enzyme replacement therapy ( ert ) is currently the major form of treatment for a number of lysosomal storage diseases , although its efficacy varies among the individual disorders . most of these inherited disorders arise from the lack of activity of a single lysosomal enzyme , which leads to the accumulation of the material normally degraded by the enzyme .
the build - up of the storage material in the lysosome eventually results in cell and organ dysfunction .
the goal of ert is to introduce sufficient amounts of normal enzyme into the lysosomes of the deficient cells to clear the storage material and restore lysosome function .
this form of therapy was first used in patients with type 1 gaucher disease , who lack acid -glucocerebrosidase ( gba ) activity and accumulate glucosylceramide primarily in macrophage type cells .
the replacement enzyme , containing n - linked glycans with terminal mannose residues , is infused intravenously and taken up by macrophages via cell surface mannose receptors . the endocytosed enzyme is then transported via endosomes to lysosomes , where it functions with good clinical results in this disorder . because most cell types lack mannose receptors , the replacement enzymes used to treat lysosomal storage disorders that involve cell types other than macrophages use binding to the cation - independent mannose 6-phosphate receptor ( ci - mpr ) at the cell surface for subsequent delivery to lysosomes . in this regard ,
studies of a mouse model of gaucher disease have produced evidence for the involvement of multiple cell lineages other than macrophages in the pathophysiology of this disorder , particularly osteoblasts , dendritic cells , and t  cells .
therefore , a therapy that included these cell types may be of clinical benefit in this disorder .
the enzymes used for ert are purified from the secretions of mammalian cells , mostly chinese hamster ovary cells , engineered to produce high levels of the enzyme of interest .
the feasibility of this approach is dependent upon the ability of the endogenous glcnac-1-phosphotransferase to phosphorylate mannose residues of the n - glycans of the expressed lysosomal enzyme .
some of the replacement enzymes produced by this technique are highly phosphorylated and bind well to the ci - mpr .
this includes the pompe disease enzyme ( acid -glucosidase [ gaa ] ) and the alpha - mannosidosis enzyme ( lysosomal acid -mannosidase [ laman]).5 , 6 in addition , the gba currently used in the treatment of gaucher disease contains a very low level of man-6-p .
first , the activity of the endogenous glcnac-1-phosphotransferase in the producing cells may be insufficient to effectively phosphorylate the high levels of these enzymes being synthesized .
if this were the case , co - transfection of glcnac-1-phosphotransferase with the lysosomal enzyme would be expected to enhance phosphorylation .
one potential limitation to this approach is that overexpression of wild - type ( wt ) glcnac-1-phosphotransferase in mammalian cells can result in a portion of the expressed enzyme remaining inactive because of lack of proteolytic cleavage by the site-1 protease ( s1p ) , which is essential for catalytic activation of the protein .
a second possibility is that these two lysosomal enzymes are poor substrates for wt glcnac-1-phosphotransferase compared with other lysosomal enzymes .
gba differs from the other lysosomal enzymes in that it is known to be transported to lysosomes by a man-6-p - independent pathway .
this fact , plus its very low content of man-6-p , has led to the conclusion that it is not a substrate for glcnac-1-phosphotransferase . during the course of studies to understand the roles of the various domains of the / subunits of glcnac-1-phosphotransferase , we engineered a modified enzyme that bypasses the requirement for proteolytic cleavage , is highly expressed and has good activity toward a panel of endogenous lysosomal enzymes .
we then compared the ability of the wt and modified glcnac-1-phosphotransferase to act on a number of poorly phosphorylated lysosomal enzymes when co - transfected into cells .
our findings document that it is possible to substantially enhance the phosphorylation of lysosomal enzymes , including gba , that are poor substrates for glcnac-1-phosphotransferase using our modified glcnac-1-phosphotransferase .
acid hydrolases synthesized by this procedure display increased binding to man-6-p receptors and enhanced uptake by target cells compared with the enzymes produced by the current procedure .
we have reported that the / subunits are able to phosphorylate most lysosomal enzymes in the absence of . therefore , we used a construct encoding only the / precursor as the starting source of mannose phosphorylating activity .
proteolytic cleavage of this precursor at k928 to give rise to the  and  subunits is mediated by the site-1 protease in the golgi , and this process is essential for catalytic competency of the protein.8 , 9 the  subunit contains two notch modules and a dna methyltransferase - associated protein ( dmap ) interaction domain that mediate the specific recognition of protein determinants on the lysosomal enzyme substrates ( figure  1a ) .
in addition , there are four so - called spacer domains in the / precursor whose functions are beginning to be elucidated .
thus , we have reported that spacer-1 ( residues 86322 ) determines the site of cleavage of the / precursor and serves to limit  the phosphorylation of non - lysosomal glycoproteins , whereas spacer-2 contains the -subunit binding site.12 , 14 besides these domains , the  and  subunits also harbor four stealth domains that together form the catalytic core of the protein .
the stealth domains of all eukaryotic glcnac-1-phosphotransferases are highly conserved and resemble sequences within bacterial proteins that encode sugar - phosphate transferases involved in cell wall polysaccharide biosynthesis .
in contrast to the mammalian enzymes , however , the bacterial proteins such as the n.  meningitidis glcnac-1-phosphotransferase ( figure  s1 ) lack the other domains , with no evidence that proteolytic cleavage is necessary for catalytic activation . thus
, we asked if it was possible to engineer a human glcnac-1-phosphotransferase that is not cleaved but retained high catalytic activity toward the n - linked glycans of lysosomal enzymes .
we have previously reported that a construct ( n1-d ; figure  1a ) lacking the region from the notch1 module to the end of dmap ( residues 438819 ) is well expressed and has good catalytic activity toward the simple sugar -methylmannoside ( mm ) but is unable to phosphorylate lysosomal enzymes . when deletion of this region was combined with removal of spacer-1 ( construct s1-d ) , the expressed protein ( figure  1b , lane 5 ) had similar activity as n1-d toward mm but slightly greater phosphorylation activity toward the lysosomal enzyme panel ( figures 1c and 1d ) , despite only a small amount of the  subunit product resulting from this construct ( figure  1b , compare lanes 4 and 5 ) .
this is in agreement with our recent study that upon removal of spacer-1 , the uncleaved / precursor retains some catalytic activity . to determine
if it was possible to bypass the requirement for cleavage altogether , construct n1-s3 was made , which extended the deletion from notch1 up to the site-1 protease cleavage site in spacer-3 ( residues 438928 ) .
this construct , which remained a single - chain molecule , was very highly expressed ( figure  1b , lane 6 ) , properly localized to the golgi ( figure  s2 ) , and was 17-fold more active toward mm than observed with wt / ( figure  1c ) . however , it only phosphorylated the panel of lysosomal enzymes about 30%40% as well as the wt transferase ( figure  1e ) .
we also tested the ability of n1-s3 and wt enzyme to phosphorylate the total cellular pool of soluble glycoproteins , which would include proteins other than lysosomal enzymes .
for this experiment , cells transfected with either the wt or the modified construct were labeled with [ 2-h]mannose for 2  hr .
the cells were then lysed , and following removal of the membrane fraction by high - speed centrifugation , the supernatants were incubated with ci - mpr beads to bind the man-6-p - containing proteins , and the percentage of total counts bound was determined . as shown in figure  1f , both constructs phosphorylated about 2.6% of the total soluble glycoprotein pool .
because n1-s3 had less activity toward lysosomal enzymes than wt , this result indicated that it acted upon non - lysosomal glycoproteins to a greater extent than does the wt enzymes .
the final construct ( s1-s3 ) had spacer-1 deleted along with the region from notch1 to the cleavage site in spacer-3 ( figure  1a ) .
this construct , like the n1-s3 , was expressed at a much higher level than the wt construct ( figure  1b , lane 7 ) , localized to the golgi ( figure  s2 ) , and displayed a 21-fold increase in catalytic activity toward mm over wt / ( figure  1c ) .
it also phosphorylated the lysosomal enzyme panel better than observed with n1-s3 , ranging from 56%117% of wt level ( figure  1e ) .
strikingly , we noted that s1-s3 phosphorylated the total pool of cellular soluble glycoproteins 3-fold greater than n1-s3 and the wt enzyme , consistent with increased phosphorylation of lysosomal enzymes that are poorly phosphorylated by the wt enzyme as well as an enhanced ability to phosphorylate non - lysosomal glycoproteins ( figure  1f ) . to test the latter possibility , we determined the ability of the various constructs to  phosphorylate a panel of non - lysosomal glycoproteins , two of which were known to be weak substrates of wt glcnac-1-phosphotransferase , namely , renin and protein o - fucosyltransferase 2 ( pofut2).17 , 18 phosphorylation was determined by binding to ci - mpr beads followed by western blotting to detect the proteins .
as shown in figure  1 g , the s1-s3 construct phosphorylated renin , pofut2 , glycopepsinogen ( gp ) , and the von willebrand factor ( vwf ) a1a2a3 domain , whereas the wt construct only phosphorylated renin weakly in this assay .
the n1-s3 construct acted on these proteins as well but to a lesser degree than s1-s3 .
in an attempt to further reduce human glcnac-1-phosphotransferase to resemble the n.  meningitidis enzyme ( figure  s1 ) , a series of deletions within spacer-4 were generated .
however , none of these mutants displayed enzyme activity toward mm ( data not shown ) .
on the basis of the above findings , we selected construct s1-s3 along with the wt / precursor construct to co - transfect with plasmids encoding the three poorly phosphorylated lysosomal enzymes ( laman , gaa , and gba ) and gla as a control for a well - phosphorylated enzyme . in the initial experiment ,
the constructs were co - expressed in gnptab hela cells , followed by incubation with [ 2-h]mannose to label the n - linked glycans of the lysosomal enzymes .
the media was collected and the secreted proteins were immunoprecipitated and analyzed for their content of glycans containing one or two man-6-p residues . as shown in figure  2 , the s1-s3 construct produced 2.1- to 4.9-fold greater phosphorylation of the glycans present on the panel of lysosomal enzymes that were co - expressed with this truncated / precursor relative to wt .
next we co - transfected either expi293 cells or mouse d9  l cells ( lacking the ci - mpr ) with the same plasmids and collected the media after 4872  hr to use as a source of secreted lysosomal enzymes for receptor binding and cell uptake experiments . as a control for phosphorylation mediated by the endogenous glcnac-1-phosphotransferase
, cells were transfected with plasmids encoding the lysosomal enzymes but not with the cdnas for the / precursors .
a representative coomassie blue - stained sds gel of the media from expi293 cells is shown in figure  s3 . in all instances ,
the enzyme secreted by cells co - transfected with the s1-s3 construct bound to the ci - mpr - beads to a much greater extent than observed with the lysosomal enzymes expressed alone in the cells ( figure  3 ) .
further , with the exception of laman , co - transfection with s1-s3 resulted in significantly greater lysosomal enzyme binding to the ci - mpr - beads than obtained with the wt enzyme .
the impact of the increased man-6-p content of the various lysosomal enzymes on their uptake by hela cells is shown in table 1 . in all four instances ,
the enzymes secreted by cells co - transfected with the plasmid encoding the truncated / precursor were internalized many fold better than enzyme secreted by cells using only the endogenous glcnac-1-phosphotransferase .
similar results were obtained with co - transfection of the wt / precursor with the exception of gaa , consistent the ci - mpr - bead binding results .
most of the uptake was blocked by the presence of 5  mm man-6-p in the media , showing that the uptake is mediated by the ci - mpr .
the results with laman were particularly striking , with man-6-p - inhibitable uptake being stimulated by 130- to 153-fold .
it is also notable that the gba was internalized to a great extent in a man-6-p - dependent manner .
the findings presented here establish that phosphorylation of expressed lysosomal enzymes can be substantially increased by co - transfection with an engineered truncated / precursor of glcnac-1-phosphotransferase and , in some instances , with the wt / precursor .
importantly , the  subunit , encoded by a different gene , is not required for this effect .
the enhanced phosphorylation of the lysosomal enzymes increases their binding to the ci - mpr and uptake by cells .
this effect even occurs with lysosomal enzymes such as gla that are well phosphorylated by the endogenous glcnac-1-phosphotransferase .
but most important is the finding that this method enhances the phosphorylation and uptake of human laman and gaa , two lysosomal enzymes that are poorly phosphorylated by endogenous glcnac-1-phosphotransferase .
in addition , the production of gba containing high levels of man-6-p offers the opportunity to restore enzyme activity to cell types in patients with gaucher disease who lack the mannose receptor .
this is of particular interest in light of the reported findings in a mouse model of gaucher disease that cell types other than macrophages are dysfunctional in this disorder .
the availability of highly phosphorylated gba could potentially provide additional benefit to the current therapy that is targeted specifically to macrophages . in conclusion ,
the methods described here have the potential to significantly improve the effectiveness of lysosomal enzymes in ert .
in addition to providing better cell uptake , these preparations may allow lower doses to be administered to patients , perhaps at less frequent intervals .
the method should be applicable to the production of phosphorylated lysosomal enzymes for other lysosomal storage diseases that may be amenable to ert .
expi293 cells ( life technologies ) were grown in suspension in expi293 expression medium ( life technologies ) . the gnptab hela cell line has been described in detail elsewhere .
parental and gnptab hela cells were maintained as a monolayer in dmem ( life technologies ) containing 0.11 g / l sodium pyruvate and 4.5  g / l glucose , supplemented with 10% ( vol / vol ) fetal bovine serum ( fbs ) ( atlanta biologicals ) , 100,000  u / l penicillin , 100  mg / l streptomycin ( life technologies ) , and 2  mm l - glutamine ( life technologies ) .
the ci - mpr negative mouse d9 l - cell line has been  described .
d9 l- cells were maintained as a monolayer in -mem ( life technologies ) containing 100,000  u / l penicillin and 100  mg / l streptomycin ( life technologies ) .
human gnptab - v5/his 8 in pcdna6 and the n1-d deletion construct has been described .
constructs s1-d and s1-s3 were generated in two steps as follows : in the first step , the human spacer-1 sequence was replaced with d.  discoideum spacer-1 using a 0.5 kb gblocks gene fragment ( idt ) in a two - stage overlap extension pcr ( oe - pcr ) .
nucleotides corresponding to amino acids 438819 or 438928 were subsequently removed in the second step to generate s1-d and s1-s3 , respectively .
the construct n1-s3 was generated by deleting nucleotides encoding amino acids 438928 from the wt construct .
the laman - myc - flag cdna was purchased from origene , while the gaa cdna was a kind gift of eline van meel ( leiden university ) .
a c - terminal myc - tag was appended to the gba and gaa c - dna .
renin - ha cdna was purchased from addgene ( cambridge , ma ) , while the plasmid , vwf - a1a2a3-strep - pcdna6 , was kindly provided by joshua muia and j. evan sadler ( washington university school of medicine , st .
constructs encoding the wt and s1-s3 / precursors were expressed in gnptab hela by transfection with lipofectatime 3000 ( life technologies ) according to the manufacturer s protocol .
forty - eight hours post - transfection , cells in six - well plates were harvested and lysed in 250  l of buffer a ( 25  mm tris - cl [ ph 7.2 ] , 150  mm nacl , 1% triton x-100 , and protease inhibitor cocktail ) .
either10  l ( wt ) or 2  l ( s1-s3 ) of cell extract was incubated in buffer b ( 50  mm tris - cl [ ph 7.4 ] , 10  mm mgcl2 , 10  mm mncl2 , 2  mg / ml bsa , 2  mm atp ) in the presence of 75  m udp - glcnac , 1  ci udp-[h]glcnac , and 100  mm -mm in a final volume of 50  l for 1  hr at 37c .
the reactions were stopped by the addition of 1  ml of 2  mm edta [ ph 8.0 ] , and the samples were subjected to qae - sephadex chromatography as previously described .
proteins resolved by sds - page under reducing conditions were transferred to nitrocellulose membrane and detected with antibodies as described in the figure legends .
the indicated amounts of whole - cell extracts were loaded on the gels . to visualize the subcellular localization of wt / and the s1-s3 mutant , the constructs were transfected into gnptab hela cells using lipofectamine 3000 according to the manufacturer s protocol .
twenty - four hours post - transfection , the cells were fixed with 4% formaldehyde ( sigma - aldrich ) and the / subunits were detected with mouse anti - v5 monoclonal antibody ( life technologies ) . the golgi marker golph4 was detected with rabbit anti - golph4 polyclonal antibody ( abcam ) .
the processed cells were mounted in prolong gold antifade mounting medium ( life technologies ) , and the images were acquired with an lsm880 confocal microscope ( carl zeiss ) .
labeling experiments were performed with transfected gnptab hela cells as follows : 48  hr post - transfection , cells in 60  mm tissue culture plates were incubated with 50150  ci of [ 2-h]mannose ( perkin elmer ) for 2  hr , followed by the addition of complete medium containing 5  mm glucose , 5  mm mannose , and 10  mm nh4cl to stop mannose uptake and induce secretion .
the cells were incubated for an additional 3  hr before the medium was collected for analysis .
acid hydrolases secreted into the media were immunoprecipitated , and oligosaccharides isolated and analyzed essentially as described in detail previously . because the laman , gaa , and gba cdnas contained a c - terminal myc - tag , 20  l anti - myc monoclonal antibody ( santa cruz biotechnology ) was pre - bound to 100  l protein g - agarose - plus beads ( santa cruz biotechnology ) prior to immunoprecipitation of these lysosomal hydrolases from the media . in the case of gla ,
the secreted enzyme was immunoprecipitated with protein g - agarose - plus beads pre - bound to anti--gal antibody ( amicus therapeutics ) .
immunoprecipitated material was treated with endo h ( neb ) and filtered with ultracel-10k ( emd millipore ) .
the filtrate containing the released neutral and phosphorylated high mannose glycans was treated with mild acid to remove any n - acetylglucosamine residues still attached to the phosphate moieties and applied to a qae - column matrix to separate the oligosaccharides bearing zero , one or two man-6-p residues .
the retentate containing endo h - resistant complex oligosaccharides was treated with pronase ( roche diagnostics ) and fractionated on cona - sepharose 4b ( ge healthcare ) .
the [ 2-h]-mannose content of the various fractions was determined , and the percentage phosphorylation was calculated as described .
labeling experiments were performed with transfected gnptab hela cells as follows : 48  hr post - transfection , cells in six - well plates were incubated with 10  ci of [ 2-h]mannose ( perkin elmer ) for 2  hr .
following the 2  hr pulse , cells were rinsed twice with pbs and harvested , then resuspended in detergent - free buffer containing 25  mm tris - cl ( ph 7.2 ) and 150  mm nacl at 4c with a protease inhibitor cocktail ( life technologies ) .
cell were lysed by sonication , then subjected to ultracentrifugation at 100,000   g for 1  hr to separate the membrane proteins from the soluble fraction .
one hundred microliters of the soluble fraction was then incubated with purified ci - mpr that was covalently conjugated to cyanogen bromide - activated - sepharose 4b in order to pellet the mannose - phosphorylated glycoproteins , while 10  l of the soluble fraction was precipitated by 1.5% phosphotungstic acid to obtain total [ 2-h]mannose label incorporation into the soluble proteins .
this method allowed the accurate quantification of all the mannose - labeled glycoproteins that were phosphorylated by either wt or mutant glcnac-1-pt .
l - cells were co - transfected with gaa cdna , along with empty vector , wt / precursor , or s1-s3 mutant .
expi293 cells were co - transfected with laman , gba , or gla cdnas , along with either empty vector , wt / precursor , or s1-s3 mutant .
the media was harvested after 23  days . for the production of gba , 10  m of compound at3375 ( amicus therapeutics ) was added to the media to stabilize the secreted enzyme .
soluble bovine ci - mpr was purified from fbs and covalently conjugated to cyanogen bromide - activated - sepharose 4b ( sigma - aldrich ) as described .
aliquots of media from 2- to 3-day transfected expi293 cells or mouse d9 l - cells were diluted with buffer a ( 25  mm tris - cl [ ph 7.2 ] , 150  mm nacl , and 1% triton x-100 ) and incubated with the ci - mpr beads at 4c for 1  hr to bind the phosphorylated lysosomal enzymes . the beads were then sedimented , washed with buffer a , and assayed for lysosomal enzyme activity as described.12 , 22 , 23 the amount of the starting enzyme recovered on the beads was calculated .
parental hela cells were plated on a 12-well plate at approximately 80% density 1  day prior to the cell uptake experiment .
aliquots of media containing each enzyme from the producing cells were added to the parental hela cells in a final volume of 500  l . for competition experiments ,
the cells were incubated with the media for 24  hr , and then the cells and media were collected separately .
the media and cell extracts were centrifuged at 20,000   g , and the supernatants were assayed for their content of lysosomal enzyme activity .
drafted the manuscript , and all authors contributed to editing it to produce the final version .
 several lysosomal enzymes currently used for enzyme replacement therapy in patients with lysosomal storage diseases contain very low levels of mannose 6-phosphate , limiting their uptake via mannose 6-phosphate receptors on the surface of the deficient cells . 
 these enzymes are produced at high levels by mammalian cells and depend on endogenous glcnac-1-phosphotransferase / precursor to phosphorylate the mannose residues on their glycan chains . 
 we show that co - expression of an engineered truncated glcnac-1-phosphotransferase / precursor and the lysosomal enzyme of interest in the producing cells resulted in markedly increased phosphorylation and cellular uptake of the secreted lysosomal enzyme . 
 this method also results in the production of highly phosphorylated acid -glucocerebrosidase , a lysosomal enzyme that normally has just trace amounts of this modification . 
Introduction
Results
Discussion
Materials and Methods
Author Contributions
Conflicts of Interest