α-Defensins in Enteric Innate Immunity
2009; Elsevier BV; Volume: 284; Issue: 41 Linguagem: Inglês
10.1074/jbc.m109.050773
ISSN1083-351X
AutoresJennifer R. Mastroianni, André J. Ouellette,
Tópico(s)Immune Response and Inflammation
ResumoPaneth cells are a secretory epithelial lineage that release dense core granules rich in host defense peptides and proteins from the base of small intestinal crypts. Enteric α-defensins, termed cryptdins (Crps) in mice, are highly abundant in Paneth cell secretions and inherently resistant to proteolysis. Accordingly, we tested the hypothesis that enteric α-defensins of Paneth cell origin persist in a functional state in the mouse large bowel lumen. To test this idea, putative Crps purified from mouse distal colonic lumen were characterized biochemically and assayed in vitro for bactericidal peptide activities. The peptides comigrated with cryptdin control peptides in acid-urea-PAGE and SDS-PAGE, providing identification as putative Crps. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry experiments showed that the molecular masses of the putative α-defensins matched those of the six most abundant known Crps, as well as N-terminally truncated forms of each, and that the peptides contain six Cys residues, consistent with identities as α-defensins. N-terminal sequencing definitively revealed peptides with N termini corresponding to full-length, (des-Leu)-truncated, and (des-Leu-Arg)-truncated N termini of Crps 1–4 and 6. Crps from mouse large bowel lumen were bactericidal in the low micromolar range. Thus, Paneth cell α-defensins secreted into the small intestinal lumen persist as intact and functional forms throughout the intestinal tract, suggesting that the peptides may mediate enteric innate immunity in the colonic lumen, far from their upstream point of secretion in small intestinal crypts. Paneth cells are a secretory epithelial lineage that release dense core granules rich in host defense peptides and proteins from the base of small intestinal crypts. Enteric α-defensins, termed cryptdins (Crps) in mice, are highly abundant in Paneth cell secretions and inherently resistant to proteolysis. Accordingly, we tested the hypothesis that enteric α-defensins of Paneth cell origin persist in a functional state in the mouse large bowel lumen. To test this idea, putative Crps purified from mouse distal colonic lumen were characterized biochemically and assayed in vitro for bactericidal peptide activities. The peptides comigrated with cryptdin control peptides in acid-urea-PAGE and SDS-PAGE, providing identification as putative Crps. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry experiments showed that the molecular masses of the putative α-defensins matched those of the six most abundant known Crps, as well as N-terminally truncated forms of each, and that the peptides contain six Cys residues, consistent with identities as α-defensins. N-terminal sequencing definitively revealed peptides with N termini corresponding to full-length, (des-Leu)-truncated, and (des-Leu-Arg)-truncated N termini of Crps 1–4 and 6. Crps from mouse large bowel lumen were bactericidal in the low micromolar range. Thus, Paneth cell α-defensins secreted into the small intestinal lumen persist as intact and functional forms throughout the intestinal tract, suggesting that the peptides may mediate enteric innate immunity in the colonic lumen, far from their upstream point of secretion in small intestinal crypts. Antimicrobial peptides (AMPs) 2The abbreviations used are: AMPantimicrobial peptideCrpcryptdinMMP-7matrix metalloproteinase-7pro-Crppro-cryptdinrhBDrhesus macaque β-defensinhBDhuman β-defensinHPLChigh-performance liquid chromatographyAUacid-ureaMALDI-TOFmatrix-assisted laser desorption ionization time-of-flightTSBtrypticase soy brothCFUcolony forming unit(s)MSmass spectrometryTricineN-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine. are released by epithelial cells onto mucosal surfaces as effectors of innate immunity (1Lehrer R.I. Ganz T. Curr. Opin. Hematol. 2002; 9: 18-22Crossref PubMed Scopus (280) Google Scholar, 2Lehrer R.I. Ganz T. Curr. Opin. Immunol. 2002; 14: 96-102Crossref PubMed Scopus (596) Google Scholar, 3Schutte B.C. McCray Jr., P.B. Annu. Rev. Physiol. 2002; 64: 709-748Crossref PubMed Scopus (222) Google Scholar, 4Selsted M.E. Ouellette A.J. Nat. Immunol. 2005; 6: 551-557Crossref PubMed Scopus (977) Google Scholar, 5Mukherjee S. Vaishnava S. Hooper L.V. Cell Mol. Life Sci. 2008; 65: 3019-3027Crossref PubMed Scopus (109) Google Scholar). In mammals, most AMPs derive from two major families, the cathelicidins and defensins (6Zasloff M. Nature. 2002; 415: 389-395Crossref PubMed Scopus (6943) Google Scholar). The defensins comprise the α-, β-, and θ-defensin subfamilies, which are defined by the presence of six cysteine residues paired in characteristic tridisulfide arrays (7Ganz T. Nat. Rev. Immunol. 2003; 3: 710-720Crossref PubMed Scopus (2411) Google Scholar). α-Defensins are highly abundant in two primary cell lineages: phagocytic leukocytes, primarily neutrophils, of myeloid origin and Paneth cells, which are secretory epithelial cells located at the base of the crypts of Lieberkühn in the small intestine (8Selsted M.E. Miller S.I. Henschen A.H. Ouellette A.J. J. Cell Biol. 1992; 118: 929-936Crossref PubMed Scopus (285) Google Scholar, 9Patil A. Hughes A.L. Zhang G. Physiol. Genomics. 2004; 20: 1-11Crossref PubMed Scopus (137) Google Scholar, 10Ouellette A.J. Cordell B. Gastroenterology. 1988; 94: 114-121Abstract Full Text PDF PubMed Scopus (29) Google Scholar). Neutrophil α-defensins are stored in azurophilic granules and contribute to non-oxidative microbial cell killing in phagolysosomes (11Ganz T. Selsted M.E. Szklarek D. Harwig S.S. Daher K. Bainton D.F. Lehrer R.I. J. Clin. Invest. 1985; 76: 1427-1435Crossref PubMed Scopus (1162) Google Scholar, 12Selsted M.E. Ouellette A.J. Trends Cell Biol. 1995; 5: 114-119Abstract Full Text PDF PubMed Scopus (118) Google Scholar), except in mice whose neutrophils lack defensins (13Eisenhauer P.B. Lehrer R.I. Infect. Immun. 1992; 60: 3446-3447Crossref PubMed Google Scholar). In the small bowel, α-defensins and other host defense proteins (14Harwig S.S. Tan L. Qu X.D. Cho Y. Eisenhauer P.B. Lehrer R.I. J. Clin. Invest. 1995; 95: 603-610Crossref PubMed Google Scholar, 15Qu X.D. Lloyd K.C. Walsh J.H. Lehrer R.I. Infect. Immun. 1996; 64: 5161-5165Crossref PubMed Google Scholar, 16Porter E.M. Bevins C.L. Ghosh D. Ganz T. Cell Mol. Life Sci. 2002; 59: 156-170Crossref PubMed Scopus (334) Google Scholar, 17Cash H.L. Whitham C.V. Behrendt C.L. Hooper L.V. Science. 2006; 313: 1126-1130Crossref PubMed Scopus (1099) Google Scholar, 18Peeters T. Vantrappen G. Gut. 1975; 16: 553-558Crossref PubMed Scopus (134) Google Scholar) are released apically as components of Paneth cell secretory granules in response to cholinergic stimulation and after exposure to bacterial antigens (19Ayabe T. Satchell D.P. Wilson C.L. Parks W.C. Selsted M.E. Ouellette A.J. Nat. Immunol. 2000; 1: 113-118Crossref PubMed Scopus (862) Google Scholar). Therefore, the release of Paneth cell products into the crypt lumen is inferred to protect mitotically active crypt cells from colonization by potential pathogens and confer protection against enteric infection (7Ganz T. Nat. Rev. Immunol. 2003; 3: 710-720Crossref PubMed Scopus (2411) Google Scholar, 20Ganz T. C R Biol. 2004; 327: 539-549Crossref PubMed Scopus (155) Google Scholar, 21Ouellette A.J. Springer Semin. Immunopathol. 2005; 27: 133-146Crossref PubMed Scopus (53) Google Scholar). antimicrobial peptide cryptdin matrix metalloproteinase-7 pro-cryptdin rhesus macaque β-defensin human β-defensin high-performance liquid chromatography acid-urea matrix-assisted laser desorption ionization time-of-flight trypticase soy broth colony forming unit(s) mass spectrometry N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine. Under normal, homeostatic conditions, Paneth cells are not found outside the small bowel, although they may appear ectopically in response to local inflammation throughout the gastrointestinal tract (22Sommers S.C. Gastroenterology. 1966; 51: 841-850Abstract Full Text PDF PubMed Google Scholar, 23Boulton R.A. Usselmann B. Mohammed I. Jankowski J. World J. Surg. 2003; 27: 1014-1017Crossref PubMed Scopus (8) Google Scholar). Paneth cell numbers increase progressively throughout the small intestine, occurring at highest numbers in the distal ileum (24Trier J.S. Gastroenterology. 1966; 51: 560-562Abstract Full Text PDF PubMed Google Scholar). Mouse Paneth cells express numerous α-defensin isoforms, termed cryptdins (Crps) (25Ouellette A.J. Hsieh M.M. Nosek M.T. Cano-Gauci D.F. Huttner K.M. Buick R.N. Selsted M.E. Infect. Immun. 1994; 62: 5040-5047Crossref PubMed Google Scholar), that have broad spectrum antimicrobial activities (6Zasloff M. Nature. 2002; 415: 389-395Crossref PubMed Scopus (6943) Google Scholar, 26Kolls J.K. McCray Jr., P.B. Chan Y.R. Nat. Rev. Immunol. 2008; 8: 829-835Crossref PubMed Scopus (272) Google Scholar). Collectively, α-defensins constitute approximately seventy percent of the bactericidal peptide activity in mouse Paneth cell secretions (19Ayabe T. Satchell D.P. Wilson C.L. Parks W.C. Selsted M.E. Ouellette A.J. Nat. Immunol. 2000; 1: 113-118Crossref PubMed Scopus (862) Google Scholar), selectively killing bacteria by membrane-disruptive mechanisms (27Satchell D.P. Sheynis T. Kolusheva S. Cummings J. Vanderlick T.K. Jelinek R. Selsted M.E. Ouellette A.J. Peptides. 2003; 24: 1795-1805Crossref PubMed Scopus (50) Google Scholar, 28Satchell D.P. Sheynis T. Shirafuji Y. Kolusheva S. Ouellette A.J. Jelinek R. J. Biol. Chem. 2003; 278: 13838-13846Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 29Tanabe H. Qu X. Weeks C.S. Cummings J.E. Kolusheva S. Walsh K.B. Jelinek R. Vanderlick T.K. Selsted M.E. Ouellette A.J. J. Biol. Chem. 2004; 279: 11976-11983Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 30Hadjicharalambous C. Sheynis T. Jelinek R. Shanahan M.T. Ouellette A.J. Gizeli E. Biochemistry. 2008; 47: 12626-12634Crossref PubMed Scopus (39) Google Scholar). The role of Paneth cell α-defensins in gastrointestinal mucosal immunity is evident from studies of mice transgenic for human enteric α-defensin-5, HD-5, which are immune to infection by orally administered Salmonella enterica sv. typhimurium (S. typhimurium) (31Salzman N.H. Ghosh D. Huttner K.M. Paterson Y. Bevins C.L. Nature. 2003; 422: 522-526Crossref PubMed Scopus (652) Google Scholar). The biosynthesis of mature, bactericidal α-defensins from their inactive precursors requires activation by lineage-specific proteolytic convertases. In mouse Paneth cells, inactive ∼8.4-kDa Crp precursors are processed intracellularly into microbicidal ∼4-kDa Crps by specific cleavage events mediated by matrix metalloproteinase-7 (MMP-7) (32Wilson C.L. Ouellette A.J. Satchell D.P. Ayabe T. López-Boado Y.S. Stratman J.L. Hultgren S.J. Matrisian L.M. Parks W.C. Science. 1999; 286: 113-117Crossref PubMed Scopus (923) Google Scholar, 33Ayabe T. Wulff H. Darmoul D. Cahalan M.D. Chandy K.G. Ouellette A.J. J. Biol. Chem. 2002; 277: 3793-3800Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). MMP-7 null mice exhibit increased susceptibility to systemic S. typhimurium infection and decreased clearance of orally administered non-invasive Escherichia coli (19Ayabe T. Satchell D.P. Wilson C.L. Parks W.C. Selsted M.E. Ouellette A.J. Nat. Immunol. 2000; 1: 113-118Crossref PubMed Scopus (862) Google Scholar, 32Wilson C.L. Ouellette A.J. Satchell D.P. Ayabe T. López-Boado Y.S. Stratman J.L. Hultgren S.J. Matrisian L.M. Parks W.C. Science. 1999; 286: 113-117Crossref PubMed Scopus (923) Google Scholar). Although the α-defensin proregions are sensitive to proteolysis, the mature, disulfide-stabilized peptides resist digestion by their converting enzymes in vitro, whether the convertase is MMP-7 (32Wilson C.L. Ouellette A.J. Satchell D.P. Ayabe T. López-Boado Y.S. Stratman J.L. Hultgren S.J. Matrisian L.M. Parks W.C. Science. 1999; 286: 113-117Crossref PubMed Scopus (923) Google Scholar), trypsin (34Ghosh D. Porter E. Shen B. Lee S.K. Wilk D. Drazba J. Yadav S.P. Crabb J.W. Ganz T. Bevins C.L. Nat. Immunol. 2002; 3: 583-590Crossref PubMed Scopus (364) Google Scholar), or neutrophil serine proteinases (35Kamdar K. Maemoto A. Qu X. Young S.K. Ouellette A.J. J. Biol. Chem. 2008; 283: 32361-32368Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). Because α-defensins resist proteolysis in vitro, we hypothesized that Paneth cell α-defensins resist degradation and remain in a functional state in the large bowel, a complex, hostile environment containing varied proteases of both host and microbial origin. Here, we report on the isolation and characterization of a population of enteric α-defensins from the mouse colonic lumen. Full-length and N-terminally truncated Paneth cell α-defensins were identified and are abundant in the distal large bowel lumen. Native Crp4, disulfide-null (6C/A)-Crp4, proCrp4, (M19L)-Crp1, (M19L)-Crp6, and rhesus macaque β-defensin-4 (rhBD-4) were produced by recombinant methods and purified to homogeneity as described previously (27Satchell D.P. Sheynis T. Kolusheva S. Cummings J. Vanderlick T.K. Jelinek R. Selsted M.E. Ouellette A.J. Peptides. 2003; 24: 1795-1805Crossref PubMed Scopus (50) Google Scholar, 36Maemoto A. Qu X. Rosengren K.J. Tanabe H. Henschen-Edman A. Craik D.J. Ouellette A.J. J. Biol. Chem. 2004; 279: 44188-44196Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). Recombinant peptides were expressed in E. coli as N-terminal His6-tagged fusion proteins from the EcoRI and SalI sites of the pET-28a expression vector (Novagen, Inc., Madison, WI) and subsequently purified as described (29Tanabe H. Qu X. Weeks C.S. Cummings J.E. Kolusheva S. Walsh K.B. Jelinek R. Vanderlick T.K. Selsted M.E. Ouellette A.J. J. Biol. Chem. 2004; 279: 11976-11983Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 36Maemoto A. Qu X. Rosengren K.J. Tanabe H. Henschen-Edman A. Craik D.J. Ouellette A.J. J. Biol. Chem. 2004; 279: 44188-44196Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar, 37Shirafuji Y. Tanabe H. Satchell D.P. Henschen-Edman A. Wilson C.L. Ouellette A.J. J. Biol. Chem. 2003; 278: 7910-7919Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). Peptide homogeneity was assessed by analytical reversed-phase HPLC, acid-urea polyacrylamide gel electrophoresis (AU-PAGE) (40Selsted M.E. Genet. Eng. 1993; 15: 131-147Crossref PubMed Scopus (32) Google Scholar), and peptide masses were confirmed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS, Voyager DE, PE Biosystems, Foster City, CA) (39Weeks C.S. Tanabe H. Cummings J.E. Crampton S.P. Sheynis T. Jelinek R. Vanderlick T.K. Cocco M.J. Ouellette A.J. J. Biol. Chem. 2006; 281: 28932-28942Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Synthetic human β-defensins 1 and 3 (hBD-1 and hBD-3) were generously provided by Drs. Michael Selsted and Gÿorgÿ Ösapay (Dept. of Pathology and Laboratory Medicine, University of California, Irvine). Synthetic Crp2 and Crp3 peptides were a gift from Dr. Wuyuan Lu (Institute of Human Virology, University of Maryland School of Medicine). All procedures on mice were performed with approval and in compliance with the policies of the Institutional Animal Care and Use Committee of the University of California, Irvine. Outbred Swiss Webster mice were 45-day-old males from Charles River Breeding Laboratories, Inc. (Wilmington, MA). Mice were housed under 12-h cycles of light and dark and had free access to standard rat chow and water. Peptides were isolated using procedures described previously except where noted (8Selsted M.E. Miller S.I. Henschen A.H. Ouellette A.J. J. Cell Biol. 1992; 118: 929-936Crossref PubMed Scopus (285) Google Scholar). Segments of ileum and distal large bowel, consisting of the colon and rectum only, were excised from mice immediately following euthanasia by halothane inhalation followed by cervical dislocation. Protein extracts were prepared from "complete" tissue, consisting of tissue plus luminal contents. Alternatively, extracts were generated from tissue and luminal contents processed separately. To remove the luminal contents, intestinal segments were opened by longitudinal incisions, and the luminal contents were washed away from the segments by dipping the organ into ice-cold water. Samples were homogenized in 100 ml of ice-cold 60% acetonitrile plus 1% trifluoroacetic acid and incubated at 4 °C overnight prior to clarification by centrifugation and lyophilization. Lyophilized samples were resuspended in 5 ml of 5% acetic acid and chromatographed on a 10 × 60-cm Bio-Gel P-60 column (Bio-Rad), and 350 drop fractions were collected. Fractions containing α-defensins were identified by the presence of rapidly migrating peptides in AU-PAGE stained with Coomassie Brilliant Blue, a characteristic of α-defensins in this system (8Selsted M.E. Miller S.I. Henschen A.H. Ouellette A.J. J. Cell Biol. 1992; 118: 929-936Crossref PubMed Scopus (285) Google Scholar, 40Selsted M.E. Genet. Eng. 1993; 15: 131-147Crossref PubMed Scopus (32) Google Scholar). The fraction of total extracted protein in α-defensin containing fractions was determined by Bradford Assay (Bio-Rad Protein Assay). Samples of combined α-defensins were purified further by cation-exchange chromatography before analysis by SDS-PAGE, in bactericidal peptide assays, and by peptide sequencing. Lyophilized α-defensin pools were resuspended in 100 mm ammonium formate loading buffer (pH 6.2) and applied to a 3-ml CM-Sepharose column equilibrated with the same buffer and washed with 10 ml of loading buffer. Resin-bound peptides were eluted using 10-ml volumes of 0.2 m, 1.25 m, and 2.0 m ammonium acetate (pH 5.2) followed by a final wash of 2.0 m ammonium acetate. α-Defensins eluted from the resin with 1.25 m ammonium acetate. Fractions were lyophilized, resuspended in 5 ml of 5% acetic acid, dialyzed against 5 liters of 5% acetic acid, and lyophilized again. Samples were separated by C18 reversed-phase HPLC that was developed with an aqueous 15–35% gradient of acetonitrile using 0.1% trifluoroacetic acid as the ion-pairing agent (41Ouellette A.J. Satchell D.P. Hsieh M.M. Hagen S.J. Selsted M.E. J. Biol. Chem. 2000; 275: 33969-33973Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar) delivered in 90 min at 1 ml/min. Samples of HPLC fractions were mixed with an equal volume of 10 mg/ml α-cyano-4-hydroxycinnamic acid in 60% acetonitrile containing 0.1% trifluoroacetic acid and left to dry completely prior to analysis by MALDI-TOF MS (Voyager-DE, PE Biosystems) in the Mass Spectroscopy Facility of the Dept. of Chemistry at the University of California, Irvine. HPLC fractions with masses consistent with known or predicted mouse α-defensins were subjected to performic oxidation to detect the modification of cysteine and methionine residues. Performic acid reagent was prepared by mixing 1 part hydrogen peroxide with 19 parts 97% formic acid and incubating on ice for 1 h. Lyophilized samples were dissolved in this reagent and incubated at ambient temperature for 30 min, dried in vacuo, washed once with 50 μl of ice-cold H2O, and twice washed with 20 μl of H2O. Samples of oxidation reactions were mixed 1:1 with 10 mg/ml α-cyano-4-hydroxycinnamic acid in 60% acetonitrile containing 0.1% trifluoroacetic acid prior to analysis by MALDI-TOF MS as before. Upon oxidation, cystine and cysteine are converted to cysteic acid by addition of three oxygen atoms per cysteine; methionine residues are converted to methionine sulfone by the addition of two oxygen atoms to each methionine sulfur atom. Consequently modification of peptides or proteins containing 6 cysteine residues results in a predicted increase in mass of 288 atomic mass units. Performic acid oxidation of Crps containing 6 Cys and 1 or 2 Met residues would increase peptide mass by 320 or 352 atomic mass units, respectively. SDS-PAGE was performed using 10–20% Tris-Tricine precast ready-gels (Bio-Rad) with samples that were diluted 1:2 with SDS-PAGE sample buffer (Bio-Rad) prepared according to manufacturer's instructions with 1% β-mercaptoethanol and boiled for 10 min. 5 μl of Kaleidoscope (Bio-Rad) prestained molecular weight standards were run on each gel. α-Defensin preparations from mouse complete ileum and complete colon were run in AU-PAGE and electroblotted to 0.1 μm polyvinylidene difluoride membrane (Millipore Immobilon PSQ) using 5% acetic acid as the transfer buffer. Individual peptide bands were excised from Coomassie-stained membranes prior to N-terminal sequencing. Eleven rounds of Edman degradation were performed on an ABI 494-HT Procise Edman Sequencer by the Molecular Structure Facility at the University of California, Davis. E. coli ML35, Listeria monocytogenes 10403S, and Salmonella enterica sv. typhimurium ΔphoP were target organisms for assays testing bactericidal activity of α-defensin preparations as described previously (42Ayabe T. Satchell D.P. Pesendorfer P. Tanabe H. Wilson C.L. Hagen S.J. Ouellette A.J. J. Biol. Chem. 2002; 277: 5219-5228Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). Bacteria growing exponentially in Trypticase soy broth (TSB) at 37 °C were collected by centrifugation, washed, and resuspended in 10 mm PIPES (pH 7.4) supplemented with 0.01 vol. of (1% v/v) TSB (PIPES-TSB) (28Satchell D.P. Sheynis T. Shirafuji Y. Kolusheva S. Ouellette A.J. Jelinek R. J. Biol. Chem. 2003; 278: 13838-13846Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 37Shirafuji Y. Tanabe H. Satchell D.P. Henschen-Edman A. Wilson C.L. Ouellette A.J. J. Biol. Chem. 2003; 278: 7910-7919Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). Bacteria (5 × 106/ml) were exposed to varied peptide concentrations for 1 h at 37 °C in 50 μl of PIPES-TSB. Samples were diluted 1:100 with 10 mm PIPES (pH 7.4) and plated on Trypticase soy agar plates using an Autoplate 4000 (Spiral Biotech, Inc., Bethesda, MD). Surviving bacteria were quantified as colony-forming units per milliliter (CFUs/ml) after 10–18 h of incubation. Recombinant and synthetic α-defensins and β-defensins were digested with trypsin (Sigma, T-8253) or α-chymotrypsin (Sigma, C-9135) as described below and analyzed for susceptibility to proteolysis by AU-PAGE and MALDI-TOF MS as above. Samples (5 μg) were incubated with each proteinase at 37 °C for 2 h at a substrate to enzyme molar ratio of 50:1 in 50 mm ammonium bicarbonate (pH 8.0). Samples consisting of 85% of each digest were analyzed by AU-PAGE, and the remainder of each digest was subjected to MALDI-TOF MS. Paneth cell α-defensins were tentatively identified in protein extracts from mouse distal colonic lumen following protein separation by gel-permeation chromatography. Because mouse enteric α-defensins are highly mobile in AU-PAGE, Bio-Gel P-60 (P-60) fractions containing apparent α-defensins were subjected to AU-PAGE analysis to identify fractions containing rapidly migrating peptides of low molecular weight (40Selsted M.E. Genet. Eng. 1993; 15: 131-147Crossref PubMed Scopus (32) Google Scholar). As expected (8Selsted M.E. Miller S.I. Henschen A.H. Ouellette A.J. J. Cell Biol. 1992; 118: 929-936Crossref PubMed Scopus (285) Google Scholar), peptides with mobilities characteristic of α-defensins were detected in P-60 separations of complete (tissue plus luminal contents) mouse ileum (boxed region, Fig. 1A). In AU-PAGE, proteins resolve on the basis of charge-to-size ratio rather than as a more direct function of molecular weight alone. In mice, production of α-defensin mRNAs and peptides occurs exclusively in Paneth cells in the small intestine (8Selsted M.E. Miller S.I. Henschen A.H. Ouellette A.J. J. Cell Biol. 1992; 118: 929-936Crossref PubMed Scopus (285) Google Scholar, 9Patil A. Hughes A.L. Zhang G. Physiol. Genomics. 2004; 20: 1-11Crossref PubMed Scopus (137) Google Scholar, 10Ouellette A.J. Cordell B. Gastroenterology. 1988; 94: 114-121Abstract Full Text PDF PubMed Scopus (29) Google Scholar, 31Salzman N.H. Ghosh D. Huttner K.M. Paterson Y. Bevins C.L. Nature. 2003; 422: 522-526Crossref PubMed Scopus (652) Google Scholar, 43Ouellette A.J. Greco R.M. James M. Frederick D. Naftilan J. Fallon J.T. J. Cell Biol. 1989; 108: 1687-1695Crossref PubMed Scopus (196) Google Scholar, 44Ouellette A.J. Miller S.I. Henschen A.H. Selsted M.E. FEBS Lett. 1992; 304: 146-148Crossref PubMed Scopus (75) Google Scholar). In contrast, α-defensin production in the colon has not been detected at any level (8Selsted M.E. Miller S.I. Henschen A.H. Ouellette A.J. J. Cell Biol. 1992; 118: 929-936Crossref PubMed Scopus (285) Google Scholar, 9Patil A. Hughes A.L. Zhang G. Physiol. Genomics. 2004; 20: 1-11Crossref PubMed Scopus (137) Google Scholar, 10Ouellette A.J. Cordell B. Gastroenterology. 1988; 94: 114-121Abstract Full Text PDF PubMed Scopus (29) Google Scholar, 31Salzman N.H. Ghosh D. Huttner K.M. Paterson Y. Bevins C.L. Nature. 2003; 422: 522-526Crossref PubMed Scopus (652) Google Scholar, 43Ouellette A.J. Greco R.M. James M. Frederick D. Naftilan J. Fallon J.T. J. Cell Biol. 1989; 108: 1687-1695Crossref PubMed Scopus (196) Google Scholar, 44Ouellette A.J. Miller S.I. Henschen A.H. Selsted M.E. FEBS Lett. 1992; 304: 146-148Crossref PubMed Scopus (75) Google Scholar). Nevertheless, when protein extracts of complete large intestine were separated as shown for the complete ileum sample, highly mobile peptides of low molecular weight were readily apparent in AU-PAGE (boxed region, Fig. 1B). In replicate experiments, putative α-defensin-containing fractions from complete ileum and complete large bowel were combined, as were the remaining fractions containing all other extracted proteins. Less total protein was consistently extracted from complete large intestine compared with the complete ileum; however, the measurements also showed that the combined α-defensin-containing fractions represented approximately an equivalent ∼4% of all protein extracted from both sources. Additionally, putative α-defensins were visualized when similar experiments were performed for samples of complete rectum, the most distal portion of the large bowel (data not shown). Putative α-defensins among proteins extracted from complete mouse large bowel derive from colonic lumen. Proteins extracted separately from large bowel tissue and luminal contents were analyzed by gel-permeation chromatography and AU-PAGE. Consistent with the absence of both Paneth cells and α-defensin mRNAs in mouse colon (10Ouellette A.J. Cordell B. Gastroenterology. 1988; 94: 114-121Abstract Full Text PDF PubMed Scopus (29) Google Scholar), no apparent α-defensin peptides were detected in extracts of colonic tissue (Fig. 1C). In contrast, putative α-defensins were both evident and abundant among proteins extracted from the luminal contents of the distal colon (boxed region, Fig. 1D). Thus, the finding of putative α-defensins in the colonic lumen is consonant with previous studies showing that α-defensins are not produced in the colonic tissue and must derive from another source. Given that α-defensins have been isolated and characterized from both the mouse and human small intestinal lumen (34Ghosh D. Porter E. Shen B. Lee S.K. Wilk D. Drazba J. Yadav S.P. Crabb J.W. Ganz T. Bevins C.L. Nat. Immunol. 2002; 3: 583-590Crossref PubMed Scopus (364) Google Scholar, 41Ouellette A.J. Satchell D.P. Hsieh M.M. Hagen S.J. Selsted M.E. J. Biol. Chem. 2000; 275: 33969-33973Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar) and Paneth cells are the only cells that express α-defensin genes in mouse small bowel, Paneth cell secretions released into small intestinal crypts are the most likely source of these peptides. To test the possibility that colonic luminal α-defensins may contain cryptic intrastrand scissions, preparations of combined ileal α-defensins and of putative colonic α-defensins were analyzed by SDS-PAGE (Fig. 2). Although the α-defensin canonical disulfide array (Fig. 3) confers resistance to proteolysis in vitro (35Kamdar K. Maemoto A. Qu X. Young S.K. Ouellette A.J. J. Biol. Chem. 2008; 283: 32361-32368Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar, 36Maemoto A. Qu X. Rosengren K.J. Tanabe H. Henschen-Edman A. Craik D.J. Ouellette A.J. J. Biol. Chem. 2004; 279: 44188-44196Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar), we considered that the complex milieu of host and microbial-derived proteases in the colonic lumen could cleave within the α-defensin polypeptide backbone, but such intrastrand scissions might not be evident in peptides stabilized by disulfide bonds. The presence of intrastrand scissions would result in the peptides disintegrating into multiple fragments upon reduction and their disappearance from the gel. However, under reducing conditions, putative α-defensins from the colon showed no evidence of cryptic scissions, co-migrating at 4 kDa with native, intact ileal α-defensins and with Crp3 and Crp4 control peptides (Fig. 2). This finding shows that the apparent α-defensins in colon are intact and resistant to intrastrand cleavage events in the highly proteolytic environment of the colonic lumen.FIGURE 3Primary structures of colonic α-defensins deduced by MALDI-TOF MS. Masses of colonic α-defensins were determined by MALDI-TOF MS ("Experimental Procedures"). Experimental masses were compared with the theoretical masses deduced from previously characterized mouse Paneth cell α-defensin peptide and mRNA sequences and used to deduce peptide primary structures and identities. All masses are given in atomic mass units (A.M.U.). Dash characters were introduced into the alignment of certain colonic α-defensin primary structures to maintain cysteine spacing. The canonical α-d
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