Regulation of Intestinal α-Defensin Activation by the Metalloproteinase Matrilysin in Innate Host Defense

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For immunohistochemistry, frozen sections stored at −80°C were air dried and incubated in Bouin's fixative for 15 min at room temperature. After a phosphate-buffered saline (PBS) wash, endogenous peroxidases were quenched with 0.6% H₂O₂ in methanol for 15 min. Slides were rinsed in tris-buffered saline and then blocked for 1 hour in buffer [10 mM tris-HCl (pH 7.5), 0.1 M MgCl₂, 0.5% Tween-20, 1% bovine serum albumin (BSA), and 5% goat serum]. Sections were incubated overnight at 4°C with 1:5000 (antibody to matrilysin) or 1:1000 (antibody to cryptdin-1) dilutions of antibody in blocking solution. Sections were developed with the rabbit Vectastain Elite ABC kit (Vector Laboratories). Protein was visualized with True Blue substrate, and nuclei were counterstained with Contrast Red (Kirkegaard and Perry).

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For generation of recombinant PC-1, nucleotides +58 to +280 of PC-1 cDNA were amplified from a full-length clone with primers 5′-GCGCGAATTCA TCGAGGGAAGGATCCTATCCAAAACACA-3′ (forward cryptdin sequence underlined) and 5′-ATATATGTCGACTCAGCGACAGCAGAGCGTGTACAATAAATG3′ (reverse cryptdin sequence underlined). PC-1 was expressed in E. coli as a fusion protein to maltose-binding protein with the pMalc2 expression vector (New England Biolabs). Bacteria were lysed in buffer containing 10 mM tris-HCl (pH 7.4), 200 mM NaCl, 1 mM EDTA, deoxyribonuclease I (50 μg/ml), 50 mM MgCl₂, and 30% (v/v) B-PER reagent (Pierce). The soluble fusion protein was purified by amylose resin affinity chromatography and subjected to Factor Xa digestion to liberate PC-1, which was further purified by C-18 rpHPLC on a Vydac 218TP510 column. Protein homogeneity was assessed by AU-PAGE (12.5% polyacrylamide) and tris-tricine SDS-PAGE (10 to 20% polyacrylamide gradient). To produce the chimeric procryptdin (proCC), we amplified preprocryptdin-15 cDNA from −42 to +274 with forward primer 5′-GCGGATCCATTGAGCTCCTGCTCA-3′ and reverse primer 5′-GGCCTAGGACAGCAGAGCGTGTACAATAAATG-3′. The reverse primer encoded a threonine residue substitution (characteristic of cryptdin-1) for the methionine normally at position 73 in procryptdin-15. A 40–base pair fragment encoding the last three COOH-terminal residues of cryptdin-4 (PRR) and the 6xHis tag was ligated to the amplified product before cloning into the baculovirus transfer vector pVL1393. Sf9 cells were transfected with this construct and BaculoGold DNA (Pharmingen) to produce recombinant baculovirus. After a single round of amplification, High Five insect cells (Stratagene) were infected and harvested 4 days later. Cell pellets were lysed in denaturing buffer [8 M urea, 100 mM sodium phosphate (pH 8), 10 mM tris-HCl (pH 8), and 1% Triton X-100] and clarified by passage through an 18-G needle and centrifugation. proCC was purified from the supernatant by nickel–nitrilotriacetic acid metal-affinity chromatography (Qiagen) and elution at low pH. Purity was assessed by tris-tricine SDS-PAGE and Coomassie blue staining.

Single-letter abbreviations for the amino acids are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr.

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The difference between MAT^+/+ and MAT^−/− cryptdin peptide profiles was consistently seen in four independent, blind analyses with extracts from over 30 animals. Neither the genetic background of the mice (C57BL/6 or 129/Sv-C57BL/6 hybrid) nor the housing environment (barrier facilities at Vanderbilt University and Washington University) affected the results.

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Residues 1 to 39 of the cryptdin-1 pro segment (Fig. 2A) were deduced from cDNA sequence (15). The peptide was synthesized in solid phase and purified to homogeneity by rpHPLC by Quality Controlled Biochemicals (Hopkinton, MA). The sequence was verified by mass spectrometry and amino acid analysis. After conjugation of the peptide to BSA, polyclonal antiserum to pro segment was produced in a sheep by administration of three to four dorsal subcutaneous injections of BSA-conjugated peptide mixed with an equal volume of complete Freund's adjuvant. Injections were repeated twice, and the antiserum titer was evaluated by enzyme-linked immunosorbent assay by Quality Controlled Biochemicals. The antibody also recognizes proCC, suggesting that it is immunoreactive against multiple procryptdins. The pro segment sequence is highly conserved among the family.

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Crypts were isolated from MAT^+/+ and MAT^−/− males (original 129/Sv-C57BL/6 hybrid backcrossed to C57BL/6 for 10 generations) as described (27). Eluted crypts resuspended in ice-cold Ca²⁺- and Mg²⁺-free Hank's balanced salt solution were transferred singly to siliconized microcentrifuge tubes to obtain 1, 5, or 20 crypts per tube; to transfer 100 crypts per tube, we measured crypt numbers with a hemacytometer and transferred appropriate volumes. Crypts were centrifuged at 700g for 5 min and resuspended in 30 μl of isotonic PIPES buffer [10 mM PIPES (pH 7.4) and 0.8% NaCl] containing 100 μM CCh to stimulate selectively Paneth cell degranulation (28). After a 30-min incubation at 37°C in a rotary shaker at 150 cycles/min, crypts were centrifuged for 5 min at 700g, and supernatants containing CCh-induced Paneth cell secretions were harvested aseptically and stored at −20°C.

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Male and female MAT^+/+ and MAT^−/− mice (C57BL/6 background) were infected at 5 weeks of age. Escherichia coli KBC-236 (in 150 ml of Luria-Bertani broth containing ampicillin and kanamycin) was grown statically for 48 hours at 37°C to induce pili formation. Bacteria were pelleted by centrifugation at 10,000 rpm for 10 min and washed with cold sterile PBS. After recentrifugation, bacteria were resuspended in 1.5 ml of PBS, and the approximate concentration of bacteria was determined by optical density at 600 nm. The actual number of colony-forming units (CFUs) was measured by plating serial dilutions. Mice were made to fast for 2 hours before oral administration of bacteria. Animals were lightly anesthetized with methoxyfluorane and then given a 0.1-ml dose of 0.1 M sodium bicarbonate (to neutralize stomach acids) immediately followed by a 0.1-ml bolus of bacteria (1 to 2 × 10¹⁰ CFUs) in PBS with a 1-ml syringe fitted with a stainless steel feeding needle. Mice were given access to food 30 min after inoculation. After 2 hours, mice were anesthetized and killed by cervical dislocation. The small intestine was removed en bloc and cut into three portions of equal length (proximal, mid, and distal segments). Each segment was homogenized in 3 ml of PBS and serially diluted for plating on kanamycin for determination of CFUs per milliliter.

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Paneth cell secretory granules were prepared from enriched crypt fractions from duodenum and ileum. Crypts dissociated from the lamina propria with EDTA (27) were resuspended in ice-cold Ca²⁺- and Mg²⁺-free Hank's balanced salt solution, and cells were lysed in a Parr cavitation bomb. After an initial centrifugation of cell lysate at 700g, granules were pelleted from the supernatant at 27,000g for 40 min.

We thank R. P. Mecham, T. J. Broekelmann, and A. H. Henschen for protein sequencing; M. E. Selsted for the antibody to cryptdin-1, the cryptdin-4 peptide standard, and helpful discussions; M. A. Mulvey for help with bacteria and critical review of the manuscript; F. Heffron for the virulent S. typhimurium strain 14028s; and S. Shapiro and J. Lohi for insightful comments on the manuscript. All animal experiments were performed with the approval of the Washington University School of Medicine Animal Studies Committee. This work was supported by a Barnes-Jewish Hospital Foundation grant (to C.L.W.) and by grants from NIH (to W.C.P., A.J.O., L.M.M., and S.J.H.).