteolytic fragments still bound to the HAP and those released into the solution were identified by LC-MALDI MS/MS.

#### **EFFECT OF BARE HAP ON MMP20 ACTIVITY**

To determine whether HAP affects MMP20 activity, we added different amount of HAP into the 96-well plate that contained MMP20 and its substrate, a quenched fluorescent peptide [Mca-Pro-Leu-Gly-Leu-Dap (Dnp)-Ala-Arg-NH2; Bachem, CA, USA] in 200µL of MMP20 reaction buffer. The digestion was conducted at the enzyme/susbtrate ratio of 1:100. We measured hydrolysis of the substrate by monitoring fluorescence every 5 min for 1 h at 37◦C with 320 nm excitation and 405 nm emission.

#### **RESULTS**

### **MMP20 AND KLK4 DIGESTION PROGRESSIVELY AND EXTENSIVELY REMOVE AMELOGENIN FROM HAP**

The amount of protein retained on HAP after digestion for various periods of time is illustrated in **Figure 1**. Both MMP20 and KLK4 hydrolysis could gradually remove the adsorbed amelogenin from HAP (**Figures 1A,C**) into the reaction buffer solution (**Figures 1B,D**). After 36 h of digestion by MMP20, only about 12% of amelogenin still remained on HAP. In comparison, the removal rate of adsorbed amelogenin was much faster by KLK4 digestion. Nearly 98% of amelogenin was released into the surrounding solution by merely 12 h processing. During the entire period of incubation with shaking, bound amelogenin could also be desorbed without digestion, but this loss appeared to be negligible compared to that observed in digested samples (**Figures 1A,C**). Because UV detection showed only a trace amount of MMP20 and KLK4 bound to HAP during digestion process, their effects on the measurement of amelogenin amount were not included from our analysis.

#### **BINDING OF AMELOGENIN TO HAP ACCELERATED THE RATE OF MMP20 OR KLK4 HYDROLYSIS**

The SDS-PAGE data showed that amelogenin bound to HAP was hydrolyzed by MMP20 faster than the proteins in solution (**Figure 2**). The amelogenin substrate on HAP progressively disappeared during the incubation. The intermediate digestion product at an apparent molecular weight of 23 kDa was further hydrolyzed by MMP20 into smaller fragments, which could not be detected by SDS-PAGE analysis. After 3 h of digestion, both the amelogenin at 25 kDa and derivative fragments at 23 kDa were almost completely degraded on HAP, but still partially remained in the samples without HAP.

Further validation and comparison were achieved through HPLC analysis to observe the loss of the 25 kDa protein peak and concomitant generation of peptide peaks. The results of HPLC demonstrated that the digestion of amelogenin on HAP or in solution immediately produced one product peak (labeled "23 kDa" in **Figure 3A**). The corresponding HPLC fraction of this peak was collected and analyzed by MALDI TOF MS. The measured mass (m/z = 18,616) suggested that the 23 kDa band seen on SDS-PAGE corresponded to amelogenin truncated at P164/S (theoretical m/z = 18,615). The areas of individual peaks measured on HPLC traces before and after digestion were used

to analyze the rate of hydrolysis. As shown in **Figure 3B**, the full-length amelogenin in solution was hydrolyzed significantly slower than the protein attached to the crystal surface. The same trend was observed for the 23 kDa proteolytic fragment, which was digested much slower in solution than in its HAPbound form (**Figure 3C**). Interestingly, we note that the 23 kDa

amelogenin fragment rapidly increases in the first hour of the digestion. This result indicates that MMP20 rapidly cleaves off amelogenin C-terminus, causing the accumulation of the truncated hydrophobic peptide in solution.

Similarly, SDS-PAGE data showed that KLK4 hydrolyzed amelogenin much faster on HAP than that in solution (**Figure 4**). At the end of 180 min of KLK4 hydrolysis, there is much less HAP-bound 25 kDa amelogenin remained as compared with amelogenin digested in solution.

# **BINDING OF AMELOGENIN TO HAP INCREASED THE ACCESSIBLE CLEAVAGE SITES FOR MMP20/KLK4**

Previous *in vitro* proteolysis experiments have shown that unbound amelogenin is cleaved by MMP20 primarily at its Nand C-termini (Fincham et al., 1981; Tanimoto et al., 2008a,b). To detect whether the adsorption to HAP affects amelogenin cleavage pattern by proteases, samples digested in the presence or absence of HAP were analyzed by LC-MALDI MS/MS. **Table 1A** lists the cleavage sites of MMP20 and KLK4 in amelogenins adsorbed on HAP and in solution, identified by mass spectrometric analyses. Amelogenin residing on HAP was cleaved by MMP20 at 54 sites, out of which 30 appeared to be unique for the HAP-bound substrate (**Table 1B**). Similarly, KLK4 produced more cleavages within amelogenin bound to HAP (78 sites), in comparison to samples without HAP (53 sites) (**Table 1C**). These results suggested that HAP indeed influences the cleavage pattern of adsorbed amelogenin and the larger number of utilized cleavage sites might thus explain an enhanced proteolysis after the proteins adsorb to HAP. In addition, interaction with HAP may also expose additional cleavage sites within the central region, the most hydrophobic part in amelogenin (**Tables 1B,C**).

# **REDUCED MMP20 ACTIVITY BY BARE HAP**

The quenched-peptide assay showed that the addition of bare HAP into the digestion reaction resulted in a significant decrease

**FIGURE 4 | KLK4 digestion of amelogenin in solution and on HAP shown by SDS-PAGE. (A)** Amelogenin digestion in solution. **(B)** KLK4 hydrolysis of amelogenin adsorbed on HAP. M, standard molecular weight.

**(B) CLEAVAGE SITES IN HUMAN AMELOGENIN FOR MMP20**

MPL\*PPHPGHPGY/IN/F/S/YE/VLTP/LK/W/YQS\*IRPPYP/S\*Y\*G\*YEP\* MGG\*W/L\*HH\*Q\*IIP\*V\*L\*S\*Q\*QHPPTHT\*LQPHHHIPVVP\*A\*Q\*QP/ VIPQQP\*MMPVP\*GQ/H/S\*MTP\*IQ/HHQPNLPPP\*A/Q\*QP/YQPQP/VQ\* PQPHQP\*MQPQPPVHP/MQ\*P/L/PPQPPLPPMFP/MQ\*PLPPMLP\*DL/T/ LEAWP/STDKTKREEVD

#### **(C) CLEAVAGE SITES IN HUMAN AMELOGENIN FOR KLK4**

MPLPPHPGHPG\*Y/I\*N/F/S\*Y/E/V/LTP\*L\*K/W/Y/Q/S/IR/PPYPS/YGYEP/M/ G/G/W/L/H/H/Q/IIPVLS/Q\*QHPPT/H\*T/L\*Q\*P\*H/HHIP/VVP\*A/QQP/VI/ PQ/Q\*PM\*MPVPG\*QH/S/MTP/IQ\*H/H\*QPN\*LPPPA\*QQPY\*QPQP/ VQPQPH\*QP\*M/QPQPP\*VHP/M/Q\*PL/PPQ\*PPLPPM/FP/M/QP/L/PP/ MLP/D/L/T\*L/E/AW/PSTDKTK/REEVD

*The / in (B) and (C) indicates the MMP20 or KLK4 cleavage sites identical in both amelogenins adsorbed on HAP and in solution. The \* indicates the cleavage sites of MMP20 or KLK4 unique for amelogenins adsorbed on HAP.*

in the enzymatic activity of MMP20 in a dose-dependent manner (**Figure 5**).

# **DISCUSSION**

The highly mineralized enamel crystals develop from a layer of enamel protein matrix that is predominated by amelogenins (*>*90%), which form special nanostructures to modulate crystal formation (Fincham and Moradian-Oldak, 1995; Fincham et al., 1999; Moradian-Oldak et al., 2001). The multilayer of amelogenin nanospheres in the matrix surrounding the thin and long crystals not only provide physical protection and mechanical support to these newly formed and extremely fragile crystals, but also modulate and inhibit their growth (Moradian-Oldak et al., 2001). During transition and maturation stages of enamel development, amelogenins are gradually processed by enamel proteinases, and mineralization simultaneously increases to form a fully mineralized enamel matrix (Fukae et al., 1998; Bartlett and Simmer, 1999; Li et al., 1999; Ryu et al., 1999, 2002; Simmer and Hu, 2002; Bartlett et al., 2003).

It has previously been evidenced that full-length amelogenin proteins strongly adsorb to apatite crystals and retard their further growth (Doi et al., 1984; Aoba and Moreno, 1987). Therefore, it is not difficult to understand that the removal of the proteins from matrix not only opens the space but also eliminates the possible inhibitory effects of amelogenin for enamel crystal growth. The question is which part of the matrix will be degraded and removed first, the proteins bound to crystals or unbound to appetite? If the amelogenins in the matrix (unbound to crystals) were digested faster or even at the same rates as those adhered to the crystal, the newly formed crystals with extreme thin and long structures would lose the protection and support. Consequently, they would be easy to collapse, break apart or fuse

together. Therefore, the layers of amelogenin adsorbed on crystal surface need to be preferentially removed to open up the space for the growth of a new layer of crystals, while still having relatively intact surrounding protein matrix to support the thin and fragile nascent crystals.

The amelogenin-crystal interactions may contribute to this preferential degradation by proteinases. Proteins usually alter their conformation when bound to solid surfaces (Hlady and Buijs, 1996; Gray, 2004). In the recently published report by the Shaw group, they analyzed self-assembled amelogenin residing on single fluoroapatite (FAP) crystals, whose structures are the same as those of HAP crystals. They found that protein layer on the FAP surfaces were much thinner than the nanospheres originally present in solution. Their studies provide the strong evidence that amelogenin nanospheres undergo structural changes upon interacting with apatite surfaces (Tarasevich et al., 2009a,b). By using Fourier transform infrared (FTIR), the Beniash group also reported on the reduction in structural organization of amelogenin proteins upon their interactions with minerals at neutral pH (Beniash et al., 2012). In addition, it was shown that amelogenin nanospheres disassembled into oligomers and monomers when they adsorbed onto positively or negatively charged mica surfaces (Chen et al., 2012). The adsorption-induced conformational changes of the amelogenin and its assemblies can further alter their interactions with other enamel proteins, including proteinases (Smolarczyk et al., 2005; Sakiyama et al., 2006). The results of the present work clearly demonstrated that the binding of amelogenin to apatite crystals exerts significant effects on the proteolysis of the protein. Compared to the unbound proteins, the HAP-bound amelogenins were more rapidly hydrolyzed by both MMP20 and KLK4. Moreover, the additional proteinases-susceptible cleavage sites, primarily in the central region, were uncovered upon amelogenin binding to HAP. Previous studies suggested that the hydrophobic central region of amelogenins forms a dense core of nanosphere surrounded by the hydrophilic C-terminal tails in solution (Margolis et al., 2006). The buried central region is not readily accessible to the proteinases, and hence MMP20 hydrolysis of amelogenin occurs primarily at its termini. The interactions of HAP and amelogenins result in the disassembly of nanospheres into monomers (Chen et al., 2012), which may effectively exposes the central region of the protein. As a result, more cleavage sites become accessible and a higher rate of hydrolysis was observed.

The present study identified that MMP20 and KLK4 hydrolyzed amelogenin in solution at 25 and 54 cleavage sites respectively. These numbers are significantly higher than those previously reported (Nagano et al., 2008, 2009). In addition, we detected seven MMP20-susceptible cleavage sites within the sequence of tyrosine rich amelogenin peptide (TRAP). Previous studies indicated that TRAP was not susceptible to MMP20 hydrolysis (Nagano et al., 2008, 2009). The differences in experiment results among laboratories may result from several reasons. First, as we aimed at investigating the complete enzymatic degradation of amelogenin, beyond the initial generation of large proteolytic fragments, we used relatively higher enzyme-to-substrate ratios, longer incubation time and also replenished enzyme after 24 h of incubation. Second, in order to maintain similar conditions when comparing digestion of amelogenin in solution and bound to HAP, all digestion reactions utilized constant and vigorous shaking to keep HAP particles suspended in solution. The shaking procedure is very critical in these experiments, because HAP easily forms big clumps, especially after amelogenin adsorption. In addition, we used both amelogenin and proteinases from human species that is different from other reported ones, which may be another reason for these different cleavage activities.

Proteins and peptides usually inhibit the crystal growth by blocking the attaching sites and they should be completely or partially detached to facilitate the further crystal growth (De Yoreo and Vekilov, 2003; De Yoreo et al., 2007; Friddle et al., 2010, 2011). Our proteolytic experiments (**Figure 1**) showed that MMP20 or KLK4 hydrolysis of amelogenin resulted in gradual loss of its binding affinity to HAP and nearly complete release of the protein from HAP. Furthermore, we found that KLK4 digested amelogenin more efficiently and more completely than MMP20, demonstrating its major role in the clearance of amelogenin from enamel matrix at the maturation stage.

A previous publication reported that HAP reduced amelogenin hydrolysis by MMP20 and KLK4 (Sun et al., 2010), which seems to contradict our current finding at the first glance. However, after further comparisons, we found that the difference was due to the different experimental systems and techniques used in these two studies. In Sun's study, HAP was the last component to be added into the reaction mixture containing amelogenin and proteinases. In contrast, in our system, amelogenin was first adsorbed onto HAP in a pre-binding step. We used the pre-binding procedure to saturate the HAP surfaces with amelogenins to mimic the physiological and developmental conditions *in vivo*. As we have known, in the secretory stage, enamel matrix contains a large amount of amelogenins and the majority of these amelogenins remain intact or are only partially hydrolyzed by proteinases (Smith et al., 1989). The amelogenin protein matrix surrounding the enamel crystals will interact with the surface of HAP, which would alter the conformation of the bound protein and in turn affect protein hydrolysis and crystal growth. The result that emerged from the experiments that utilized a pre-binding step prior to proteolysis indicated that the adsorption-induced changes of amelogenin hydrolysis should mainly result from the interactions between amelogenin and HAP crystal surfaces, rather than from the proteinase-crystal interactions as shown in our quenched peptide experiment (**Figure 5**).

**FIGURE 6 | Proposed model for enamel crystal growth guided by crystal-amelogenin-proteinase interactions. (A)** HAP crystal interacts with amelogenins and causes their structural changes; **(B)** The structural changes of adsorbed amelogenins result in preferential amelogenin degradation on crystal surface; **(C)** Removal of the amelogenin on the crystal surface releases

the space for HAP crystal growth; **(D)** Summary of the proposed model of enamel crystal growth guided by crystal-amelogenin-proteinase interactions. Amelogenin interactions with crystal surface change its conformation, which increases its susceptibility to proteinase. The direct effect of proteinase-crystal interactions will change the structure of proteinase, showing inhibitory effects. In this experiment, different amounts of HAP were directly added into mixtures of MMP20 and its fluorescent substrate. The data showed dose-dependent inhibitory effects, indicating that the bare HAP can directly interact with MMP20 and inhibit its enzymatic activity.

Based on the results obtained from this study, we propose a model to explain how the enamel crystal growth is mediated by interactions among crystal, amelogenin, and proteinases. As shown in **Figure 6A**, the amelogenins secreted into enamel matrix first bind to the nascent HAP crystals and the adsorption of amelogenins onto the crystal surface results in conformational changes of the bound proteins. The conformational changes may expose more cleavage sites to proteinases. As a result, cleavage of amelogenins by MMP20 or KLK4 is enhanced (**Figure 6B**). The preferential removal of amelogenins from the crystal surface opens up the space surrounding the crystals for their further growth (**Figures 6C,D**). When the nascent crystals growing in this new space come into contact with the next layer of amelogenin nanospheres, yet another cycle of the interaction-mediated preferential removal of bound amelogenin and crystal growth is initiated. The cycle of *binding-growth-digestion* repeats until the HAP crystals grow to fill up the entire enamel space during tooth enamel development.

#### **ACKNOWLEDGMENTS**

This study was supported by National Institute of Health grant R01DE015821 and R01DE015821S1 to Wu Li. The UCSF SMMS Core Facility acknowledges the support of the Sandler Family Foundation and the Gordon & Betty Moore Family Foundation.

# **REFERENCES**

imaging (ESI) and electron spectroscopic diffraction (ESD). *Cell Tissue Res.* 277, 151–158. doi: 10.1007/BF00303091

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 28 May 2014; paper pending published: 09 June 2014; accepted: 26 June 2014; published online: 24 July 2014.*

*Citation: Zhu L, Liu H, Witkowska HE, Huang Y, Tanimoto K and Li W (2014) Preferential and selective degradation and removal of amelogenin adsorbed on hydroxyapatites by MMP20 and KLK4 in vitro. Front. Physiol. 5:268. doi: 10.3389/fphys. 2014.00268*

*This article was submitted to Craniofacial Biology, a section of the journal Frontiers in Physiology.*

*Copyright © 2014 Zhu, Liu, Witkowska, Huang, Tanimoto and Li. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Kallikrein-related peptidase-4 (KLK4): role in enamel formation and revelations from ablated mice

# *John D. Bartlett 1,2\* and James P. Simmer 3*

*1 Harvard School of Dental Medicine, Boston, MA*

*2 Department of Mineralized Tissue Biology, The Forsyth Institute, Cambridge, MA*

*3 Department of Biological and Material Sciences, University of Michigan School of Dentistry, Ann Arbor, MI, USA*

#### *Edited by:*

*Megan Pugach-Gordon, The Forsyth Insitute, USA*

#### *Reviewed by:*

*Tom Diekwisch, University of Illinois at Chicago, USA Michael Paine, University of Southern California, USA*

#### *\*Correspondence:*

*John D. Bartlett, Harvard School of Dental Medicine; Department of Mineralized Tissue Biology, The Forsyth Institute, 245 First Street, Cambridge, MA 02142, USA e-mail<EMAIL_ADDRESS>
Enamel development occurs in stages. During the secretory stage, a soft protein rich enamel layer is produced that expands to reach its final thickness. During the maturation stage, proteins are removed and the enamel matures into the hardest substance in the body. KLK4 is expressed during the transition from secretory to the maturation stage and its expression continues throughout maturation. KLK4 is a glycosylated chymotrypsin-like serine protease that cleaves enamel matrix proteins prior to their export out of the hardening enamel layer. Mutations in *KLK4* can cause autosomal recessive, non-syndromic enamel malformations in humans and mice. *Klk4* ablated mice initially have normal-looking teeth with enamel of full thickness. However, the enamel is soft and protein-rich. Three findings are notable from *Klk4* ablated mice: first, enamel rods fall from the interrod enamel leaving behind empty holes where the enamel fractures near the underlying dentin surface. Second, the ∼10,000 crystallites that normally fuse to form a solid enamel rod fail to grow together in the ablated mice and can fall out of the rods. Third, and most striking, the crystallites grow substantially in width and thickness (a- and b-axis) in the ablated mice until they almost interlock. The crystallites grow in defined enamel rods, but interlocking is prevented presumably because too much protein remains. Conventional thought holds that enamel proteins bind specifically to the sides of enamel crystals to inhibit growth in width and thickness so that the thin, ribbon-like enamel crystallites grow predominantly in length. Results from *Klk4* ablated mice demonstrate that this convention requires updating. An alternative mechanism is proposed whereby enamel proteins serve to form a mold or support structure that shapes and orients the mineral ribbons as they grow in length. The remnants of this support structure must be removed by KLK4 so that the crystallites can interlock to form fully hardened enamel.

**Keywords: enamel development, MMP20, amelogenin, ameloblastin, enamelin, enamel crystallites, enamel rods**

# **INTRODUCTION**

The enamel layer covers the crown of the tooth and is unique because it is an epithelially-derived calcified tissue and becomes the hardest substance in the body. Its hardness is between that of iron and carbon steel, but enamel has a higher elasticity (Newbrun and Pigman, 1960). Enamel hardness is a function of its high mineral content. Unlike bone and dentin (20–30% organic material by weight), fully formed enamel contains very little protein (less than 1% organic material) (Lefevre and Manly, 1938; Deakins and Volker, 1941). Enamel mineral is very similar to hydroxyapatite (HAP) [Ca5OH (PO4)3], but also contains low percentages of carbonate, sodium, and magnesium. Therefore within the body, teeth are the most resistant to deterioration and have been examined extensively for anthropological studies.

But, what are the developmental and mechanistic processes that make enamel harder than the cementum formed along the tooth root, the dentin underlying the enamel layer, and the skeletal bones? The ameloblasts are a single cell layer that cover the developing enamel and are responsible for enamel composition. Enamel development (amelogenesis) can be broken down into three defined stages: secretory, transition and maturation. The stages are defined by the morphology and function of the ameloblasts. Dentin mineralizes first and pre-ameloblasts transform into secretory stage ameloblasts by elongating into tall columnar cells and by forming Tomes' processes at their apical ends nearest the forming enamel. The Tomes' process is a conical structure that points toward the forming enamel matrix. Enamel matrix proteins are primarily secreted from one side of the Tomes' process (secretory face) and all ameloblasts within a row secrete protein from the same side of their Tomes' processes. The first formed enamel ribbons grow between the dentin crystals perhaps by mineralizing around dentin proteins such as collagen. At their growing tips near the Tomes' process, secretory stage enamel ribbons are only about 1.5 nm thick and 15 nm wide (Daculsi and Kerebel, 1978; Cuisinier et al., 1992) and these ribbons are extended until they span the entire thickness of the enamel layer. Approximately 10,000 parallel mineral ribbons are present in each enamel rod (Daculsi et al., 1984). A rod is about 5μm in cross-sectional diameter (Skobe and Stern, 1980) and each is generated by a single ameloblast (Skobe, 1977). Enamel ribbons elongate at the mineralization front where enamel proteins are secreted (Ronnholm, 1962). As the ameloblasts secrete large amounts of enamel matrix proteins, they move away from the dentin surface so that the nascent enamel layer can thicken. The mineral ribbons crystallize into HAP within the rod and will grow progressively in c-axis length parallel to one another as the ameloblasts move progressively away from the dentin surface. The crystallites are surrounded with abundant proteins that prevent them from fusing into a solid rod. The secretory stage enamel is therefore protein rich and has a soft cheese-like consistency.

During the secretory stage, ameloblasts not only move away from the dentin as the enamel thickens, but they also move in groups that slide by one another and this movement culminates in the characteristic decussating enamel prism pattern observed in rodent incisors (Reith and Ross, 1973) or the entwined gnarled prism pattern seen in human molars (Boyde, 1989). Secretory stage ameloblasts secrete four different proteins into the enamel matrix. Three are "structural" proteins and one is a proteinase. The structural proteins are amelogenin (AMELX), ameloblastin (AMBN), enamelin (ENAM), and the proteinase is matrix metalloproteinase-20 (MMP20, enamelysin). Amelogenin comprises approximately 80–90% of the organic matter within the secretory stage enamel matrix and ameloblastin and enamelin comprise roughly 5 and 3–5%, respectively (Fincham et al., 1999; Hu et al., 2007). MMP20 is present in trace amounts. The precise function of these proteins remains unclear. However, human mutations in *AMELX* (Hu et al., 2012), *ENAM* (Rajpar et al., 2001), and *MMP20* (Kim et al., 2005; Ozdemir et al., 2005; Lee et al., 2010; Gasse et al., 2013) genes and mouse knockout models (Gibson et al., 2001; Caterina et al., 2002; Fukumoto et al., 2004; Masuya et al., 2005; Seedorf et al., 2007; Hu et al., 2008) have definitively demonstrated that each of these proteins are absolutely required for proper enamel formation. This conclusion is supported by the observation that the genes encoding secretory stage enamel proteins are consistently pseudogenized in vertebrates that have lost the ability to make teeth, or specifically dental enamel, during evolution (Meredith et al., 2009, 2011, 2013). By the end of the secretory stage the enamel layer has achieved its full thickness. It is not until the end of the maturation stage when the proteins are almost completely removed, that the enamel achieves its final hardened form.

As the ameloblasts enter the transition stage, they no longer move relative to each other. They retract their Tomes' processes and transition into shorter and fatter maturation stage cells and, at the enamel surface, start modulating between ruffle and smooth-ended cells (Smith, 1998). It is during the maturation stage that ameloblasts actively secrete kallikrein-related peptidase-4 (KLK4) to help remove the mass of previously secreted and partially hydrolyzed (by MMP20) matrix proteins from the enamel layer so that the crystallites can expand, coalesce and fuse with adjacent crystals (Simmer et al., 2009). This strengthens the enamel rods and forms the most highly mineralized substance in the body. Therefore, unlike cementum, dentin and bone, the proteins responsible for mineralization are removed from enamel so that the enamel can attain its final hardened form. Also, relative to the other mineralized tissues, the entwined rod and interrod enamel forms a more highly ordered 3D structure that is highly resistant to occlusal forces. The stages of enamel development and the developmental processes as the enamel forms are remarkably similar in different mammalian species (Weinmann et al., 1942; Robinson et al., 1988). Rodents have continuously erupting incisors so every developmental stage is always present in specific locations along the forming rodent incisor.

# **ENAMEL PROTEINASES**

The proteinase expressed during the secretory through early maturation stage is MMP20 (Begue-Kirn et al., 1998) and the proteinase expressed from the transition through maturation stages is KLK4 (Hu et al., 2002). To date, these are the only two proteinases proven to be secreted into the enamel matrix (Bartlett, 2013). Both proteinases are present in small amounts during enamel development and each proteinase was separately cloned by performing PCR-based homology cloning (Bartlett et al., 1996; Simmer et al., 1998). KLK4 was originally named enamel matrix serine proteinase-1 (EMSP1) (Simmer et al., 1998), but its name was changed to KLK4 because the gene encoding KLK4 locates within the kallikrein gene cluster.

Human and mouse mutations in both *MMP20* (Caterina et al., 2002; Kim et al., 2005; Ozdemir et al., 2005; Lee et al., 2010; Gasse et al., 2013) and *KLK4* (Hart et al., 2004; Simmer et al., 2009; Wang et al., 2013) cause severe enamel malformations and therefore demonstrate that no other proteinase has an extensive overlapping function with either of these proteinases. If this were the case, no severe enamel phenotype would likely occur if the activity of MMP20 or KLK4 were compromised. Furthermore, accumulated porcine secretory stage enamel protein cleavage products have been extensively characterized and MMP20 specifically cleaves recombinant enamel proteins *in vitro* to generate the full spectrum of cleavages that occur *in vivo* (Ryu et al., 1999; Nagano et al., 2009). Others have proposed that MMP9 is present within secretory stage developing enamel (Feng et al., 2012). However, this claim is uncertain as MMP20 activity can explain all of the secretory stage amelogenin cleavages and MMP9 mutations cause metaphyseal anadysplasia, which is not associated with enamel defects (Lausch et al., 2009). Similarly, chymotrypsin C is associated with enamel formation (Lacruz et al., 2011). However, although loss of *CTRC* function is a risk factor for pancreatitis, an associated enamel phenotype has not been described (Zhou and Sahin-Toth, 2011). Signal-peptide-peptidase-like 2a (SPPL2A) is a membrane bound protease in lysosomes/late endosomes that is expressed by enamel epithelium during the secretory and maturation stages of amelogenesis. Spll2a null mice show defective enamel, highlighting the importance of intracellular degradation of enamel proteins reabsorbed by endocytosis (Bronckers et al., 2013). Although it is likely that several proteases degrade enamel proteins within ameloblast lysosomes, MMP20, and KLK4 remain the only proteinases that are known to be secreted into the enamel matrix (Bartlett, 2013).

#### **DISCOVERY OF KLK4**

In 1977 a protease was purified from pig enamel (Fukae et al., 1977) that was later demonstrated to be inhibited by serine proteinase inhibitors phenylmethylsulfonyl fluoride (PMSF) and diisopropylfluoro phosphate (DIFP) (Shimizu et al., 1979). This protease was expressed during the early maturation stage when the enamel proteins are reabsorbed from the hardening enamel (Overall and Limeback, 1988). KLK4 was eventually cloned by PCR-based homology cloning from porcine cDNA with subsequent screening of a porcine cDNA library (Simmer et al., 1998). The porcine KLK4 preproenzyme is composed of 254 amino acids while the proenzyme has 230 residues and the active form has 224 amino acids (Simmer et al., 1998). The *KLK4* genes of both mouse and human have six exons the first of which is noncoding. The mouse *Klk4* gene is approximately 10 kb in size and locates in cytogenic region B2 on mouse chromosome 7 (Hu et al., 2000b). The human *KLK4* gene is approximately 7 kb in size and is located near the telomere of chromosome 19 (19q13.3–19q13.4) in a cluster of genes including the KLK family of serine proteases. Its gene exon/intron structure and protein domain structure is identical to that of the mouse (Hu et al., 2000b). Thus, because KLK4/EMSP1 was cloned after MMP20, it became the second proteinase identified by name that is secreted into the developing enamel matrix.

# **KLK4 TISSUE LOCALIZATION**

KLK4 is a glycosylated, chymotrypsin-like serine protease that is expressed and secreted by transition to maturation stage ameloblasts (Hu et al., 2000a,b, 2002). KLK4 protein has not been isolated from any tissue other than from developing teeth (Ryu et al., 2002; Nagano et al., 2009). However, several studies have performed immunoassays or qPCR techniques to identify KLK4 in various tissues and many of these studies conflict with one another as to exactly where KLK4 is expressed (reviewed in Simmer et al., 2011b). To definitively identify where KLK4 is expressed, a gene targeted mouse strain was developed. These mice have a *LacZ* reporter gene with a mouse nuclear localization signal (NLS-βgal) inserted at the natural *Klk4* translation initiation site. Therefore, with these mice, locations of KLK4 expression were identified within tissues by using β-galactosidase histochemistry (Simmer et al., 2009). KLK4 was expressed highly in maturation stage ameloblasts (**Figure 1**) and low levels of KLK4 expression were observed in the striated ducts of the submandibular salivary gland and in small patches of prostate epithelia. Furthermore, in these *Klk4 LacZ* knock-in mice, no obvious morphological abnormalities were observed in any of the non-dental tissues examined suggesting that their normal development is not *Klk4* dependent (Simmer et al., 2011b). As is true for MMP20, it appears that the only essential, non-overlapping function of KLK4 is in enamel development.

#### **KLK4 ACTIVATION**

It is not known how KLK4 is activated *in vivo.* Removal of the KLK4 propeptide is essential for activation because it allows a salt linkage to form between the new N-terminus and the side chain of Asp194 and this is essential for enzyme activity (Scully et al., 1998; Debela et al., 2006). Unlike the other kallikrein-related peptidases, KLK4 has a Gln as the last residue of its propeptide and not an Arg or Lys which means that KLK4 cannot be activated by trypsin-like enzymes (Lundwall and Brattsand, 2008). KLK4 cannot activate itself, but can be activated by MMP20 and thermolysin *in vitro* (Ryu et al., 2002). However, KLK4 is active in *Mmp20* ablated mice (Yamakoshi et al., 2011) so MMP20 cannot be the sole KLK4 activator. Previously it was shown that dipeptidyl peptidase I (Cathepsin C, CTSC) activates KLK4 *in vitro* (Tye et al., 2009). In the enamel organ, CTSC is expressed at progressively increasing levels as development progresses to the early maturation stage when KLK4 begins its expression. Therefore, it remains a possibility that this cysteine aminopeptidase is the primary enzyme that activates KLK4.

# **KLK4 SUBSTRATE SPECIFICITY**

KLK4 was assessed for its substrate specificity by using recombinant KLK4 to screen tetrapeptide positional scanning synthetic combinatorial libraries (PS-SCL) (Matsumura et al., 2005). The identified preferred P1–P4 positions were:

**maxillary first molars of wild-type (Wt) heterozygous (***Klk4***+***/***lacZ) and homozygous (***Klk***4***lacZ/lacZ* **) mice at postnatal days 5, 8, 11, and 14.** LacZ histochemistry shows nuclear localized β-galactosidase activity where KLK4 is normally expressed. At the 5-h incubation used, no endogenous (lysosomal) β-gal activity was observed and the only observed in transition and maturation ameloblasts. No expression was observed in odontoblasts. B, Bone; Od, odontoblasts; D, dentin; E, enamel; Am, ameloblasts. Scale bars = 100μm. This figure was reprinted with permission (S. Karger AG, Basel) from Simmer et al. (2011a).

P1-Arg; P2-Gln/Leu/Val; P3-Gln/Ser/Val and P4-Ile/Val. The first report demonstrating that KLK4 cleaves amelogenin, used native porcine KLK4 incubated with recombinant pig amelogenin and this resulted in the generation of twelve cleavage products which were characterized by N-terminal sequencing (Ryu et al., 2002). It was subsequently demonstrated that the primary MMP20 N-terminal cleavage product, tyrosine-rich amelogenin polypeptide (TRAP), was further cleaved by KLK4 which was consistent with the notion that KLK4 cleaves enamel matrix proteins into small peptides to facilitate their export out of the enamel as the enamel hardens (Nagano et al., 2009). Porcine ameloblastin was stably expressed and secreted from HEK293-N cells and was purified for digestion by KLK4. The cleavage products were characterized by N-terminal sequencing and KLK4 was shown to cleave ameloblastin at nine different sites (Chun et al., 2010). The 32 kDa enamelin is presumed to be an MMP20 cleavage product and it is the only domain of the parent protein that accumulates in the deeper, more mature enamel layer. Native porcine KLK4 was incubated with native porcine 32 kDa enamelin and the digestion products were fractionated by reverse-phase high-performance liquid chromatography (RP-HPLC) and characterized by Edman sequencing, amino acid analysis, and mass spectrometry. KLK4 digestion of the 32-kDa enamelin generated nine major cleavage products (Yamakoshi et al., 2006). Therefore, KLK4 cleaves all the structural enamel matrix proteins that are known to be secreted into the enamel matrix. Recently, it was confirmed that MMP20 activates pro-KLK4 and strikingly, that active KLK4 cleaves and inactivates MMP20 (Yamakoshi et al., 2013). In effect, by activating KLK4, MMP20 inactivates itself. This mechanism of MMP20 inactivation is supported by evidence demonstrating that in *Klk4* ablated mice, MMP20 is active well into the maturation stage when MMP20 activity has normally ceased (Yamakoshi et al., 2011).

# **HUMAN** *KLK4* **MUTATIONS**

Two different human *KLK4* mutations are known to cause autosomal recessive hypomaturation *amelogenesis imperfecta*. The first discovered is a nonsense mutation occurring upstream of the KLK4 catalytic domain (p.Trp153∗). This tryptophan residue is completely conserved in mouse and pig KLK4 and expression of this mutated gene would result in a truncated protein lacking the final 101 amino acids which includes the catalytic triad (His71, Asp116, and Ser207). This homozygous mutation occurred in two female siblings and both their primary and permanent dentitions were similarly affected. The sibling's teeth were yellow-brown in color and were excessively sensitive to hot and cold. The enamel was normal in thickness, but radiographically showed only a slight increase in opacity over that of the underling dentin indicating a decreased enamel mineral content. This soft enamel fractured from the occlusal surfaces of the primary molars (Hart et al., 2004). No other phenotype resulted from this nonsense mutation in *KLK4*. The second human *KLK4* mutation was recently discovered by use of whole exome sequencing which identified a single nucleotide deletion (p.Gly82Alafs∗87) in both alleles of a 9 year-old female. The frameshift was in the third of five coding exons so the mutant *KLK4* transcripts may have been degraded by nonsense-mediated decay. If translated, the mutant protein would lack the same catalytic triad that was also lacking in the first discovered *KLK4* mutation. As for the previously discovered KLK4 mutation, the enamel covering this proband's teeth appeared normal in size and shape, but was discolored yellowbrown and chipped on multiple teeth. This proband was also secondarily affected with dental caries (Wang et al., 2013). No other phenotype was observed due to the nucleotide deletion in *KLK4*. Therefore, in humans KLK4 is essential for enamel to achieve its final hardened form, and that just as for MMP20, the only non-overlapping function of KLK4 is in dental enamel development.

# **THE** *KLK4 KNOCKOUT/LACZ* **KNOCKIN MOUSE**

As stated above under KLK4 tissue localization, gene targeting was used to generate a mouse strain carrying a null allele of *Klk4* that has a nuclear *LacZ* reporter gene inserted directly into the *Klk4* translation initiation site. Therefore, the *LacZ* code was positioned in the same genomic context as wild-type *Klk4* and so provided a sensitive tissue reporter for native *Klk4* expression (Simmer et al., 2009). Other than a tooth phenotype, the *Klk4* ablated mice were normal. The teeth were normal, the enamel attained normal thickness and no abnormalities were observed until the enamel reached the transition to early maturation stage of development. At this point, the normal export of enamel matrix proteins from the matrix back to the ameloblasts destined for lysosomal degradation was impeded. The enamel retained proteins that were normally removed and the soft, protein-rich enamel abraded from the mouse teeth (**Figures 2A–C**). This strongly supports the belief that KLK4 functions to cleave enamel matrix proteins to facilitate their export out of the hardening enamel (Simmer et al., 2009). Unexpectedly, the rod enamel sometimes pulled away from interrod enamel. This left holes in the interrod enamel that were once filled by enamel rods (**Figures 2D,E**) (Simmer et al., 2009, 2011a). Backscatter scanning electron microscopy revealed that the enamel layer of *Klk4* null mice is reasonably well-mineralized at the surface, but is progressively less mineralized with depth (Hu et al., 2011; Smith et al., 2011). This pattern suggests that extracellular degradation of enamel proteins by KLK4 facilitates the movement of proteins in the deeper enamel toward the surface for ameloblast endocytosis.

Another observation in the *Klk4* null mice was that the lack of KLK4 activity prevented the individual crystals within the rod from growing sufficiently in width and thickness so that they could interlock with adjacent crystals. Strikingly, bunches of crystals appeared to fall out of the rods and separate into individual crystals (**Figure 3**) similar in appearance to strands (crystals) of "uncooked angel hair spaghetti" falling out of a circular bundle (rod). Although the normal rod pattern was present in the *Klk4* ablated enamel, the ∼10,000 crystallites within the rod failed to interlock properly and the crystallites fell from the rods (Simmer et al., 2009). Developmental analyses of *Klk4* null incisor enamel showed that percent mineral by weight increased almost identically to that occurring in wild-type mice until mid-maturation when a level of about 80% mineral by weight was attained. In contrast to wild-type enamel, the mineral content of the null mouse enamel remained unchanged as development progressed

**FIGURE 2 | Scanning electron micropscopy of the mandibular molars (A,B) and mandibular incisor (C–E) of a** *Klk4* **null mouse at 7 weeks.** The enamel of all molars showed a significant loss of enamel from all working surfaces (buccal cusps, occlusal surface, and marginal ridges) **(A,B)**. Similarly, the enamel layer was abraded at the working (buccal) surface of the mandibular incisor at its tip **(C)**. Higher magnification of the chipped area near the tip of the incisor showed the break was in the enamel layer, close to, but not at the DEJ. The broken surface appears to be composed of interrod (ir) enamel with holes where enamel rods (r) had pulled out and separated **(D)** from the initial deposit of interrod enamel near the DEJ. The holes are too numerous to be made by odontoblastic processes penetrating the enamel (enamel spindles). The orientation of the crystallites on the walls of the holes is parallel to the direction of the tubular holes and to the crystallites between the holes **(E)**. This figure was originally published by the American Society for Biochemistry and Molecular Biology in Simmer et al. (2009).

**FIGURE 3 | Comparison of enamel rods from (A) wild-type and (B)** *Klk4* **null mice.** Enamel rods in wild-type mice have tightly packed crystallites that lose some aspect of their individuality. Enamel rods in the *Klk4* null mice are composed of distinctly individual crystallites resembling angel hair spaghetti. Holes or vacancies in some rods give the impression that smaller bundles of crystallites broke at a slightly deeper level and slid out of the rod. This figure was originally published by the American Society for Biochemistry and Molecular Biology in Simmer et al. (2009).

through the maturation stage (Smith et al., 2011). This seminal finding indicated that enamel maturation advanced normally, even in the presence of abundant protein, but arrested when the residual protein physically blocked final crystal maturation by occupying the shrinking space between crystals. If amelogenins and/or the other proteins inhibited crystal maturation by selectively binding to the sides of enamel crystals, the inhibition would have occurred earlier and would have resulted in a more severe enamel phenotype. All-in-all, the *Klk4* knockin/knockout mouse has revealed surprises about enamel formation and has forced us to reexamine some of our more firmly held beliefs about how crystallites grow in width and thickness and interlock to form an enamel rod.

# **A MODIFIED THEORY OF ENAMEL DEVELOPMENT**

During the secretory stage, proteins are secreted into the enamel matrix and are quickly cleaved by MMP20. Selected enamel protein cleavage products accumulate within the matrix and as new proteins are secreted they also are cleaved so that an abundance of MMP20 cleavage products are present throughout the enamel layer as the entire enamel layer grows away (thickens) from the dentin. Previously it was demonstrated that enamel mineral first forms as amorphous calcium phosphate (ACP) (Bodier-Houlle et al., 2000; Beniash et al., 2009). ACP has no defined structure. It can be thought of as "grains of sand" that require a mold if it is to have defined 3D structure. However, as described above, the enamel crystallites have a very specific shape. They grow into long thin ribbons. So it is postulated that the MMP20 cleavage products form a mold to define the shape of each crystallite ribbon so that the ACP can attain the proper shape prior to its conversion into HAP. Therefore, the nucleation event for mineral formation would occur within the protein mold so that the ACP will form a proper 3D ribbon structure prior to when it crystallizes into HAP. It is envisioned that the crystallite ribbon molds protect the crystallite ribbons, much as packaging material protects the contents of a box, as the enamel crystallites elongate from the dentin surface to the eventual outer surface of the enamel layer. This new theory (Simmer et al., 2012) represents a departure from previous beliefs that amelogenin by itself initiates enamel formation and from the thought that amelogenin inhibits crystallite growth in width and thickness (Bartlett and Simmer, 1999). In summary, results from the *Klk4* knockin/knockout mouse have led us to reevaluate our theories of enamel formation because we now have attained a better understanding of the importance of KLK4 activity and why it is so critical in enamel development.

## **ACKNOWLEDGMENTS**

Research reported in this publication was supported by the National Institute of Dental and Craniofacial Research of the National Institutes of Health under award numbers RO1DE016276 (John D. Bartlett) and R01DE019775 (James P. Simmer).

# **REFERENCES**

the developing permanent porcine dentition. *Caries Res.* 22, 321–326. doi: 10.1159/000261131

**Conflict of Interest Statement:** The Associate Editor declares that, despite being affiliated to the same institution as one of the authors, John D. Bartlett, the review process was handled objectively and no conflict of interest exists. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 06 May 2014; paper pending published: 23 May 2014; accepted: 10 June 2014; published online: 04 July 2014.*

*Citation: Bartlett JD and Simmer JP (2014) Kallikrein-related peptidase-4 (KLK4): role in enamel formation and revelations from ablated mice. Front. Physiol. 5:240. doi: 10.3389/fphys.2014.00240*

*This article was submitted to Craniofacial Biology, a section of the journal Frontiers in Physiology.*

*Copyright © 2014 Bartlett and Simmer. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Mapping residual organics and carbonate at grain boundaries and the amorphous interphase in mouse incisor enamel

# *Lyle M. Gordon and Derk Joester\**

*Department of Materials Science and Engineering, Northwestern University, Evanston, IL, USA*

#### *Edited by:*

*Bernhard Ganss, University of Toronto, Canada*

#### *Reviewed by:*

*Michel Goldberg, University Paris Descartes, France Victor E. Arana-Chavez, University of São Paulo, Brazil*

#### *\*Correspondence:*

*Derk Joester, Department of Materials Science and Engineering, Northwestern University, 2220 Campus Drive, Evanston, IL 60208, USA e-mail<EMAIL_ADDRESS>Dental enamel has evolved to resist the most grueling conditions of mechanical stress, fatigue, and wear. Adding insult to injury, it is exposed to the frequently corrosive environment of the oral cavity. While its hierarchical structure is unrivaled in its mechanical resilience, heterogeneity in the distribution of magnesium ions and the presence of Mg-substituted amorphous calcium phosphate (Mg-ACP) as an intergranular phase have recently been shown to increase the susceptibility of mouse enamel to acid attack. Herein we investigate the distribution of two important constituents of enamel, residual organic matter and inorganic carbonate. We find that organics, carbonate, and possibly water show distinct distribution patterns in the mouse enamel crystallites, at simple grain boundaries, and in the amorphous interphase at multiple grain boundaries. This has implications for the resistance to acid corrosion, mechanical properties, and the mechanism by which enamel crystals grow during amelogenesis.

**Keywords: dental enamel, caries, atom probe tomography, chemical imaging, grain boundaries, interphases**

# **INTRODUCTION**