Title: Structural Determinants of Procryptdin Recognition and Cleavage by Matrix Metalloproteinase-7
Abstract: The bactericidal activity of mouse Paneth cell αdefensins, or cryptdins, is dependent on processing of cryptdin precursors (pro-Crps) by matrix metalloproteinase-7 (MMP-7) (Wilson, C. L., Ouellette, A. J., Satchell, D. P., Ayabe, T., Lopez-Boado, Y. S., Stratman, J. L., Hultgren, S. J., Matrisian, L. M., and Parks, W. C. (1999) Science 286, 113–117). To investigate the mechanisms of pro-Crp processing by this enzyme, recombinant pro-Crp4, a His-tagged chimeric pro-Crp (pro-CC), and site-directed mutant precursors of each were digested with MMP-7, and the cleavage products were analyzed by NH2-terminal peptide sequencing. Proteolysis of pro-Crp4 with MMP-7 activated in vitrobactericidal activity to the level of the mature Crp4 peptide by cleaving pro-Crp4 at Ser43↓Ile44 and Ala53↓Leu54 in the proregion and near the Crp4 peptide NH2 terminus between Ser58↓Leu59. Because the Crp4 NH2terminus occurs at Gly61, not Leu59, MMP-7 is necessary but insufficient to complete the processing of Crp4. Crp activating proteolysis at S58↓L59 was unaffected by I44S/I44D or L54S/L54D loss-of-function mutations in pro-Crp4, and a (L59S)-pro-CC mutant was cleaved normally at Ser43↓Val44and Ser53↓Leu54 sites but not at the peptide NH2 terminus. C57BL/6 mice contain an abundant (L59S)-Crp4 mutant peptide with Leu54 at its NH2 terminus resulting from Ala53↓Leu54 cleavage and loss-of-function at the Ser58↓Ser59 cleavage site. Thus, α-defensins resulting from mutations at MMP-7 cleavage sites exist in mouse populations. A pro-CC substrate containing both L54S and L59S mutations resisted cleavage at Ser43↓Val44 completely, showing that cleavage at one or both downstream sites must precede proteolysis at Ser43↓Val44. These findings show that MMP-7 activation of pro-Crps can occur without proteolysis of the proregion, and prosegment fragmentation depends, at least in part, on the release of the Crp peptide from the precursor. The bactericidal activity of mouse Paneth cell αdefensins, or cryptdins, is dependent on processing of cryptdin precursors (pro-Crps) by matrix metalloproteinase-7 (MMP-7) (Wilson, C. L., Ouellette, A. J., Satchell, D. P., Ayabe, T., Lopez-Boado, Y. S., Stratman, J. L., Hultgren, S. J., Matrisian, L. M., and Parks, W. C. (1999) Science 286, 113–117). To investigate the mechanisms of pro-Crp processing by this enzyme, recombinant pro-Crp4, a His-tagged chimeric pro-Crp (pro-CC), and site-directed mutant precursors of each were digested with MMP-7, and the cleavage products were analyzed by NH2-terminal peptide sequencing. Proteolysis of pro-Crp4 with MMP-7 activated in vitrobactericidal activity to the level of the mature Crp4 peptide by cleaving pro-Crp4 at Ser43↓Ile44 and Ala53↓Leu54 in the proregion and near the Crp4 peptide NH2 terminus between Ser58↓Leu59. Because the Crp4 NH2terminus occurs at Gly61, not Leu59, MMP-7 is necessary but insufficient to complete the processing of Crp4. Crp activating proteolysis at S58↓L59 was unaffected by I44S/I44D or L54S/L54D loss-of-function mutations in pro-Crp4, and a (L59S)-pro-CC mutant was cleaved normally at Ser43↓Val44and Ser53↓Leu54 sites but not at the peptide NH2 terminus. C57BL/6 mice contain an abundant (L59S)-Crp4 mutant peptide with Leu54 at its NH2 terminus resulting from Ala53↓Leu54 cleavage and loss-of-function at the Ser58↓Ser59 cleavage site. Thus, α-defensins resulting from mutations at MMP-7 cleavage sites exist in mouse populations. A pro-CC substrate containing both L54S and L59S mutations resisted cleavage at Ser43↓Val44 completely, showing that cleavage at one or both downstream sites must precede proteolysis at Ser43↓Val44. These findings show that MMP-7 activation of pro-Crps can occur without proteolysis of the proregion, and prosegment fragmentation depends, at least in part, on the release of the Crp peptide from the precursor. matrix metalloproteinase-7 cryptdin procryptdin matrix-assisted laser desorption ionization-time of flight mass spectrometry reverse-phase high performance liquid chromatography acid urea-polyacrylamide gel electrophoresis nickel-nitrilotriacetic acid N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine 1,4-piperazinediethanesulfonic acid colony forming units chimeric pro-Crp The release of endogenous antimicrobial peptides by mammalian epithelial cells contributes to innate mucosal immunity (1Huttner K.M. Bevins C.L. Pediatr. Res. 1999; 45: 785-794Google Scholar, 2Lehrer R.I. Ganz T. Curr. Opin. Immunol. 1999; 11: 23-27Google Scholar). In the small intestine of most mammals, Paneth cells that reside at the base of the crypts synthesize and secrete microbicidal α-defensins, termed cryptdins in mice, as components of apical secretory granules (3Porter E.M. Bevins C.L. Ghosh D. Ganz T. Cell. Mol. Life Sci. 2002; 59: 156-170Google Scholar, 4Ouellette A.J. Satchell D.P. Hsieh M.M. Hagen S.J. Selsted M.E. J. Biol. Chem. 2000; 275: 33969-33973Google Scholar, 5Ayabe T. Satchell D.P. Pesendorfer P. Tanabe H. Wilson C.L. Hagen S.J. Ouellette A.J. J. Biol. Chem. 2002; 277: 5219-5228Google Scholar, 6Porter E.M. Liu L. Oren A. Anton P.A. Ganz T. Infect. Immun. 1997; 65: 2389-2395Google Scholar). The granules are released by Paneth cells in response to cholinergic agonists or when exposed to bacterial stimuli (7Peeters T. Vantrappen G. Gut. 1975; 16: 553-558Google Scholar, 8Peeters T.L. Vantrappen G.R. Experientia. 1976; 32: 1125-1126Google Scholar, 9Ayabe T. Satchell D.P. Wilson C.L. Parks W.C. Selsted M.E. Ouellette A.J. Nat. Immunol. 2000; 1: 113-118Google Scholar, 10Cunliffe R.N. Rose F.R. Keyte J. Abberley L. Chan W.C. Mahida Y.R. Gut. 2001; 48: 176-185Google Scholar). Cryptdin peptides constitute ∼70% of the bactericidal peptide activity released by mouse Paneth cells, and cryptdin concentration at the point of secretion is at least 1000 times greater than the minimal bactericidal concentration of the peptides (9Ayabe T. Satchell D.P. Wilson C.L. Parks W.C. Selsted M.E. Ouellette A.J. Nat. Immunol. 2000; 1: 113-118Google Scholar). The production of functional α-defensins involves proteolytic processing of inactive precursor forms by mechanisms that differ between mice and humans (5Ayabe T. Satchell D.P. Pesendorfer P. Tanabe H. Wilson C.L. Hagen S.J. Ouellette A.J. J. Biol. Chem. 2002; 277: 5219-5228Google Scholar,11Ghosh 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: 583-590Google Scholar). α-Defensins are processed from inactive proforms by specific proteolytic cleavage steps. Both neutrophil and Paneth cell α-defensins derive from ∼10-kDa prepropeptides that contain canonical signal sequences, acidic proregions, and a ∼3.5-kDa mature α-defensin peptide in the COOH-terminal portion of the precursor. Most pro-α-defensins are fully processed in mature phagocytic leukocytes (12Valore E.V. Ganz T. Blood. 1992; 79: 1538-1544Google Scholar, 13Ganz T. Liu L. Valore E.V. Oren A. Blood. 1993; 82: 641-650Google Scholar), and processing of myeloid α-defensin precursors occurs within 4–24 h after synthesis by apparently sequential events that produce major intermediates of 75 and 56 amino acids (12Valore E.V. Ganz T. Blood. 1992; 79: 1538-1544Google Scholar, 13Ganz T. Liu L. Valore E.V. Oren A. Blood. 1993; 82: 641-650Google Scholar, 14Michaelson D. Rayner J. Couto M. Ganz T. J. Leukocyte Biol. 1992; 51: 634-639Google Scholar). Deletions in the prosegment adjacent to the proregion-defensin junction impaired post-translational processing of human neutrophil pro-α-defensins when expressed heterologously in mouse 32DCL3 cells (13Ganz T. Liu L. Valore E.V. Oren A. Blood. 1993; 82: 641-650Google Scholar). The anionic propeptide segments also appear to neutralize the cationic COOH-terminal defensin peptides, as suggested by the inhibition of in vitro α-defensin bactericidal activity when intact proregions are added in trans (5Ayabe T. Satchell D.P. Pesendorfer P. Tanabe H. Wilson C.L. Hagen S.J. Ouellette A.J. J. Biol. Chem. 2002; 277: 5219-5228Google Scholar, 14Michaelson D. Rayner J. Couto M. Ganz T. J. Leukocyte Biol. 1992; 51: 634-639Google Scholar, 15Valore E.V. Martin E. Harwig S.S. Ganz T. J. Clin. Invest. 1996; 97: 1624-1629Google Scholar). Human Paneth cells store HD-5 α-defensin in precursor form that is converted rapidly by trypsin to the mature HD-5 peptide after secretion (10Cunliffe R.N. Rose F.R. Keyte J. Abberley L. Chan W.C. Mahida Y.R. Gut. 2001; 48: 176-185Google Scholar, 11Ghosh 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: 583-590Google Scholar, 16Porter E.M. Poles M.A. Lee J.S. Naitoh J. Bevins C.L. Ganz T. FEBS Lett. 1998; 434: 272-276Google Scholar), but mouse Paneth cell pro-Crps are processed and activated by matrix metalloproteinase-7 (matrilysin, MMP-7,1 EC 3.4.24.23) by intracellular processing events that precede secretion (5Ayabe T. Satchell D.P. Pesendorfer P. Tanabe H. Wilson C.L. Hagen S.J. Ouellette A.J. J. Biol. Chem. 2002; 277: 5219-5228Google Scholar). In mouse small intestine, MMP-7 is expressed only by Paneth cells (17Wilson C.L. Heppner K.J. Rudolph L.A. Matrisian L.M. Mol. Biol. Cell. 1995; 6: 851-869Google Scholar), where the enzyme activates all α-defensins from 8.4-kDa proforms (18Wilson 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: 113-117Google Scholar). Previously, both procryptdin-1 (pro-Crp1) and a COOH-terminal His6 tagged pro-Crp chimera (pro-CC) containing sequence from pro-Crp1, pro-Crp4, and pro-Crp15 were shown to be activated to 3.5 kDa α-defensins in vitro by MMP-7-catalyzed cleavage at conserved sites in the proregion and at the junction of the propeptide and the NH2 terminus of the mature cryptdin peptide (5Ayabe T. Satchell D.P. Pesendorfer P. Tanabe H. Wilson C.L. Hagen S.J. Ouellette A.J. J. Biol. Chem. 2002; 277: 5219-5228Google Scholar, 18Wilson 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: 113-117Google Scholar). In those studies, MMP-7 was found to cleave between Ser43↓Val44 in the prosegment and at Ser58↓Leu59, where Leu59 is the NH2-terminal residue for all known mouse cryptdins except Crp4 and Crp5 (5Ayabe T. Satchell D.P. Pesendorfer P. Tanabe H. Wilson C.L. Hagen S.J. Ouellette A.J. J. Biol. Chem. 2002; 277: 5219-5228Google Scholar). Additional preliminary evidence showed that MMP-7 cleaved pro-Crp4 at Ala53↓Leu54 (5Ayabe T. Satchell D.P. Pesendorfer P. Tanabe H. Wilson C.L. Hagen S.J. Ouellette A.J. J. Biol. Chem. 2002; 277: 5219-5228Google Scholar), a site that corresponds to processing intermediates isolated from mouse small intestine (19Putsep K. Axelsson L.G. Boman A. Midtvedt T. Normark S. Boman H.G. Andersson M. J. Biol. Chem. 2000; 275: 40478-40482Google Scholar). Thus, mouse pro-Crps contain conserved sites within the precursor proregion that MMP-7 recognizes and cleaves, but their role in pro-Crp activation is uncharacterized. In this study, cryptdin biosynthesis was investigated by analyzing the processing of recombinant pro-Crps by MMP-7 in vitro. We focused on Crp4, because it is the most bactericidal mouse α-defensin, and comparisons of pro-Crp4 cleavage with cleavage of the pro-Crp chimera pro-CC allowed the generality of MMP-7 site usage to be assessed. The products of in vitro pro-Crp cleavage by MMP-7 and the effects of eliminating the proregion processing sites by site-directed mutagenesis have been determined. The results show that cryptdin activation by cleavage of the peptide bond at the peptide NH2 terminus is independent of proteolysis within the prosegment; however, cleavage upstream at Ser43↓Val44 is dependent on cleavage downstream at the Ser58↓Leu59 or Ala/Ser53↓Leu54 sites. Also, C57BL/6 mice accumulate Crp4-related processing intermediates that result from loss-of-function mutations at MMP-7 processing sites. All procedures on mice were performed with approval and in compliance with the policies of the Institutional Animal Care and Use Committees of the University of California, Irvine (UCI), and Washington University School of Medicine. Six-week-old adult male C57BL/6 mice were purchased from Charles River Breeding Laboratories, Inc. (North Wilmington, MA). MMP-7 null mice were 6–8-week-old males backcrossed for 10 generations onto the C57BL/6 background. Mice were housed in a specific pathogen-free facility under 12 h cycles of light and dark and had free access to standard rat chow and water. Recombinant pro-Crp4 and pro-Crp4 variants with mutated MMP-7 recognition sites were prepared using the pET-28a expression system to produce NH2-terminal His6-tagged fusion proteins (Novagen, Madison, WI). By PCR amplification, a Met-coding trideoxynucleotide was incorporated 5′ of codon 20 in the Crp4 precursor cDNA and cloned in-frame with the amino-terminal His6 in the EcoRI/SalI sites of pET-28a. For cloning pro-Crp4, forward primer pETPCr4-F (5′-GCGCGAATTCATGGATCCTATCCAAAACACA) was paired with reverse primer SLpMALCrp4R (5′-ATATATGTCGACTGTTCAGCGGCGGGGGCAGCAGTACAA), corresponding to nucleotides 104–119 and 301–327 in prepro-Crp4 cDNA (20Ouellette A.J. Darmoul D. Tran D. Huttner K.M. Yuan J. Selsted M.E. Infect. Immun. 1999; 67: 6643-6651Google Scholar). Reactions were performed using the GeneAmp PCR Core Reagents (Applied Biosystems, Foster City, CA) by incubating the reaction mixture at 95 °C for 5 min, followed by successive cycles at 60 °C for 1 min, 72 °C for 1 min, and 94 °C for 1 min for 40 cycles. The underlined codon in the pETPCr4-F primer denotes a Met codon that was introduced immediately upstream of the pro-Crp4 NH2-terminal Asp residue to incorporate a CNBr cleavage site. Following PCR amplification, samples (25 μl) of individual reactions were gel purified using 2% agarose gels, and extracted using QIAEX II (Qiagen Inc., Valencia CA). In most cases, amplification products were cloned in pCR2.1-TOPO, sequenced, digested with EcoRI andSalI, and gel-purified EcoRI/SalI inserts were ligated into EcoRI and SalI-digested pET-28a plasmid DNA, and transformed into bothEscherichia coli XL-2 Blue and BL21(DE3) Codon Plus cells (Stratagene Cloning Systems, Inc., La Jolla, CA). Recombinant proteins were expressed for 6 h at 37 °C inE. coli BL21(DE3) Codon Plus cells growing exponentially in Terrific Broth medium by induction with 0.2 mmisopropyl-1-thio-β-d-galactopyranoside under kanamycin selection. Bacterial cells were harvested by centrifugation and stored at −20 °C. Cells were lysed in 6 m guanidine HCl, 100 mm Tris-Cl (pH 8), and clarified by centrifugation in a Sorvall SA-600 rotor at 30,000 × g for 30 min at 4 °C. Fusion proteins were purified immediately after lysate clarification. Recombinant precursor fusion proteins were purified by nickel-nitrilotriacetic acid (Ni-NTA, Qiagen) resin affinity chromatography and recovered from fusions after CNBr cleavage. His-tagged fusion proteins were eluted from Ni-NTA resin with 2 column volumes of buffer consisting of 6 m guanidine HCl, 1m imidazole, and 100 mm Tris-HCl (pH 6.0). Fusion proteins, containing an NH2-terminal His6 tag, a 26-amino acid spacer, and pro-Crp4, were dialyzed against 5% acetic acid and lyophilized. Lyophilized proteins dissolved in 80% formic acid were adjusted to 10 mg/ml CNBr in 80% formic acid and incubated under N2 overnight at room temperature. Cleavage was terminated by addition of 10 volumes of H2O, proteins were lyophilized, dissolved in 5% acetic acid, and purified by C-18 RP-HPLC by eluting peptides over 120 min with an 20–40% acetonitrile gradient. The identities of recombinant pro-Crp4 molecules were verified by MALDI-TOF MS at the UCI Biomedical Protein and Mass Spectrometry Resource Facility and by acid-urea PAGE (21Selsted M.E. Setlow J.K. Genetic Engineering: Principles and Methods. Plenum Press, New York1993Google Scholar). Mutations were introduced into recombinant pro-Crp4 molecules by PCR. In reaction 1, the mutant forward primer, e.g. pc4I44Dfor, containing the mutant codon flanked by three natural codons was paired with SLpMALCrp4R, the normal reverse primer at the 3′-end of the desired sequence. In reaction 2, the mutant reverse primer, e.g.pc4I44Drev, the exact complement of the mutant forward primer, was paired with the normal forward primer pETPCr4-F at the 5′-end of the desired sequence, and sequences were amplified from the pET-28a pro-Crp4 construct as described above: 95 °C for 5 min, followed by successive cycles at 60 °C for 1 min, 72 °C for 1 min, and 94 °C for 1 min for 40 cycles. Products from reactions 1 and 2 were purified electrophoretically, and 0.5-μl samples of gel-purified DNA were combined as templates in PCR reaction 3, using normal external primers, SLpMALCrp4R and pETPCr4-F, as amplimers. The full-length, mutated pro-Crp4 product of reaction 3 was cloned sequentially into the vectors pCR2.1-TOPO and pET-28a as noted above, and all mutations were verified by DNA sequencing prior to expression. The following forward and reverse internal primers were used to introduce mutations into pro-Crp4. To eliminate the Ser43↓Ile44 MMP-7 cleavage site, the following mutant constructs were prepared: (I44D)-pro-Crp4 using forward primer pc4I44Dfor (5′-CAGGCTGTGTCTGACTCCTTTGGAGGC) and reverse primer pc4I44Drev (5′-GCCTCCAAAGGAGTCAGACACAGCCTG); (I44S)-pro-Crp4 using forward primer pc4I44Sfor (5′-CAGGCTGTGTCTTCCTCCTTTGGAGGC) and reverse primer pc4I44Srev (GCCTCCAAAGGAGGAAGACACAGCCTG); (V42D-S43E-I44D-S45E-F46D)-pro-Crp4 ((DEDED)-pro-Crp4) using forward primer petPC4DEDEDfor (5′-GAGGACCAGGCTGACGAAGACGAAGACGGAGGCCAAGAA) and reverse primer petPC4D EDEDrev (5′-TTCTTGGCCTCCGTCTTCGTCTTCGTCAGCCTGGTC CTC). To eliminate the MMP-7 cleavage site at Ala53-Val54, the following mutant pro-Crp4 constructs were prepared: (L54D)-pro-Crp4, using forward primer pc4L54Dfor (5′-GAAGGGTCTGCTGA CCATGAAAAATCT) and reverse primer pc4L54Drev (5′-AGATTTTTCATGGTCAGCAGACCCTTC); (L54S)-pro-Crp4, using forward primer pc4L54Sfor (5′-GAAGGGTCTGCTTCCCATGAAAAATCT) and reverse primer pc4L54Srev (AGATTTTTCATGGGAAGCAGACCCTTC). To ablate both the Ser43-Ile44 and Ala53-Leu54 MMP-7 cleavage sites, (DEDED)-pro-Crp4 was used as template for mutagenesis of the Ala53-Leu54 site using the (L54D)-pro-Crp4 primers pc4L54Dfor and pc4L54Drev described above to produce (DEDED/L54D)-pro-Crp4. The pro-CC construct for production in the baculovirus expression system has been described (18Wilson 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: 113-117Google Scholar). Briefly, prepro-Crp15 cDNA was amplified using a forward primer derived from sequence encoding the signal peptide and a reverse primer that changed a Met residue in the COOH terminus of the mature peptide to a Thr (characteristic of Crp1). In addition, the COOH-terminal Arg residue of the precursor was converted to Pro-Arg-Arg-His-His-His-His-His-His; Pro-Arg-Arg is the Crp4 COOH-terminal sequence. The amplified sequence was cloned into the transfer vector pVL1393 and transfected into Sf9 insect cells along with BaculoGold DNA (BD Pharmingen, La Jolla, CA) to produce recombinant baculovirus. Following a 4–5-day infection of HighFive insect cells (Stratagene) with recombinant baculovirus, cells were harvested by centrifugation and stored at −20 °C. Pellets were thawed and lysed in 6 m guanidine HCl, 100 mmsodium phosphate (pH 8), and 10 mm Tris-HCl (pH 8). Lysates were passed several times through an 18-gauge needle and centrifuged at 4 °C in a Sorvall SS-34 rotor at 12,000 × g. Supernatants were incubated batchwise at room temperature with Ni-NTA resin (Qiagen) for 1–3 h with mixing. Bound His-tagged precursor was eluted with 8 m urea, 100 mm sodium phosphate (pH 4.5), 10 mm Tris-HCl (pH 4.5), and 1% Triton X-100 in 0.5–1.0-ml fractions. Peak fractions were pooled and dialyzed against 50 mm NaCl, 20 mm Tris-HCl (pH 7.5). Protein concentration and purity were assessed by reducing Tris-Tricine SDS-PAGE and GelCode Blue (Pierce) staining. If further purification was required, proteins were concentrated to 0.6 ml using Centricon YM-3 centrifugal filter devices and subjected to gel filtration chromatography on a Superdex 75 HR/10/30 column connected to anÄKTAFPLC system (Amersham Pharmacia Biotech). As with pro-Crp4, mutations were introduced into recombinant pro-CC by PCR. To generate the L59S mutation, the forward primer used originally to construct pro-CC, CRPBam-F (5′-GCGGATCCTCCTGCTCACCAATCCTCCA-3′) was paired with the mutant reverse primer CRP-L59S (5′-ACCAGATCTCTCGACGATTCCTCT-3′), where the underlined sequence corresponds to BamHI and BglII restriction sites, respectively. Conditions for amplification from the pro-CC template were as follows: 94 °C for 5 min, 30 cycles of 94 °C for 1 min, 53 °C for 1 min, 72 °C for 2 min, and a final extension at 72 °C for 15 min. The 220-bp product was cloned into the pCR 2.1-TOPO vector (Invitrogen); theBamHI-BglII fragment from this plasmid construct was used to replace the BamHI-BglII fragment in the pro-CC cDNA in pGEM-7Zf(+) (Promega). The full-length (L59S)-pro-CC cDNA, flanked by BamHI andEcoRI sites, was then transferred to the pVL1393 baculovirus expression vector using these two restriction enzymes. To add the L54S mutation, pGEM7-(L59S)-pro-CC was used as a template for forward primer CRPBam-F and mutant reverse primer CRPL54/59SBglII-R containing aBglII restriction site (underlined) (5′-AGATCTCTCGACGATTCCTCTTGACTAGAAGAGCC-3′). Amplification parameters were similar to those used for (L59S)-pro-CC except that the annealing temperature was increased to 65 °C and the number of cycles was reduced to 25. The 220-bp product was subcloned into pVL1393 as described above for pro-CCL59S. To eliminate only the Ser43↓Val44 and Ser53↓Leu54 MMP-7 cleavage sites in pro-CC, a two-step process was used. First, the forward primer CRPBam-F was paired with the mutant reverse primer CRPL54SBglII-R (5′-AGATCTCTCAACGATTCCTCTTGACTAGAAG AGCC-3′), where theBglII restriction is underlined. The PCR product was subcloned and transferred to pGEM7-pro-CC as described above. In the second step, this new construct, pGEM7-(L54S)-pro-CC, was used as a template for the mutant forward primer CRPV44SbbsI-F (5′-GAAGACGACCAGGCTGTGTCTTCCTCTTTTGGAGAC-3′), where theBbsI restriction site is underlined, and a normal downstream primer containing a PstI restriction site (underlined), CRPPstI-R (5′-CTGCAGGTCCCATTTATGTGT-3′). The 130-bp product was cloned into pCR2.1-TOPO; theBbsI-PstI fragment from this construct was used to replace the BbsI-PstI fragment in pGEM7-(L54S)-pro-CC. Full-length cDNA from the resulting plasmid, pGEM7-(V44SL54S)-pro-CC, was subcloned into the BamHI andEcoRI sites of pVL1393. All mutations were verified by DNA sequencing prior to expression. A naturally existing α-defensin that has a L59S mutation was purified from C57BL/6 mice, and the corresponding precursor to that Crp4(B6a) variant was isolated from MMP-7 null mouse small intestine by extraction with 30% acetic acid as described (5Ayabe T. Satchell D.P. Pesendorfer P. Tanabe H. Wilson C.L. Hagen S.J. Ouellette A.J. J. Biol. Chem. 2002; 277: 5219-5228Google Scholar,18Wilson 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: 113-117Google Scholar). Protein samples were applied to analytical C-18 RP-HPLC columns (Vydac 218TP54) in aqueous 0.1% trifluoroacetic acid and eluted at ∼35 min using a 10–45% acetonitrile gradient developed over 55 min. Protein fractions containing apparent pro-Crp4 were analyzed by acid urea-(AU)-PAGE as described (18Wilson 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: 113-117Google Scholar, 21Selsted M.E. Setlow J.K. Genetic Engineering: Principles and Methods. Plenum Press, New York1993Google Scholar), and their identities were deduced from a combination of NH2-terminal peptide sequencing, MALDI-TOF-MS, and comparisons with the corresponding cDNA sequence in RIKEN (accession number AK008107). Purification of the Crp4 mutant and corresponding pro-Crp was completed subsequently by C-18 reverse phase-HPLC using a 120-min, 20–40% acetonitrile gradient, from which cryptdin precursors eluted between 23 and 30% acetonitrile. Peptide concentrations were determined using bicinchoninic acid assay (Pierce, Rockford, IL). Peptide samples were lyophilized, dissolved in 20 μl of 5% acetic acid containing 3.0 m urea, and electrophoresed on 12.5% AU-PAGE for 1 h at 100 V and for 3.5 h at 250 V (21Selsted M.E. Setlow J.K. Genetic Engineering: Principles and Methods. Plenum Press, New York1993Google Scholar). Resolved proteins were visualized by staining with Coomassie R-250 after fixation in formalin-containing acetic acid/methanol. Crp4 and pro-Crp4 were identified by co-migration with authentic mouse Crps and pro-Crps in AU-PAGE (>0.6 × R F of methyl green dye) as described (22Selsted M.E. Becker 3rd, H.W. Anal. Biochem. 1986; 155: 270-274Google Scholar) and confirmed by MALDI-TOF-MS and NH2- terminal sequencing. For reduction and alkylation, recombinant peptides dissolved at 500 μg/ml in 6m guanidine HCl, 100 mm Tris-HCl (pH 8.0) were reduced with dithiothreitol at 50 °C for 3–4 h using 5 mol of dithiothreitol per mol of polypeptide cysteine. After cooling, a 3-fold mass excess of iodoacetic acid to dithiothreitol was added to the reduced peptide solution, incubated for 10 min, and residual iodoacetic acid was reacted with excess dithiothreitol. The native and alkylated peptides were purified on C-18 RP-HPLC, and the molecular masses were determined by MALDI-TOF MS, followed by NH2-terminal sequencing in the UCI Biomedical Protein and Mass Spectrometry Resource Facility. Recombinant pro-Crp4 and pro-CC molecules were digested with MMP-7, analyzed by AU-PAGE and SDS-PAGE, and samples of the proteolytic digests were analyzed by NH2-terminal sequencing. Samples (1 μg) of pro-Crp4 and all variants, as well as pro-CC and corresponding variants, were incubated with activated recombinant human MMP-7 (0.3–1.0 μg) catalytic domain (Calbiochem, La Jolla, CA, or Chemicon International, Inc., Temecula, CA) in buffer containing 10 mm HEPES (pH 7.4), 150 mm NaCl, 5 mm CaCl2 for 18–24 h at 37 °C. Samples of pro-Crp4 digests were analyzed by AU-PAGE, and ∼200 ng quantities of complete digests were subjected to 5 or more cycles of NH2-terminal peptide sequencing at the UCI Biomedical Protein and Mass Spectrometry Resource Facility. Pro-CC digests were analyzed by Tris-Tricine SDS-PAGE (15% polyacrylamide), and bands were visualized using GelCodeTM Blue staining reagent (Pierce). For NH2-terminal sequencing by Edman degradation, 3 μg of precursor incubated with MMP-7 were separated by Tris-Tricine SDS-PAGE and transferred to and visualized on mini-ProBlott polyvinylidene difluoride membranes (Applied Biosystems) according to the manufacturer's instructions. Also, samples of the Crp1 prosegment corresponding to residues 19–58 in prepro-Crp1 (23Huttner K.M. Selsted M.E. Ouellette A.J. Genomics. 1994; 19: 448-453Google Scholar, 24Ouellette 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: 5040-5047Google Scholar) were digested with MMP-7 and sequenced. The Crp1 proregion consisting of the primary structure, DPIQNTDEETKTEEQPGEDDQAVSVSFGDPEGTSLQEES, was synthesized by Quality Controlled Biochemicals, Inc., (Hopkinton, MA). The composition and properties of the synthetic prosegment has been reported previously (5Ayabe T. Satchell D.P. Pesendorfer P. Tanabe H. Wilson C.L. Hagen S.J. Ouellette A.J. J. Biol. Chem. 2002; 277: 5219-5228Google Scholar). The activation of pro-Crp4 was assayed by conducting bactericidal peptide assays with pro-Crp4 following digestion with MMP-7 under conditions of quantitative cleavage at Leu59(above). Samples consisting of exponentially growing bacterial cells (∼1 × 106 colony forming units (CFU)/ml) were incubated with 0–20 μg/ml Crp4 or pro-Crp4 with or without MMP-7-mediated proteolysis (above). After 60 min at 37 °C, 20 μl of each incubation mixture was diluted 1:1000 with 10 mmPIPES (pH 7.4), and 50 μl of the diluted samples were plated on trypticase soy agar using a Spiral Biotech Autoplate 4000 (Spiral Biotech Inc., Bethesda, MD). Surviving bacteria were quantitated as colony forming units on plates after incubation at 37 °C for 12 h. Pro-Crp4 is cleaved by MMP-7 at sites corresponding to those identified previously in natural pro-Crps (5Ayabe T. Satchell D.P. Pesendorfer P. Tanabe H. Wilson C.L. Hagen S.J. Ouellette A.J. J. Biol. Chem. 2002; 277: 5219-5228Google Scholar). Recombinant pro-Crp4 molecules expressed in E. coli were purified to homogeneity by RP-HPLC (Fig.1). Because the pro-Crp4 protein lacks methionine, CNBr provided a means for quantitative chemical cleavage of affinity purified fusion proteins from which the pro-Crp4 component could be separated from the His-tagged fusion partner by sequential RP-HPLC fractionation. The mass of the pro-Crp4 molecule was verified by MALDI-TOF-MS to be 8231 atomic mass units, and its homogeneity was judged by analytical RP-HPLC, AU-PAGE, and NH2-terminal sequencing. The sequence DPIQNT … , the consensus for mouse pro-α-defensins, was the only NH2terminus detected. Also, cleavage of recombinant pro-Crp4 with MMP-7in vitro produced a single evident product that migrated only slightly slower than Crp4 in AU-PAGE gels and was comparable with Crp4 in bactericidal activity (Fig. 1). Previously, Paneth cell α-defensin precursor substrates isolated from MMP-7 null mouse small intestine were cleaved in vitro with MMP-7, identifying cleavage sites at Ser43↓Val44 and Ser58↓Leu59,