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• W2058761441 abstract "Paneth cells at the base of small intestinal crypts secrete apical granules that contain antimicrobial peptides including α-defensins, termed cryptdins. Using an antibody specific for mouse cryptdin-1, -2, -3, and -6, immunogold-localization studies demonstrated that cryptdins are constituents of mouse Paneth cell secretory granules. Several cryptdin peptides have been purified from rinses of adult mouse small intestine by gel filtration and reverse-phase high performance liquid chromatography. Their primary structures were determined by peptide sequencing, and their antimicrobial activities were compared with those of the corresponding tissue forms. The isolated luminal cryptdins included peptides identical to the tissue forms of cryptdin-2, -4, and -6 as well as variants of cryptdin-1, -4, and -6 that have N termini truncated by one or two residues. In assays of antimicrobial activity againstStaphylococcus aureus, Escherichia coli, and the defensin-sensitive Salmonella typhimurium phoP − mutant, full-length cryptdins had the same in vitro antibacterial activities whether isolated from tissue or from the lumen. In contrast, the N-terminal-truncated (des-Leu), (des-Leu-Arg)-cryptdin-6, and (des-Gly)-cryptdin-4 peptides were markedly less active. The microbicidal activities of recombinant cryptdin-4 and (des-Gly)-cryptdin-4 peptides against E. coli, and S. typhimurium showed that the N-terminal Gly residue or the length of the cryptdin-4 N terminus are determinants of microbicidal activity. Innate immunity in the crypt lumen may be modulated by aminopeptidase modification of α-defensins after peptide secretion. Paneth cells at the base of small intestinal crypts secrete apical granules that contain antimicrobial peptides including α-defensins, termed cryptdins. Using an antibody specific for mouse cryptdin-1, -2, -3, and -6, immunogold-localization studies demonstrated that cryptdins are constituents of mouse Paneth cell secretory granules. Several cryptdin peptides have been purified from rinses of adult mouse small intestine by gel filtration and reverse-phase high performance liquid chromatography. Their primary structures were determined by peptide sequencing, and their antimicrobial activities were compared with those of the corresponding tissue forms. The isolated luminal cryptdins included peptides identical to the tissue forms of cryptdin-2, -4, and -6 as well as variants of cryptdin-1, -4, and -6 that have N termini truncated by one or two residues. In assays of antimicrobial activity againstStaphylococcus aureus, Escherichia coli, and the defensin-sensitive Salmonella typhimurium phoP − mutant, full-length cryptdins had the same in vitro antibacterial activities whether isolated from tissue or from the lumen. In contrast, the N-terminal-truncated (des-Leu), (des-Leu-Arg)-cryptdin-6, and (des-Gly)-cryptdin-4 peptides were markedly less active. The microbicidal activities of recombinant cryptdin-4 and (des-Gly)-cryptdin-4 peptides against E. coli, and S. typhimurium showed that the N-terminal Gly residue or the length of the cryptdin-4 N terminus are determinants of microbicidal activity. Innate immunity in the crypt lumen may be modulated by aminopeptidase modification of α-defensins after peptide secretion. matrilysin piperazine-N,N′-bis(2-ethanesulfonic acid) reverse-phase high performance liquid chromatography acid-urea polyacrylamide gel electrophoresis maltose-binding protein matrix-assisted laser desorption ionization time of flight mass spectrometry The release of antimicrobial peptides onto mucosal surfaces by diverse epithelia is recognized as a conserved innate immune mechanism (1Schonwetter B.S. Stolzenberg E.D. Zasloff M.A. Science. 1995; 267: 1645-1648Crossref PubMed Scopus (369) Google Scholar, 2Boman H.G. Marsh J. Goode J.A. Antimicrobial Peptides. John Wiley & Sons Ltd., Chichester1994: 1-4Google Scholar, 3Ouellette 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, 4Reilly D.S. Tomassini N. Zasloff M. Dev. Biol. 1994; 162: 123-133Crossref PubMed Scopus (32) Google Scholar, 5Jones D.E. Bevins C.L. FEBS Lett. 1993; 315: 187-192Crossref PubMed Scopus (300) Google Scholar, 6Moore K.S. Bevins C.L. Tomassini N. Huttner K.M. Sadler K. Moreira J.E. Reynolds J. Zasloff M. J. Histochem. Cytochem. 1992; 40: 367-378Crossref PubMed Scopus (18) Google Scholar, 7Zanetti M. Gennaro R. Romeo D. FEBS Lett. 1995; 374: 1-5Crossref PubMed Scopus (608) Google Scholar, 8Quayle A.J. Porter E.M. Nussbaum A.A. Wang Y.M. Brabec C. Yip K.P. Mok S.C. Am. J. Pathol. 1998; 152: 1247-1258PubMed Google Scholar, 9Valore E.V. Park C.H. Quayle A.J. Wiles K.R. McCray P.B. Ganz T. J. Clin. Invest. 1998; 101: 1633-1642Crossref PubMed Scopus (631) Google Scholar, 10Bals R. Goldman M.J. Wilson J.M. Infect. Immun. 1998; 66: 1225-1232Crossref PubMed Google Scholar, 11Bals R. Wang X. Wu Z. Freeman T. Bafna V. Zasloff M. Wilson J.M. J. Clin. Invest. 1998; 102: 874-880Crossref PubMed Scopus (510) Google Scholar). Epithelial antimicrobial peptides are homologs or orthologs of peptides that mediate non-oxidative microbial cell killing in phagolysosomes of leukocytes of myeloid origin (12Selsted M.E. Ouellette A.J. Trends Cell Biol. 1995; 5: 114-119Abstract Full Text PDF PubMed Scopus (116) Google Scholar). These molecules have been hypothesized to function as a biochemical barrier against microbial infection by inhibiting colonization of the epithelium by pathogenic microorganisms (13Hoffmann J.A. Kafatos F.C. Janeway C.A. Ezekowitz R.A. Science. 1999; 284: 1313-1318Crossref PubMed Scopus (2153) Google Scholar, 14Ganz T. Lehrer R.I. Curr. Opin. Immunol. 1998; 10: 41-44Crossref PubMed Scopus (337) Google Scholar). In the small intestine, Paneth cells in the crypts of Lieberkühn (Fig. 1) synthesize and secrete peptides and proteins with known host defense functions (5Jones D.E. Bevins C.L. FEBS Lett. 1993; 315: 187-192Crossref PubMed Scopus (300) Google Scholar, 15Ouellette A.J. Selsted M.E. FASEB J. 1996; 10: 1280-1289Crossref PubMed Scopus (237) Google Scholar, 16Selsted M.E. Miller S.I. Henschen A.H. Ouellette A.J. J. Cell Biol. 1992; 118: 929-936Crossref PubMed Scopus (280) Google Scholar). The large apical secretory granules of these epithelial cells contain a diverse array of peptides and proteins, including lysozyme (17Deckx R.J. Vantrappen G.R. Parein M.M. Biochim. Biophys. Acta. 1967; 139: 204-207Crossref PubMed Scopus (56) Google Scholar), β-defensin (18Bals R. Wang X. Meegalla R.L. Wattler S. Weiner D.J. Nehls M.C. Wilson J.M. Infect. Immun. 1999; 67: 3542-3547Crossref PubMed Google Scholar), sPLA2 (19Qu X.D. Lloyd K.C. Walsh J.H. Lehrer R.I. Infect. Immun. 1996; 64: 5161-5165Crossref PubMed Google Scholar, 20Elsbach P. Prog. Surg. 1997; 24: 17-22Crossref Google Scholar), and α-defensins (5Jones D.E. Bevins C.L. FEBS Lett. 1993; 315: 187-192Crossref PubMed Scopus (300) Google Scholar, 16Selsted M.E. Miller S.I. Henschen A.H. Ouellette A.J. J. Cell Biol. 1992; 118: 929-936Crossref PubMed Scopus (280) Google Scholar, 21Jones D.E. Bevins C.L. J. Biol. Chem. 1992; 267: 23216-23225Abstract Full Text PDF PubMed Google Scholar). Enteric α-defensins, or cryptdins, are highly abundant cationic 3–4-kDa peptides that contain a defining tridisulfide arrangement (15Ouellette A.J. Selsted M.E. FASEB J. 1996; 10: 1280-1289Crossref PubMed Scopus (237) Google Scholar,16Selsted M.E. Miller S.I. Henschen A.H. Ouellette A.J. J. Cell Biol. 1992; 118: 929-936Crossref PubMed Scopus (280) Google Scholar, 22Porter E.M. Liu L. Oren A. Anton P.A. Ganz T. Infect. Immun. 1997; 65: 2389-2395Crossref PubMed Google Scholar), and the in vitro microbicidal activities of cryptdins implicate them in intestinal host defense (3Ouellette 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, 23Porter E.M. van Dam E. Valore E.V. Ganz T. Infect. Immun. 1997; 65: 2396-2401Crossref PubMed Google Scholar, 24Ganz T. Lehrer R.I. Pharmacol. Ther. 1995; 66: 191-205Crossref PubMed Scopus (291) Google Scholar, 25Ganz T. Lehrer R.I. Curr. Opin. Immunol. 1994; 6: 584-589Crossref PubMed Scopus (382) Google Scholar, 26Kagan B.L. Ganz T. Lehrer R.I. Toxicology. 1994; 87: 131-149Crossref PubMed Scopus (178) Google Scholar). In mouse small bowel, the bactericidal cryptdin 1–6 peptides are specific to Paneth cells, and cryptdin-4 is the most potent of the known mouse cryptdins (3Ouellette 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). Studies of mice lacking matrilysin (matrilysin-deficient), MMP-7,1 a metalloproteinase that activates Paneth cell pro-cryptdins, have shown that the resulting deficiency in activated cryptdins impairs innate mucosal immunity (27Wilson C.L. Ouellette A.J. Satchell D.P. Ayabe T. Lopez-Boado Y.S. Stratman J.L. Hultgren S.J. Matrisian L.M. Parks W.C. Science. 1999; 286: 113-117Crossref PubMed Scopus (913) Google Scholar). Matrilysin-deficient mice lack activated cryptdins, are defective in clearing intestinal infections, and succumb more rapidly and to lower doses of virulent Salmonella typhimurium compared with wild-type mice (27Wilson C.L. Ouellette A.J. Satchell D.P. Ayabe T. Lopez-Boado Y.S. Stratman J.L. Hultgren S.J. Matrisian L.M. Parks W.C. Science. 1999; 286: 113-117Crossref PubMed Scopus (913) Google Scholar). In addition to their continual release of granules into the lumen, Paneth cells also may be stimulated to degranulate by muscarinic receptor agonists that mobilize intracellular Ca2+ stores (28Ghoos Y. Vantrappen G. Histochem. J. 1971; 3: 175-178Crossref PubMed Scopus (60) Google Scholar, 29Peeters T. Vantrappen G. Gut. 1975; 16: 553-558Crossref PubMed Scopus (132) Google Scholar) by general G-protein activators such as NaF/AlCl3 (30Satoh Y. Ishikawa K. Oomori Y. Yamano M. Ono K. Anat. Rec. 1989; 225: 124-132Crossref PubMed Scopus (32) Google Scholar, 31Satoh Y. Ishikawa K. Oomori Y. Takeda S. Ono K. Cell Tissue Res. 1992; 269: 213-220Crossref PubMed Scopus (50) Google Scholar, 32Satoh Y. Habara Y. Ono K. Kanno T. Gastroenterology. 1995; 108: 1345-1356Abstract Full Text PDF PubMed Scopus (77) Google Scholar) and by live bacteria and bacterial antigens. 2Ayabe, T., Satchell, D. P., Wilson, C. L., Parks, W. C., Selsted, M. E., and Ouellette, A. J. (2000) Nat. Immunol. 1, 113–118. The objectives of these studies were to assess the distribution of cryptdins in mouse Paneth cell granules and to investigate the biochemical features of secreted cryptdins by determining the primary structures of cryptdins isolated from the mouse small intestinal lumen and by determining their antimicrobial activities. Relative to the first cysteine residue position in the α-defensins, mouse cryptdin N termini are 3–4 amino acids longer than myeloid α-defensin orthologs (3Ouellette 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, 16Selsted M.E. Miller S.I. Henschen A.H. Ouellette A.J. J. Cell Biol. 1992; 118: 929-936Crossref PubMed Scopus (280) Google Scholar), and the possibility that the N-terminal extension is an adaptation for extracellular peptide function has been proposed (12Selsted M.E. Ouellette A.J. Trends Cell Biol. 1995; 5: 114-119Abstract Full Text PDF PubMed Scopus (116) Google Scholar,15Ouellette A.J. Selsted M.E. FASEB J. 1996; 10: 1280-1289Crossref PubMed Scopus (237) Google Scholar). The results described here show that cryptdins are present in all mouse Paneth cell secretory granules and that the mouse small intestinal lumen contains full-length as well as N-terminal-truncated cryptdin variants. Cryptdin peptides with shortened N termini have attenuated antimicrobial activities, implicating N-terminal topology as a determinant of the bactericidal activity of these peptides. Outbred Swiss mice [(Crl:CD-1)(ICR)BR] were 45-day-old males (30–35 g) or timed-pregnant dams from Charles River Breeding Laboratories, Inc. (North Wilmington, MA). Mice were housed under 12-h cycles of light and dark and had free access to standard rat chow and water. Intestinal tissues were fixed with 2% formaldehyde, 0.1% glutaraldehyde in 0.1m phosphate buffer, pH 7.4 and embedded in Unicryl at −20 °C (33Hagen S.J. J. Electron Microsc. Tech. 1990; 16: 37-44Crossref PubMed Scopus (5) Google Scholar). Thin sections were placed on formvar and carbon-coated grids and stained with rabbit anti-cryptdin-1 antibody (16Selsted M.E. Miller S.I. Henschen A.H. Ouellette A.J. J. Cell Biol. 1992; 118: 929-936Crossref PubMed Scopus (280) Google Scholar), washed, and then reacted with a 1:25 dilution of protein A labeled with 10 nm gold (Ted Pella, Inc., Redding, CA) as described in the legend to Fig.2. The sections were examined in a JEOL 100 CX electron microscope and photographed.Figure 2Immunogold localization of cryptdins to Paneth cell granules. Tissue was fixed as in Fig. 1 and stained with rabbit anti-cryptdin and 10 nm gold (see “Experimental Procedures”). G, granules of the Paneth cell. Note that gold particles (arrowheads) are found in high concentration and are uniformly distributed in these apical secretory granules. Sections incubated with pre-immune serum were negative (inset, upper left). Magnification is × 27,000; bar equals 1 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Peptides were isolated from the small intestines of adult mice by a modification of a previously described protocol (3Ouellette 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, 16Selsted M.E. Miller S.I. Henschen A.H. Ouellette A.J. J. Cell Biol. 1992; 118: 929-936Crossref PubMed Scopus (280) Google Scholar). Jejunum and ileum from the small intestines of 60 outbred Swiss Webster mice were excised immediately after euthanasia. The lumens were flushed gently with 50 ml ice-cold water, and the rinses were diluted with ice-cold glacial acetic acid to a final concentration of 30% (v/v) acetic acid. Acidified intestinal rinses were clarified by centrifugation at 28,000 rpm for 30 min in the SW 28.1 rotor and lyophilized. Samples dissolved in 30% acetic acid and clarified by filtration through Whatman 541 filter paper were chromatographed on a 10 × 60-cm Bio-Gel P-60 column in 30% acetic acid at a flow rate of 100 ml/h. Fractions containing cryptdins were identified by the presence of rapidly migrating peptides in acid-urea polyacrylamide gel electrophoresis (AU-PAGE), a characteristic of α-defensins in this system (34Selsted M.E. Becker III, H.W. Anal. Biochem. 1986; 155: 270-274Crossref PubMed Scopus (39) Google Scholar). Luminal cryptdin peptides were purified to homogeneity by reverse-phase high performance liquid chromatography (RP-HPLC) (3Ouellette 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, 16Selsted M.E. Miller S.I. Henschen A.H. Ouellette A.J. J. Cell Biol. 1992; 118: 929-936Crossref PubMed Scopus (280) Google Scholar). Pooled fractions containing cryptdins were lyophilized and separated initially on a 1 × 25-cm Vydac C18 RP-HPLC column using a gradient of water and acetonitrile with 0.13% heptafluorobutyric acid. Solvents were delivered at 3 ml/min generating the following acetonitrile gradient: 0 to 28% (10 min), 28–34% (20 min), and 34 to 40% (60 min). Peptides that co-eluted under these conditions were lyophilized and resolved by C18 RP-HPLC using water/acetonitrile with 0.1% trifluoroacetic acid. A 16–21% acetonitrile gradient was delivered in 35 min at 3 ml/min. All peptides were lyophilized and quantitated by amino acid analysis prior to testing antimicrobial activity. Samples (500 pmol) of individual peptides were S-pyridylethylated, desalted by RP-HPLC (16Selsted M.E. Miller S.I. Henschen A.H. Ouellette A.J. J. Cell Biol. 1992; 118: 929-936Crossref PubMed Scopus (280) Google Scholar, 35Ouellette A.J. Miller S.I. Henschen A.H. Selsted M.E. FEBS Lett. 1992; 304: 146-148Crossref PubMed Scopus (75) Google Scholar), and sequenced on an ABI model 477 system (American Biosystems, Inc., Foster City, CA) configured with on-line phenylthiohydantoin-derivative amino acid analysis in the UCI Biomedical Protein and Mass Spectrometry Resource Facility. Alkylated peptides were sequenced completely through to the C-terminal amino acid as previously reported for full-length cryptdins isolated from mouse small intestinal tissues (3Ouellette 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, 16Selsted M.E. Miller S.I. Henschen A.H. Ouellette A.J. J. Cell Biol. 1992; 118: 929-936Crossref PubMed Scopus (280) Google Scholar) and for full-length synthetic cryptdins (16Selsted M.E. Miller S.I. Henschen A.H. Ouellette A.J. J. Cell Biol. 1992; 118: 929-936Crossref PubMed Scopus (280) Google Scholar, 36Ouellette A.J. Darmoul D. Tran D. Huttner K.M. Yuan J. Selsted M.E. Infect. Immun. 1999; 67: 6643-6651Crossref PubMed Google Scholar). In vitroantibacterial activities of cryptdins were performed in buffers containing 10 mm PIPES or 10 mm HEPES as described (3Ouellette 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). Samples (5 μl) of purified peptides dissolved at 3–300 μg/ml in 0.01% acetic acid were pipetted into wells formed in plates of 1% agarose buffered with 10 mm PIPES, pH 7.4, and containing 1 × 106 log-phase bacteria grown in trypticase soy broth. After 3 h at 37 °C, plates were overlayered with 0.8% agarose containing 2× trypticase soy broth and incubated overnight. Combined bactericidal and bacteriostatic activities were determined as the diameter of growth inhibition around each well as a function of peptide concentration. For measurements of bactericidal activity, exponentially growing bacterial cells were deposited by centrifugation, washed twice with 10 mm PIPES, pH 7.4, and 1 × 106 bacteria were exposed to peptide solutions in the PIPES buffer for 1 h at 37 °C (3Ouellette 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, 16Selsted M.E. Miller S.I. Henschen A.H. Ouellette A.J. J. Cell Biol. 1992; 118: 929-936Crossref PubMed Scopus (280) Google Scholar). Incubation mixtures were diluted 1:2000 with PIPES buffer and plated using a Spiral Biotech Autoplate 4000 (Spiral Biotech, Inc., Bethesda, MD), and surviving colony forming units (CFU) were quantitated after overnight incubation at 37 °C. Recombinant cryptdin-4 and (des-Gly)-cryptdin-4 were isolated after cloning and expression as maltose-binding protein (MBP) fusions using the pMAL bacterial expression system (New England BioLabs, Beverly, MA). A full-length cryptdin-4 cDNA clone was amplified with forward primers containing in the 5′- to 3′- orientation an EcoRI site, codons for the IEGR Factor Xa (Xa) protease cleavage site, and the 5 N-terminal codons of the mature cryptdin-4 or (des-Gly)-cryptdin-4 peptides. Those primers were paired with a reverse primer complementary to the 5 C-terminal codons of cryptdin-4, including the stop codon, and a 5′-flanking SalI site. The sequences were pMalCrp4F (5′-GCGCGAATTCATCGAGGGAAGGGGTTTGTTATGCTATTGT) pMal-desGCrp4F (5′-GCGCGAATTCATCGAGGGAAGGTTGTTATGCTATTGT), and pMalCrp4R (5′-ATATATGTCGACTCAGCGACAGCAGAGCGTGTACAATAAATG). After plasmid constructs were verified by DNA sequence analysis, plasmids were introduced into Escherichia coli BL21(DE3)pLysS cells, and synthesis of MBP-cryptdin-4 fusion protein was induced in exponentially growing cells using 50 μmisopropyl-1-thio-β-d-galactopyranoside for 14 h at 16 °C. MBP fusion proteins were affinity purified from cell lysates using amylose resin (New England BioLabs), cleaved with Xa for 96 h at 25 °C using 1 μg Xa per mg of recombinant MBP fusion. Factor Xa-digested MBP-cryptdin-4 was applied to a C4 column (Vydac, Hesperia, CA) in an aqueous 0.1% trifluoroacetic acid solution and eluted using a linear 0–40% acetonitrile gradient for 55 min at a solvent flow rate of 1 ml/min. Under these conditions, recombinant cryptdin-4 eluted with a retention time of 35 min. Recombinant cryptdin-4 was purified to homogeneity from the C-4 fraction containing cryptdin-4 by C18 RP-HPLC (Vydac). The protein sample was applied to the C18-column in aqueous 0.1% trifluoroacetic acid and eluted at a solvent flow rate of 1 ml/min with 0–10% acetonitrile over 10 min, 10–45% acetonitrile over 55 min, and 100% over 30 min. Under these conditions, cryptdin-4 had a 51-min retention time. The protein concentration of recombinant cryptdin-4 was determined using Bio-Rad Bradford reagent (Bio-Rad) and by absorbance at 280 nm (ε = 3355 mcm−1). The molecular masses of purified recombinant peptides were determined by MALDI-TOF mass spectrometry on a Perkin-Elmer Voyager instrument in the UCI Biomedical Protein and Mass Spectrometry Resource Facility. The homogeneity and appropriate folding of recombinant cryptdin-4 were assessed by comparison with natural cryptdin-4 (16Selsted M.E. Miller S.I. Henschen A.H. Ouellette A.J. J. Cell Biol. 1992; 118: 929-936Crossref PubMed Scopus (280) Google Scholar, 36Ouellette A.J. Darmoul D. Tran D. Huttner K.M. Yuan J. Selsted M.E. Infect. Immun. 1999; 67: 6643-6651Crossref PubMed Google Scholar) in 12.5% AU-PAGE gels and found to be identical to the natural peptide. Immunogold localization studies demonstrated that cryptdins are constituents of mouse Paneth cell secretory granules. In previous immunohistochemical experiments, cryptdin peptides were found only in Paneth cells (16Selsted M.E. Miller S.I. Henschen A.H. Ouellette A.J. J. Cell Biol. 1992; 118: 929-936Crossref PubMed Scopus (280) Google Scholar, 36Ouellette A.J. Darmoul D. Tran D. Huttner K.M. Yuan J. Selsted M.E. Infect. Immun. 1999; 67: 6643-6651Crossref PubMed Google Scholar), and anti-cryptdin immunoreactivity was detected in secretory granules and diffusely in the crypt lumen (16Selsted M.E. Miller S.I. Henschen A.H. Ouellette A.J. J. Cell Biol. 1992; 118: 929-936Crossref PubMed Scopus (280) Google Scholar, 37Ouellette 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). Using a rabbit polyclonal anti-cryptdin-1 antibody that reacts with cryptdins 1–3 and -6 but not with cryptdin-4 or -5, 3M. E. Selsted, and D. Tran, unpublished observations. the subcellular location of cryptdins in adult mouse Paneth cells was shown to be specific to Paneth cell granules (Fig. 2). The distribution of gold particles was uniform, and all apical granules were labeled to approximately the same extent (Fig. 2). Immunoreactive antigen was also visible in the trans-Golgi (not shown), but there was almost no cytoplasmic staining other than in secretory granules. These results are consistent with the detection of human enteric α-defensin HD-5 in human Paneth cells (22Porter E.M. Liu L. Oren A. Anton P.A. Ganz T. Infect. Immun. 1997; 65: 2389-2395Crossref PubMed Google Scholar) and suggest that cryptdin release depends on Paneth cell degranulation. To test the prediction that functional cryptdin peptides are released into the extracellular space, α-defensins were recovered from rinses of adult mouse small intestinal lumen. The lumen of adult mouse small intestine contains intact cryptdins as well as cryptdin variants with modified N termini. Peptides with AU-PAGE mobilities characteristic of α-defensins had been detected previously in saline rinses of adult mouse jejunum and ileum (16Selsted M.E. Miller S.I. Henschen A.H. Ouellette A.J. J. Cell Biol. 1992; 118: 929-936Crossref PubMed Scopus (280) Google Scholar). Comparisons of cryptdins purified from intact small bowel with putative cryptdins from luminal washes showed that certain of the luminal peptides did not co-migrate with peptides from tissue, suggesting that cryptdins may undergo modification following secretion (16Selsted M.E. Miller S.I. Henschen A.H. Ouellette A.J. J. Cell Biol. 1992; 118: 929-936Crossref PubMed Scopus (280) Google Scholar). Putative luminal cryptdins were purified by combined gel filtration and RP-HPLC (see “Experimental Procedures”). The primary structures of these peptides were determined by Edman sequencing of each cryptdin through to the C terminus in all cases. This experimental approach, reported previously in determining the primary structures of full-length synthetic cryptdins (16Selsted M.E. Miller S.I. Henschen A.H. Ouellette A.J. J. Cell Biol. 1992; 118: 929-936Crossref PubMed Scopus (280) Google Scholar, 36Ouellette A.J. Darmoul D. Tran D. Huttner K.M. Yuan J. Selsted M.E. Infect. Immun. 1999; 67: 6643-6651Crossref PubMed Google Scholar) as well as cryptdin peptides from mouse small intestinal tissue (3Ouellette 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, 16Selsted M.E. Miller S.I. Henschen A.H. Ouellette A.J. J. Cell Biol. 1992; 118: 929-936Crossref PubMed Scopus (280) Google Scholar), showed that the peptides from the lumen were full-length or N-terminally modified cryptdins. Cryptdins recovered from the intestinal lumen consisted of both full-length and N-terminal-truncated peptides. Full-length cryptdin-2, -4, and -6 were present in washings of adult mouse small bowel, and those three peptides were structurally and functionally (Figs. 4 and5) indistinguishable from the corresponding tissue forms. Variants of cryptdin-1, -4, and -6 with shortened N termini also were recovered from the intestinal lumen, and they were identified as (des-Leu)-cryptdin-6, (des-Leu-Arg)-cryptdin-6, (des-Leu)-cryptdin-1, and (des-Gly)-cryptdin-4 by peptide sequencing (Fig. 3). Thus, following secretion by Paneth cells, cryptdins may be isolated both as intact activated mature peptides and as N-terminal variants that have been altered by apparent aminopeptidase activity that modifies the peptides during or after exocytosis.Figure 5Antimicrobial activities of luminal mouse cryptdin-4 and (des-Gly)-cryptdin-4. The peptides were assayed for combined bactericidal and bacteriostatic activity as before. Circles, cryptdin-4 (filled) or (des-Gly)-cryptdin-4 (open) against S. aureus;squares, cryptdin-4 (solid) or (des-Gly)-cryptdin-4 (open) against E. coli ML35;triangles, cryptdin-4 (solid) or (des-Gly)-cryptdin-4 (open) against S. typhimurium.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3Primary structures of cryptdins from the mouse small intestinal lumen. Primary structures determined by peptide sequencing (see “Experimental Procedures”) are shown aligned with the primary structure of cryptdin-1 in which the disulfide connectivities are noted. The sequences of full-length luminal cryptdin-2, -4, and -6 are identical to the tissue forms of these peptides. Dashes (—) were introduced in the cryptdin-4 and (des-Gly)-cryptdin-4 sequences as before (3Ouellette 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, 16Selsted M.E. Miller S.I. Henschen A.H. Ouellette A.J. J. Cell Biol. 1992; 118: 929-936Crossref PubMed Scopus (280) Google Scholar) to maintain alignment of cysteine residues. Cryptdin-4 is an unusual α-defensin, because the loop formed between the Cys3–Cys5disulfide bond lacks three amino acids that are found in α-defensins as a class. Residues N-terminal of the first Cys residue areboxed.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Antimicrobial assays of cryptdin-6, cryptdin-4, and their N-terminally modified congeners showed that removal of the N-terminal residue(s) diminished antibacterial activity. The combined bactericidal and bacteriostatic activities of the peptides were measured in agar diffusion assays (see “Experimental Procedures”) and quantitated as zones of clearing (3Ouellette 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). Regardless of whether they were cellular or luminal in origin, full-length cryptdin-2 and -6 had equivalent activities against these three species of bacteria. In these assays, cryptdin-2 (not shown) and cryptdin-6 (Fig. 4) were more active against Staphylococcus aureus than against Gram-negative bacterial species. Loss of Leu and Leu-Arg residues from the cryptdin-6 N terminus caused little effect on activity against S. aureus (Fig. 4 A), but the activity of (des-Leu)-cryptdin-6 and (des-Leu-Arg)-cryptdin-6 against E. coli ML35 (not shown) and the attenuatedphoP − mutant of S. typhimurium (Fig.4 B) was greatly reduced. The limited quantity of purified (des-Leu)-cryptdin-1 precluded a comparison with full-length cryptdin-1. Comparisons of cryptdin-4 and (des-Gly)-cryptdin-4 antimicrobial activities showed further that the N terminus is a determinant of cryptdin activity. Cryptdin-4 is the most potent of the known mouse cryptdins in vitro (3Ouellette 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), and it is equally active against Gram-positive and Gram-negative bacteria (Fig. 5). On the other hand, (des-Gly)-cryptdin-4 had only ∼30% the activity of the full-length peptide against S. aureus (Fig. 5). Furthermore, when the cryptdin-4 congeners were tested against E. coli andS. typhimurium, (des-Gly)-cryptdin-4 completely lacked activity under the assay conditions, even though the peptides differ only by an N-terminal glycine (Fig. 5). Thus, the cryptdin-4 N terminus is a critical determinant of the overall antibacterial activity of this peptide. To compare the microbicidal activities of cryptdin-4 and (des-Gly)-cryptdin-4, recombinant forms of both peptides were expressed as fusions with MBP in E. coli (see “Experimental Procedures”). After purification to homogeneity by successive C4 and C18 RP-HPLC, recombinant cryptdin-4 (rCrp4) and (des-Gly)-cryptdin-4 [r(des-G)-Crp4] peptides had correct molecular masses of 3756 and 3700 atomic mass units, respectively, as determined by MALDI-TOF mass spectrometry. Also, rCrp4 and r(des-G)-Crp4 co-migrated as single bands in AU-PAGE (Fig.6), a system that resolves defensins effectively (38Selsted M.E. Genet. Eng. 1993; 15: 131-147Crossref PubMed Scopus (32) Google Scholar). The Rf values of the two recombinant peptides were indistinguishable from that of natural cryptdin-4, evidence that both peptides were homogeneous and folded correctly (Fig.6). Consistent with the diminished activity of natural (des-Gly)-cryptdin-4 isolated from the small intestinal lumen, r(des-G)-Crp4 had no in vitro bactericidal activity, whereas rCrp4 was potently microbicidal. Against E. coli ML35 and S. typhimurium (39Miller S.I. Mol. Microbiol. 1991; 5: 2073-2078Crossref PubMed Scopus (107) Google Scholar, 40Groisman E.A. Parra-Lopez C. Salcedo M. Lipps C.J. Heffron F. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 11939-11943Crossref PubMed Scopus (223) Google Scholar), r(des-G)-Crp4 had no measurable microbicidal activity under conditions (5 μg/ml or greater) in which rCrp4 sterilized the culture of 1 × 106 bacteria/ml (Fig.7). These results confirm that the recombinant molecules are equivalent to the natural forms and provide an opportunity for further investigations of mechanisms by which N-terminal residues modulate cryptdin-4 microbicidal activity. These experiments show that full-length cryptdins are released into the intestinal lumen and that functional peptides can be isolated from the lumen of the small intestine. Immunogold-detection experiments localized mouse cryptdins to Paneth cell secretory granules (Figs. 1and 2), and, therefore, luminal cryptdins result from the secretory responses of Paneth cells and not those of other enteric epithelial lineages. Also, immunohistochemical experiments provided no evidence that cryptdins were associated with apical microvilli or the brush border of the mucosal epithelium (data not shown). These findings suggest that the peptides remain soluble following secretion or that they diffuse rapidly out of the crypt. Although approximately equal quantities of full-length cryptdins and cryptdins with N-terminal truncations of 1 or 2 amino acids were isolated, truncated variants have not been detected among cryptdins purified from mouse intestinal tissue. Whether the truncations occur in the crypt lumen or after diffusion above the crypt-villus junction is unknown. Because no Paneth cell secretory stimuli (30Satoh Y. Ishikawa K. Oomori Y. Yamano M. Ono K. Anat. Rec. 1989; 225: 124-132Crossref PubMed Scopus (32) Google Scholar, 31Satoh Y. Ishikawa K. Oomori Y. Takeda S. Ono K. Cell Tissue Res. 1992; 269: 213-220Crossref PubMed Scopus (50) Google Scholar, 32Satoh Y. Habara Y. Ono K. Kanno T. Gastroenterology. 1995; 108: 1345-1356Abstract Full Text PDF PubMed Scopus (77) Google Scholar) were administered to the mice in these studies, the luminal peptides that were isolated represent molecules released by Paneth cells under steady-state conditions. In the human neutrophil α-defensins, the N terminus is known to modulate certain activities. For example, the respective N termini of human neutrophil α-defensins HNP-1–3 are ACYC, CYC, and DCYC, but otherwise the primary structures are identical (41Selsted M.E. Harwig S.S. Infect. Immun. 1987; 55: 2281-2286Crossref PubMed Google Scholar). Despite these similarities, HNP-1 and 2 are chemotactic for monocytes and T lymphocytes, but HNP-3 lacks both activities (42Territo M.C. Ganz T. Selsted M.E. Lehrer R. J. Clin. Invest. 1989; 84: 2017-2020Crossref PubMed Scopus (406) Google Scholar, 43Chertov O. Michiel D.F. Xu L. Wang J.M. Tani K. Murphy W.J. Longo D.L. Taub D.D. Oppenheim J.J. J. Biol. Chem. 1996; 271: 2935-2940Abstract Full Text Full Text PDF PubMed Scopus (495) Google Scholar). Similarly, HNP-3 is the only peptide of the three that does not kill Candida albicans (44Lehrer R.I. Ganz T. Szklarek D. Selsted M.E. J. Clin. Invest. 1988; 81: 1829-1835Crossref PubMed Scopus (181) Google Scholar). Interestingly, those comparisons showed that the charge at the N terminus affected activity but that removal of the N-terminal Ala had little effect. Perhaps these differences reflect the differential activities of peptides that function primarily in neutrophil phagolysosomes as compared with the external environment of the intestinal lumen. One or two residue truncations of cryptdin N termini attenuate or eliminate peptide antimicrobial activity against Gram-positive and Gram-negative bacteria (Figs. 4, 5, 7). For both cryptdin-6 and cryptdin-4, the effect of truncation was greater against Gram-negative bacteria than against S. aureus, a Gram-positive bacterium. Because the molecular details of cryptdin bactericidal activity are not known, it is not immediately apparent how the deletion of a single glycine residue from the cryptdin-4 N terminus could eliminate bactericidal activity so thoroughly. α-Defensin tertiary structures show that the N and C termini are juxtaposed (45Bach A.C. Selsted M.E. Pardi A. Biochemistry. 1987; 26: 4389-4397Crossref PubMed Scopus (55) Google Scholar, 46Pardi A. Zhang X.L. Selsted M.E. Skalicky J.J. Yip P.F. Biochemistry. 1992; 31: 11357-11364Crossref PubMed Scopus (111) Google Scholar, 47Zhang X.L. Selsted M.E. Pardi A. Biochemistry. 1992; 31: 11348-11356Crossref PubMed Scopus (76) Google Scholar, 48Hill C.P. Yee J. Selsted M.E. Eisenberg D. Science. 1991; 251: 1481-1485Crossref PubMed Scopus (454) Google Scholar), and we speculate that N-terminal truncation may disrupt hypothetical associations with the C terminus, leading to impaired peptide interactions with target cell membranes. The variability of N-terminal length among certain luminal cryptdins does not appear to result from inaccurate precursor processing and activation by MMP-7, the procryptdin activating enzyme in mouse Paneth cells. In vitro, MMP-7 cleaves recombinant procryptdin substrates with high fidelity (27Wilson C.L. Ouellette A.J. Satchell D.P. Ayabe T. Lopez-Boado Y.S. Stratman J.L. Hultgren S.J. Matrisian L.M. Parks W.C. Science. 1999; 286: 113-117Crossref PubMed Scopus (913) Google Scholar). Also, extensive processing of cryptdin precursors occurs intracellularly, because mouse Paneth cell granules contain high levels of fully activated cryptdins. 4D. P. Satchell, T. Ayabe, C. L. Wilson, S. J. Hagen, and A. J. Ouellette, unpublished observations. Furthermore, N-terminal cryptdin variants have not been detected in mouse intestinal tissue (3Ouellette 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, 16Selsted M.E. Miller S.I. Henschen A.H. Ouellette A.J. J. Cell Biol. 1992; 118: 929-936Crossref PubMed Scopus (280) Google Scholar). For these reasons, it is unlikely that an error-prone MMP-7 introduces variation at cryptdin N termini during post-translational activation. Current evidence supports the view that N-terminal truncation of cryptdin peptides in the lumen is caused by the modification of activated peptides following secretion. We thank Dana Frederick, Robin Huffman, and Khoa Nguyen for excellent technical assistance." @default.
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• W2058761441 title "Characterization of Luminal Paneth Cell α-Defensins in Mouse Small Intestine" @default.
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