purification of endogenous regiiiendogenous 
  regiii was purified from the small intestines of c57bl/6 mice . 
 
intestinal tissues were homogenized in 20% acetic acid solution containing 
  protease inhibitors using a pre - chilled homogenization probe and lysed by 
  sonication using a misonix xl sonicator .
the extract was dialyzed against 25 
  mm mes ph 5.0 , 25 mm nacl and was loaded onto a cation 
  exchange column ( sp - sepharose , sigma ) .
the column was washed with 25 
  mm mes ph 5.0 , 150 mm nacl , and eluted with 25 
  mm mes ph 5.0 , 500 mm nacl .
the eluate was concentrated 
  and loaded onto a sephacryl s-100 column ( ge healthcare ) . 
 
regiii-containing fractions were identified by western blot , pooled , 
  and purified by passage over immobilized anti - regiii 
  ( 8) .
the eluate was 
  concentrated , transferred to immobilon p , and subjected to edman degradation 
  on an abi494 sequencer ( pe biosystems ) to determine the n - terminal 
  sequence .
expression and purification of recombinant 
  proteins  recombinant pro - regiii ( rpro - regiii ) and 
  rpro - hip / pap were expressed and purified as previously described 
  ( 8) . to generate the 
  recombinant processed form of regiii , a 417-bp amplicon was generated 
  using the rpro - regiii expression construct ( pet3a - regiii ) 
  ( 8) as template and the 
  specific primers 
  5-attgcgaggcatatgagcagctgccccaagggctccc-3 ( forward ) 
  and 5-ctatggggatccctaggccttgaatttgcagacatagggt-3 
  ( reverse ) .
the forward primer contained an ndei restriction site ( underlined ) 
  for cloning into pet3a .
the reverse primer contained the native stop codon 
  followed by an engineered bamhi site ( underlined ) .
the amplicon was digested 
  with ndei and bamhi and ligated into ndei / bamhi - digested pet3a ( novagen ) . 
 
similarly , the rhip / pap expression construct was generated using 
  pet3a - hip / papmut ( 8) as 
  template and specific primers 
  5-attgcgaggcatatgattcgatgtccaaaaggctccaag-3 ( forward ) and 
  5-ctatggtgatcatcagtgaactttgcagacatagggtaacc-3 ( reverse ) .
the 
  forward primer contained an ndei site ( underlined ) while the reverse primer 
  contained a bcli site ( underlined ) after the native stop codon .
the amplicon 
  was digested with ndei and bcli and ligated into ndei / bamhi - digested pet3a . 
 
point mutations were introduced into rpro - hip / pap using the quikchange ii 
  site - directed mutagenesis kit ( stratagene ) and specific primers harboring the 
  desired mutations .
expression and purification of recombinant wild - type and 
  mutant proteins was carried out as previously described 
  ( 8) .
antimicrobial assays and 
  peptidoglycan binding assays were carried out as previously described 
  ( 5 ) .
proteins were stored at 
  20 c for a maximum of 1 week prior to assay . immunoblotting 
intestinal extracts were prepared from 
  wild - type c57bl/6 or mmp7 mice ( jackson 
  laboratories ) as previously described 
  ( 5 ) .
extracts from human 
  intestinal tissues were prepared by extraction in acetic acid as previously 
  described ( 9 ) .
20 g of each 
  extract was loaded onto 15% sds - page gels and blotted onto immobilon p 
  ( millipore ) .
blots were probed with anti - regiii antiserum 
  ( 8) and goat 
  anti - rabbit - horseradish peroxidase ( amersham biosciences ) , and were detected 
  by chemiluminescence using the pierce supersignal west pico chemiluminescent 
  detection kit . in vitro trypsin proteolytic processing 
recombinant 
  pro - regiii and pro - hip / pap were digested with bovine pancreatic trypsin 
  ( sigma ) at a 1:200 molar ratio of trypsin : lectin at 37 c for 2 h. 
  proteins were immediately analyzed by sds - page on 15% gels , and were subjected 
  to n - terminal edman sequencing .
nmr spectroscopy  to prepare samples for nmr experiments , 
  escherichia coli bl21-codonplus ( de3)-rilp transformed with 
  expression plasmids were grown in m9 minimal media containing 1 g / liter of 
  nh4cl for uniformly n - labeled samples , 
  further substituting unlabeled glucose with 3 g / liter of 
  c6-glucose for uniformly 
  n / c - labeled samples .
backbone n , 
  c , and h chemical shift assignments of rpro - hip / pap 
  were generated from triple resonance nmr data recorded at 25 c using a 
  cryoprobe - equipped varian inova 600 mhz spectrometer .
backbone assignments 
  were made from a 300 m sample of 
  n / c - labeled protein in 25 mm mes ph 5.5 , 
  25 mm nacl using standard methods 
  ( 10 ) .
all spectra were 
  processed using nmrpipe ( 11 ) 
  and analyzed with nmrview 
  ( 12 ) .
regiii is proteolytically processed by trypsin in 
  vivo  two distinct forms of endogenous regiii are present in 
  the mouse small intestine ( 5 ) . 
 
the higher molecular weight form co - migrates at 16.5 kda with recombinant 
  regiii that lacks its n - terminal secretion signal , while the lower 
  molecular weight form migrates at 15 kda .
the presence of the lower 
  molecular weight form suggested that regiii might be proteolytically 
  processed in vivo . to test this idea we purified endogenous 
  regiii from mouse small intestine by acid extraction , gel filtration 
  chromatography , and antibody affinity chromatography .
edman sequencing of the 
  purified proteins verified that the higher molecular weight regiii form 
  indeed harbors an n terminus ( glu ) that is generated by removal 
  of the signal sequence ( fig . 
 
the n terminus of the lower molecular weight form is 
  ser , corresponding to the predicted trypsin cleavage site located 
  at arg 
this site 
  is conserved among all mouse and human regiii family members 
  ( fig .
1b ) , and is also 
  present in other reg subfamilies including regi 
  ( 13 ) .
liquid 
  chromatography - tandem mass spectrometry ( lc - ms / ms ) of both regiii forms 
  did not reveal any further modifications of the protein ( data not shown ) . 
 
these results indicate that the n terminus of endogenous mouse regiii 
  is proteolytically processed by trypsin or a trypsin - like protease in 
  vivo .
small intestinal -defensins are a distinct family of antimicrobial 
  peptides that also undergo n - terminal proteolytic processing .
while human 
  -defensins are processed by trypsin 
  ( 9 ) , the processing enzyme for 
  mouse -defensins is matrix metalloproteinase-7 ( mmp-7 ) 
  ( 2 ) . to verify that mmp-7 is 
  not the processing enzyme for mouse regiii we performed western blots 
  on small intestinal tissue extracts from wild - type and 
  mmp7 mice .
thus , 
  regiii and -defensins are processed by distinct proteases in the 
  mouse small intestine .
a , purification and n - terminal sequencing of 
  endogenous mouse regiii reveals processing at the conserved n - terminal 
  trypsin site .
b , conserved canonical trypsin site ( indicated by 
  arrow ) is present near the n terminus of mouse and human regiii 
  family members .
20 
  g of protein extract from wild - type and mmp7 
  mice were immunoblotted with anti - regiii antibody .
20 g of human 
  intestinal protein extract was immunoblotted and probed with 
  anti - regiii antiserum .
recombinant pro - hip / pap ( rpro - hip / pap ; with the 
  n - terminal signal sequence replaced by methionine ) and recombinant processed 
  hip / pap ( rhip / pap ; with the n - terminal tryptic fragment replaced by 
  methionine ) were included for size comparison .
, small intestinal e , 
  in vitro incubation of purified recombinant pro - regiii 
  ( rpro - regiii ) and rpro - hip / pap with bovine trypsin results in 
  quantitative cleavage at the conserved trypsin site to yield a homogeneous 
  product .
proteins were digested with a 1:200 molar ratio of trypsin : lectin and 
  were analyzed by sds - page .
n - terminal sequencing verified cleavage at 
  arg  ser and 
  arg  ile , respectively .
a , purification and n - terminal sequencing of 
  endogenous mouse regiii reveals processing at the conserved n - terminal 
  trypsin site .
b , conserved canonical trypsin site ( indicated by 
  arrow ) is present near the n terminus of mouse and human regiii 
  family members .
20 
  g of protein extract from wild - type and mmp7 
  mice were immunoblotted with anti - regiii antibody .
20 g of human 
  intestinal protein extract was immunoblotted and probed with 
  anti - regiii antiserum .
recombinant pro - hip / pap ( rpro - hip / pap ; with the 
  n - terminal signal sequence replaced by methionine ) and recombinant processed 
  hip / pap ( rhip / pap ; with the n - terminal tryptic fragment replaced by 
  methionine ) were included for size comparison .
s.i . , small intestinal e , 
  in vitro incubation of purified recombinant pro - regiii 
  ( rpro - regiii ) and rpro - hip / pap with bovine trypsin results in 
  quantitative cleavage at the conserved trypsin site to yield a homogeneous 
  product .
proteins were digested with a 1:200 molar ratio of trypsin : lectin and 
  were analyzed by sds - page .
n - terminal sequencing verified cleavage at 
  arg  ser and 
  arg  ile , respectively . in the human small intestine ,
hip / pap is the predominantly expressed 
  ortholog of regiii ( 6 , 
  14 ) and performs a similar 
  antibacterial function ( 5 ) . 
 
immunoblotting of human small intestinal extract revealed two forms of hip / pap 
  with different molecular weights , suggesting that hip / pap is also expressed as 
  a pro - protein that is proteolytically processed in vivo 
  ( fig .
although 
  the small amounts of hip / pap present in human tissue presented an obstacle to 
  purification and sequencing , we established that the lower molecular weight 
  form co - migrates with recombinant hip / pap engineered to lack the 11 amino 
  acids n - terminal to the predicted arg 
the idea that the hip / pap n terminus is processed 
  in vivo by trypsin is supported by two additional observations . 
 
first , two trypsin isoforms are produced by human small intestinal epithelia 
  and have been shown to process other intestinal proteins in vivo 
  ( 9 ) .
second , in vitro 
  incubation of recombinant pro - hip / pap ( rpro - hip / pap ) with bovine trypsin ( at a 
  1:200 molar ratio of enzyme to lectin ) resulted in quantitative cleavage at 
  arg 
proteolytic processing by trypsin activates regiii lectin antimicrobial 
  activity  we next investigated the biological relevance of lectin 
  proteolytic processing . to test the hypothesis that processing regulates 
  regiii lectin antibacterial activity , we incubated recombinant 
  pro - regiii ( rpro - regiii ) with trypsin in vitro and 
  quantitated antimicrobial activity against listeria monocytogenes in 
  standard antibacterial assays 
  ( 5 )
. mock - digested 
  rpro - regiii exhibited limited antibacterial activity , with l. 
  monocytogenes numbers declining by 68% in the presence of 10 
  m rpro - regiii ( fig . 
 
however , trypsin - digested rpro - regiii 
  exhibited enhanced antibacterial potency , with l. monocytogenes 
  numbers declining by > 96% in the presence of 10 m of 
  digested regiii ( fig . 
 
a comparison of proteolytically cleaved and 
  uncleaved rpro - hip / pap yielded similar results 
  ( fig .
the 
  enhanced activity of trypsin - cleaved hip / pap is not attributable to the 
  n - terminal prosegment itself , as addition of 50 m of synthetic 
  n - terminal peptide to l. monocytogenes did not affect bacterial 
  viability ( fig . 2c ) . 
 
these data demonstrate that removal of the n - terminal prosegment by trypsin 
  enhances regiii and hip / pap antibacterial activity .
a and b , trypsin proteolysis of recombinant 
  pro - regiii ( rpro - regiii ) and recombinant pro - hip / pap 
  ( rpro - hip / pap ) activates antibacterial activity . purified 
  rpro - regiii ( a ) and rpro - hip / pap ( b ) were digested 
  with bovine trypsin as in fig . 
 
l. monocytogenes was exposed to the 
  indicated lectin concentrations at 37 c for 2 h , and surviving bacteria 
  were quantitated by dilution plating . an assay which included trypsin but no 
  lectin
c , addition of 50 m of 
  the hip / pap n - terminal peptide did not diminish l. monocytogenes 
  viability , indicating that the prosegment alone does not exhibit antibacterial 
  activity .
d and e , antibacterial activities of recombinant 
  mature regiii and hip / pap .
the 11-amino acid prosegments of 
  regiii and hip / pap were removed and replaced with methionine to yield 
  rregiii and rhip / pap , and bactericidal activity was determined in 
  comparison with rpro - regiii ( d ) and rpro - hip / pap ( e ) . 
 
f , hip / pap n - terminal prosegment does not inhibit bactericidal 
  activity in trans .
the synthetic hip / pap n - terminal peptide depicted 
  in c was added to bactericidal assays with rhip / pap .
a and b , trypsin proteolysis of recombinant 
  pro - regiii ( rpro - regiii ) and recombinant pro - hip / pap 
  ( rpro - hip / pap ) activates antibacterial activity .
purified 
  rpro - regiii ( a ) and rpro - hip / pap ( b ) were digested 
  with bovine trypsin as in fig . 
 
l. monocytogenes was exposed to the 
  indicated lectin concentrations at 37 c for 2 h , and surviving bacteria 
  were quantitated by dilution plating . an assay which included trypsin but no 
  lectin was run as a control .
c , addition of 50 m of 
  the hip / pap n - terminal peptide did not diminish l. monocytogenes 
  viability , indicating that the prosegment alone does not exhibit antibacterial 
  activity .
d and e , antibacterial activities of recombinant 
  mature regiii and hip / pap .
the 11-amino acid prosegments of 
  regiii and hip / pap were removed and replaced with methionine to yield 
  rregiii and rhip / pap , and bactericidal activity was determined in 
  comparison with rpro - regiii ( d ) and rpro - hip / pap ( e ) . 
 
f , hip / pap n - terminal prosegment does not inhibit bactericidal 
  activity in trans .
the synthetic hip / pap n - terminal peptide depicted 
  in c was added to bactericidal assays with rhip / pap . to further establish that prosegment removal activates lectin antibacterial 
  activity , we generated recombinant forms of regiii and hip / pap that 
  lacked their n - terminal prosegments .
10 m of recombinant mature 
  regiii ( rregiii ) decreased l. monocytogenes viability 
  by over 99% , as compared with a 12% decrease for rpro - regiii 
  ( fig .
similarly , 
  10 m of recombinant mature hip / pap ( rhip / pap ) killed > 99.9% 
  of organisms , as compared with 56% for rpro - hip / pap 
  ( fig .
addition of 
  the hip / pap n - terminal prosegment in trans did not reduce the 
  antibacterial activity of rhip / pap ( fig . 
 
2f ) , indicating that the prosegment is inhibitory only 
  when covalently attached ( in cis ) .
peptidoglycan pull - down assays 
  established that prosegment removal did not diminish the ability of either the 
  human or mouse protein to bind peptidoglycan 
  ( fig .
3 ) . collectively , these 
  data establish that the regiii lectin n - terminal prosegment inhibits 
  antibacterial activity in cis , but does not affect peptidoglycan 
  binding .
recombinant unprocessed and processed regiii and hip / pap were compared 
  in peptidoglycan pull - down assays .
pellet ( p ) and supernatant 
  ( s ) fractions were analyzed by sds - page .
recombinant unprocessed and processed regiii and hip / pap were compared 
  in peptidoglycan pull - down assays .
pellet ( p ) and supernatant 
  ( s ) fractions were analyzed by sds - page .
we previously reported bactericidal activity for recombinant regiii 
  and hip / pap that were expressed with an intact n - terminal prosegment 
  ( 5 ) . to determine why we were 
  able to detect antibacterial activity in these protein preparations , we 
  investigated the fate of the pro - hip / pap n terminus during storage .
we noted 
  that the rpro - hip / pap is labile and the n terminus is progressively removed 
  over time during storage at 4 c ( supplemental fig .
the presence of 
  small amounts of mature hip / pap is thus likely to account for the 
  antibacterial activity of protein preparations derived from recombinant 
  prosegment - containing lectins .
this may also explain why we detect modest 
  antibacterial activity in rpro - regiii and rpro - hip / pap following mock 
  digestion for 2 h at 37c ( fig .
n - terminal acidic residues are essential for prosegment inhibitory 
  activity  we next sought to gain insight into the inhibitory 
  mechanism of the regiii lectin n - terminal prosegment .
we noted the presence of 
  either two or three acidic residues ( glu or asp ) at conserved positions in 
  both mouse and human regiii family members 
  ( fig .
to evaluate 
  the functional importance of the three acidic residues contained within the 
  hip / pap prosegment ( glu , glu , and glu ) , 
  we mutated combinations of these residues to ala and assessed the mutant 
  rpro - hip / pap proteins for antibacterial activity 
  ( fig .
recombinant 
  pro - hip / pap - e32a ( 10 m ) reduced l. monocytogenes 
  viability by 30% , while rpro - hip / pap - ee / aa ( harboring e27a and e28a mutations ) 
  produced a 75% decline in bacterial colony forming units ( cfus ) at the same 
  concentration ( fig . 
 
in contrast , mutation of all three acidic residues 
  ( rpro - hip / pap - eee / aaa ) yielded a prosegment - containing protein that produced a 
  > 99.9% reduction in the viability of l. monocytogenes at a 10 
  m concentration ( fig . 
 
this demonstrates that multiple n - terminal acidic 
  residues are essential for hip / pap prosegment inhibitory function , and 
  suggests that the degree of inhibitory activity is dictated by charge .
a , primary structure of the pro - hip / pap n terminus 
  showing the positions of engineered mutations .
b , comparison of 
  antibacterial activity among rpro - hip / pap , rhip / pap , and rpro - hip / pap 
  harboring mutations in n - terminal glutamic acid ( e ) residues . 
 
n - terminal acidic resides are essential for prosegment inhibitory 
  activity . a , primary structure of the pro - hip / pap n terminus 
  showing the positions of engineered mutations .
b , comparison of 
  antibacterial activity among rpro - hip / pap , rhip / pap , and rpro - hip / pap 
  harboring mutations in n - terminal glutamic acid ( e ) residues . 
 
a , ribbon diagram of 
  the pro - hip / pap crystal structure ( rcsb accession : 1uv0 ) 
  ( 15 ) .
the disordered 
  n - terminal 10 amino acids are indicated by a dotted line , and the 
  locations of the n - terminal disulfide bond ( red ) and the trypsin 
  cleavage site are indicated .
b , experimental 
  nr1 and r2 relaxation rates were determined 
  for pro - hip / pap , and a plot of the r1/r2 ratio is shown . 
 
the location of the arg  ile trypsin site is 
  indicated by a dashed red line .
a , ribbon diagram of 
  the pro - hip / pap crystal structure ( rcsb accession : 1uv0 ) 
  ( 15 ) .
the disordered 
  n - terminal 10 amino acids are indicated by a dotted line , and the 
  locations of the n - terminal disulfide bond ( red ) and the trypsin 
  cleavage site are indicated .
b , experimental 
  nr1 and r2 relaxation rates were determined 
  for pro - hip / pap , and a plot of the r1/r2 ratio is shown . 
 
the location of the arg  ile trypsin site is 
  indicated by a dashed red line .
structural analysis of the hip / pap - prosegment 
  interaction  although the crystal structure of pro - hip / pap has been 
  determined ( 15 ) , it offered 
  limited information about the hip / pap - prosegment interaction .
in particular , 
  n - terminal residues 2735 are disordered and thus absent from the 
  structure ( fig .
5a ) 
  and residues 3641 appear to be involved in crystal packing 
  interactions , raising questions about the observed structure of this essential 
  region of the protein .
we therefore used nuclear magnetic resonance ( nmr ) 
  spectroscopy to structurally characterize the interaction between hip / pap and 
  its n - terminal prosegment . using uniformly 
  n ,
c - labeled pro - hip / pap and standard triple 
  resonance methods ( 16 ) we 
  assigned the chemical shifts of 98% of the backbone resonances .
we first 
  analyzed the conformational dynamics of pro - hip / pap by measuring 
  n relaxation rates , which are influenced by fast ( ps - ns ) and slow 
  ( s - ms ) motions of backbone amides throughout a protein .
in particular , we 
  measured rate constants for spin - lattice relaxation ( r1 ) , and 
  spin - spin relaxation ( r2 ) , finding that the ratio of these values 
  ( r1/r2 ) was highest in residues 2734 
  ( fig .
this 
  indicates a high degree of backbone conformational mobility at the n terminus . 
 
furthermore , talos analyses of backbone chemical shifts 
  ( 17 ) and 
  h - h noes indicated that the prosegment adopts an 
  extended structure .
together , these data suggest that the prosegment is 
  flexible and transiently interacts with the rest of the protein .
a , orientation of arg and lys side chains 
  near the hip / pap n - terminal trypsin site .
b , mutations of basic hip / pap residues 
  yield active rpro - hip / pap .
comparison of antibacterial activity among 
  rpro - hip / pap , rhip / pap , and rpro - hip / pap harboring the indicated mutations in 
  basic residues .
c , hip / pap 
  basic residues ( arg , lys , lys ) are 
  dispensable for antibacterial activity .
limited trypsin proteolysis was 
  performed on rpro - hip / pap - rkk / aaa .
sds - page analysis of the undigested and 
  digested proteins is depicted . digested and undigested proteins were analyzed 
  for bactericidal activity as outlined in 
  fig .
hip / pap basic residues are essential for prosegment inhibition of 
  antibacterial activity . a , orientation of arg and lys side chains 
  near the hip / pap n - terminal trypsin site .
b , mutations of basic hip / pap residues 
  yield active rpro - hip / pap .
comparison of antibacterial activity among 
  rpro - hip / pap , rhip / pap , and rpro - hip / pap harboring the indicated mutations in 
  basic residues .
c , hip / pap 
  basic residues ( arg , lys , lys ) are 
  dispensable for antibacterial activity .
limited trypsin proteolysis was 
  performed on rpro - hip / pap - rkk / aaa .
sds - page analysis of the undigested and 
  digested proteins is depicted . digested and undigested proteins were analyzed 
  for bactericidal activity as outlined in 
  fig .
hip / pap basic residues are essential for prosegment inhibition of 
  antibacterial activity  these findings suggested that the hip / pap 
  n - terminal peptide may inhibit antibacterial activity through dynamic 
  interactions with other residues in the hip / pap core protein . given that 
  acidic amino acids in the n terminus are essential for prosegment inhibitory 
  activity
, we hypothesized that interactions with nearby hip / pap basic residues 
  may be required for repression of antibacterial activity . because the 
  prosegment is only 11 amino acids in length
, we reasoned that it was likely to 
  be constrained to local interactions with amino acids near the trypsin site . 
 
examination of the hip / pap primary sequence revealed three basic residues 
  ( arg , lys , lys ) positioned immediately 
  c - terminal to the arg 
while 
  introduction of a single mutation ( r39a ) was insufficient to activate 
  antimicrobial activity in rpro - hip / pap , mutation of all three basic amino 
  acids to alanine ( rpro - hip / pap - rkk / aaa ) yielded a prosegment - containing 
  variant that exhibited potent antibacterial activity , producing a > 99% 
  decline in l. monocytogenes viability 
  ( fig .
critically , 
  the presence of these mutations had no effect on the antimicrobial activity of 
  the mature protein lacking the prosegment 
  ( fig .
these data 
  demonstrate that basic hip / pap residues , positioned c - terminal to the trypsin 
  site , are essential for prosegment inhibitory activity but are dispensable for 
  antibacterial function .
structural effects of activating pro - hip / pap 
  mutations  overall , these results suggested a model in which the 
  prosegment maintains hip / pap in an inactive state through transient 
  interactions between acidic n - terminal residues and basic residues positioned 
  c - terminal to the trypsin site .
mutation of these charged residues derepresses 
  hip / pap antibacterial activity in the prosegment - containing protein , thus 
  mimicking the derepression that occurs when the peptide is removed by 
  proteolysis . to further examine this interaction we compared 
  n / h heteronuclear single quantum coherence ( hsqc ) 
  spectra of rpro - hip / pap glutamic acid - to - alanine mutants with the wild - type 
  protein ( supplemental fig . s2 ) .
we observed significant chemical shift changes 
  (  > 0.05 ppm ) only in residues n - terminal to and including 
  ile ( fig . 
 
this verified that the gain - of - function phenotype 
  produced by the eee / aaa and rkk / aaa mutations did not result from gross 
  protein misfolding .
furthermore , the lack of significant chemical shift 
  changes in the main body of the protein argues against prosegment inhibitory 
  activity being related to global changes in the core hip / pap structure . 
 
notably , n / h hsqc spectra of hip / pap with and without 
  the wild - type prosegment showed no significant chemical shift changes aside 
  from those at the trypsin cleavage site , confirming that proteolysis itself 
  also does not cause a global conformational change ( data not shown ) .
analysis 
  of peak intensity did not reveal evidence of prosegment - modulated dimer or 
  oligomer formation , even at the high concentration ( 300 m ) used 
  for nmr spectroscopy ( data not shown ) .
however , as these spectra were 
  collected in the absence of ligand , the possibility remains that multimer 
  formation may occur in the presence of peptidoglycan ligands .
we noted a striking co - linear pattern of change in key n - terminal backbone 
  amide chemical shifts when comparing the n / h hsqc 
  spectra of rpro - hip / pap and single , double or triple glutamic acid - to - alanine 
  mutants .
peaks from ser , ala , arg , and 
  ile shifted in the same direction in each of the rpro - hip / pap 
  mutants ( fig .
7b ) , 
  with the triple eee / aaa mutant exhibiting chemical shift changes that are 
  close to the additive sum of those observed for the single e32a and double 
  e27a / e28a mutants ( table 1 ) 
  ( 18 ) . such a co - linear 
  chemical shift pattern signifies a molecule that is in conformational 
  equilibrium between two states , with intermediate chemical shifts deriving 
  from a population - weighted average of these states 
  ( 16 ) .
furthermore , the 
  additivity of these chemical shift changes indicates a simple , non - cooperative 
  interaction between the charges on the prosegment and the rest of the hip / pap 
  protein .
notably , similar chemical shift changes for the 
  ser  ile segment were observed in the activated 
  rpro - hip / pap - rkk / aaa mutant , suggesting that the same conformational change is 
  being triggered from the  opposite  side of the putative 
  charge / charge interaction ( fig . 
 
close inspection of the n / h 
  hsqc spectrum for this mutant shows that peaks for residues 
  ser  ile are doubled on this vector , indicative 
  of further conformational exchange occurring on a slow time scale . 
 
collectively , these data support a model in which perturbation of the 
  interaction between hip / pap and its prosegment drives a shift in the 
  conformational equilibrium toward a biologically active state 
  ( fig .
our data further 
  suggest that the degree to which the prosegment shifts the hip / pap population 
  to the inactive state is simply determined by the number of intramolecular 
  charge - charge interactions rather than any specific ordering of the prosegment 
  against the core hip / pap structure .
table 1simple additivity of chemical shift differences in hip / pap e / a 
  mutantss35a36r37i38ppmppmppmppm e / aa 
  ( e32a ) 
   0.565 
   0.265 
   0.543 
   0.399 
   ee / aaa 
  ( e27a / e28a ) 
   0.542 
   0.268 
   0.318 
   0.510 
   eee / aaaa 
  ( e27a / e28a / e32a ) 
   1.017 
   0.511 
  
0.859 
   0.872 
 
  calculated 
  eee / aaab 
  ( e / a + ee / aa ) 
 
  1.107 
 
  0.533 
 
  0.861 
 
  0.909 
  achemical shift differences ( tot ) were obtained 
  using the following equation : tot = 
  [ h ) + (   
  n ) ] ( normalized for 
  proton with the scale factor  = 0.17 , established from estimates of 
  atom - specific chemical shift ranges in a protein environment ) 
  ( 14).bcalculated chemical shift differences were determined by tot 
  = e / a + ee / aa .
simple additivity of chemical shift differences in hip / pap e / a 
  mutants chemical shift differences ( tot ) were obtained 
  using the following equation : tot = 
  [ h ) + (   
  n ) ] ( normalized for 
  proton with the scale factor  = 0.17 , established from estimates of 
  atom - specific chemical shift ranges in a protein environment ) 
  ( 14 ) .
calculated chemical shift differences were determined by tot 
  = e / a + ee / aa .
a , 
  chemical shift changes in the n / h hsqc spectra of 
  rpro - hip / pap and the activated mutant rpro - hip / pap - eee / aaa are plotted as a 
  function of residue number .
b , superimposed n / h hsqc spectra of 
  n - labeled rpro - hip / pap and activating rpro - hip / pap mutations 
  reveal colinear chemical shift perturbations among four residues surrounding 
  the trypsin cleavage site at arg / ser . 
 
arrows indicate the direction of larger chemical shift changes from 
  wild - type and progression toward enhanced hip / pap killing activity .
a , 
  chemical shift changes in the n / h hsqc spectra of 
  rpro - hip / pap and the activated mutant rpro - hip / pap - eee / aaa are plotted as a 
  function of residue number .
b , superimposed n / h hsqc spectra of 
  n - labeled rpro - hip / pap and activating rpro - hip / pap mutations 
  reveal colinear chemical shift perturbations among four residues surrounding 
  the trypsin cleavage site at arg / ser . 
 
arrows indicate the direction of larger chemical shift changes from 
  wild - type and progression toward enhanced hip / pap killing activity .
the regiii lectins constitute a vital component of the intestinal 
  epithelial antibacterial arsenal which protect the host against pathogens 
  ( 19 ) and maintain homeostasis 
  with symbiotic bacteria ( 5 ) . 
 
however , little is known about how regiii antibacterial activity is regulated . 
  in this report
we show that regiii lectins are subject to repression by an 
  inhibitory n - terminal prosegment that is removed in vivo by trypsin . 
 
the inhibitory activity of the n - terminal segment depends 
  on charge - charge interactions with the main body of the protein .
derepression 
  of antibacterial activity occurs when these interactions are perturbed , either 
  through proteolytic removal of the prosegment or by mutation of the charged 
  residues ( fig .
the hip / pap hinge region ( dashed line ) undergoes a two - state 
  conformational shift between a closed ( inhibited ) and an open 
  ( active ) form .
maintenance of the closed , inactive state depends on 
  transient interactions between negatively charged prosegment residues and 
  positively charged residues on the hip / pap core protein .
inhibition is 
  relieved either by cleavage at a conserved trypsin site in the hinge region , 
  or by mutation of either charged region , leading to a 1000-fold increase 
  in antibacterial activity .
note that the hinge region in the activating 
  mutants is in a constitutively open form , as demonstrated by nmr .
we propose 
  that disruption of prosegment - core protein interactions alleviates inhibition 
  by unmasking a region ( shown in white ) necessary for bacterial cell 
  surface damage or for multimerization that may be required for bactericidal 
  activity .
the hip / pap hinge region ( dashed line ) undergoes a two - state 
  conformational shift between a closed ( inhibited ) and an open 
  ( active ) form .
maintenance of the closed , inactive state depends on 
  transient interactions between negatively charged prosegment residues and 
  positively charged residues on the hip / pap core protein .
inhibition is 
  relieved either by cleavage at a conserved trypsin site in the hinge region , 
  or by mutation of either charged region , leading to a 1000-fold increase 
  in antibacterial activity .
note that the hinge region in the activating 
  mutants is in a constitutively open form , as demonstrated by nmr .
we propose 
  that disruption of prosegment - core protein interactions alleviates inhibition 
  by unmasking a region ( shown in white ) necessary for bacterial cell 
  surface damage or for multimerization that may be required for bactericidal 
  activity .
this regulatory mechanism may have evolved to give the host control over 
  the timing and location of the expression of regiii antibacterial activity .
as 
  regiii lectin antibacterial activity involves damage to microbial cell 
  surfaces ( 5 ) , it is possible 
  that these proteins could also damage host cell membranes .
this would suggest 
  a need to maintain regiii lectins in an inactive state prior to their luminal 
  release .
two trypsin isozymes are expressed in human gut epithelial cells and 
  are stored as inactive zymogens that are activated by proteolysis in the 
  intestinal lumen ( 9 ) .
it thus 
  seems likely that trypsin - mediated activation of regiii proteins occurs only 
  after they are secreted .
given the fact that we were able to detect processed 
  regiii and hip / pap in small intestinal tissue extracts , we can not 
  completely rule out the possibility of some intracellular cleavage .
however , 
  it is likely that this reflects extracellular processing of proteins that are 
  trapped in the mucus layer that overlies the intestinal epithelium .
thus , we 
  propose that this regulatory mechanism evolved to ensure that lectin 
  bactericidal activity is inhibited during intracellular storage , and activated 
  upon secretion into the gut lumen . in the small intestine , members of the -defensin family of 
  antimicrobial peptides also require activation through proteolytic removal of 
  an n - terminal prosegment by specific intestinal proteases 
  ( 2 , 
  9 ) . while trypsin is the 
  processing enzyme for at least one member of the human -defensin family 
  ( 9 ) , mouse -defensins 
  undergo processing by matrix metalloproteinase-7 ( matrilysin ; mmp-7 ) 
  ( 2 ) .
our findings demonstrate 
  that trypsin coordinately activates multiple innate immune effector proteins 
  in both mice and humans , suggesting that this protease may function as a 
  central regulator of diverse intestinal antibacterial responses in multiple 
  host species .
our results indicate that hip / pap prosegment inhibitory activity is 
  governed by interactions between acidic prosegment residues and cationic 
  residues on the remainder of the protein .
the prosegments of key intestinal 
  -defensins confer inhibitory activity through a similar charge - charge 
  interaction ( 20 ) . however , our 
  results suggest that the functional outcome of this interaction is quite 
  different in the two protein families .
the -defensin prosegment 
  neutralizes cationic amino acids that bind to negatively charged bacterial 
  phospholipids and are thus essential for antibacterial function 
  ( 21 ) .
in contrast , while 
  hip / pap cationic amino acids are required for peptide inhibitory activity , 
  they are dispensable for antibacterial function , indicating that the hip / pap 
  prosegment represses antibacterial activity through a distinct mechanism .
this 
  is consistent with our finding that regiii and hip / pap bind to 
  bacterial surfaces through specific recognition of peptidoglycan , and that 
  this binding interaction is not modulated by the prosegment . collectively , 
  these observations suggest that the regiii lectins may mediate their 
  antibacterial functions via mechanisms that differ from the 
  -defensins .
nmr spectroscopy has provided structural insight into the 
  hip / pap - prosegment interaction .
our analysis revealed a distinctive colinear 
  correspondence between backbone amide shifts as point mutations were made to 
  either side of a putative charge - charge interaction pair .
notably , point 
  mutants with derepressed killing activity demonstrated the greatest degree of 
  chemical shift changes .
such correspondence between structural and functional 
  changes is distinctive , and has previously been observed in several other 
  systems 
  ( 2224 ) . 
 
the linearity of this behavior further suggests that intramolecular 
  interactions between hip / pap and its n - terminal prosegment govern a shift 
  between two states with differing activities .
although the physical basis for 
  this shift is not yet clear , we envision at least three possibilities .
one is 
  that disruption of hip / pap - prosegment interactions results in allosteric 
  changes in residues that are physically distant from the n terminus .
this 
  seems very unlikely given the limited chemical shift changes observed outside 
  the n - terminal region in comparisons of the wildtype and eee / aaa 
  n / h hsqc spectra .
a second possibility is that the 
  activating mutations increase accessibility of the trypsin site to cleavage . 
 
however , the lack of hip / pap proteolysis in our in vitro 
  antibacterial assays suggests that this is unlikely to explain the enhanced 
  bactericidal activity of the activated pro - hip / pap mutants ( supplemental fig . 
 
a third possibility is that disruption of the hip / pap - prosegment 
  interaction unmasks surfaces on the hip / pap core structure that are required 
  for bactericidal activity , and that our chemical shift changes reflect a 
  hinging motion of the prosegment away from the rest of the protein .
such 
  movement could allow the hip / pap molecule to adopt an optimal conformation to 
  damage microbial cell surfaces , or could induce formation of multimers that 
  may be required for bactericidal activity 
  ( fig .
the latter of these 
  two mechanisms has been observed in other systems in which bactericidal 
  activity requires formation of protein oligomers 
  ( 2527 ) . 
 
although our nmr spectra did not show evidence of hip / pap multimerization at 
  high concentration ( 300 m ) in the absence of ligand , it is 
  possible that such association requires both the presence of peptidoglycan 
  ligands and the disruption of hip / pap - prosegment interactions . in summary
, we have uncovered a novel mechanism controlling c - type lectin 
  biological activity .
we propose that impaired regulation of regiii 
  antibacterial activity in human populations could result in compromised 
  intestinal immunity and a predisposition to enteric infections and 
  inflammation .
furthermore , the structural insights from these studies could 
  aid in the design of novel antimicrobial therapeutics .
 members of the regiii family of intestinal c - type lectins are directly 
  antibacterial proteins that play a vital role in maintaining host - bacterial 
  homeostasis in the mammalian gut , yet little is known about the mechanisms 
  that regulate their biological activity . here 
 we show that the antibacterial 
  activities of mouse regiii and its human ortholog , hip / pap , are tightly 
  controlled by an inhibitory n - terminal prosegment that is removed by trypsin 
  in vivo . 
 nmr spectroscopy revealed a high degree of conformational 
  flexibility in the hip / pap inhibitory prosegment , and mutation of either 
  acidic prosegment residues or basic core protein residues disrupted prosegment 
  inhibitory activity . 
 nmr analyses of pro - hip / pap variants revealed distinctive 
  colinear backbone amide chemical shift changes that correlated with 
  antibacterial activity , suggesting that prosegment - hip / pap interactions are 
  linked to a two - state conformational switch between biologically active and 
  inactive protein states . 
 these findings reveal a novel regulatory mechanism 
  governing c - type lectin biological function and yield new insight into the 
  control of intestinal innate immunity . 
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
Supplementary Material