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Functional Analysis of the α-Defensin Disulfide Array in Mouse Cryptdin-4*

The α-defensin antimicrobial peptide family is defined by a unique tridisulfide array. To test whether this invariant structural feature determines α-defensin bactericidal activity, mouse cryptdin-4 (Crp4) tertiary structure was disrupted by pairs of site-directed Ala for Cys substitutions. In a series of Crp4 disulfide variants whose cysteine connectivities were confirmed using NMR spectroscopy and mass spectrometry, mutagenesis did not induce loss of function. To the contrary, the in vitro bactericidal activities of several Crp4 disulfide variants were equivalent to or greater than those of native Crp4. Mouse Paneth cell α-defensins require the proteolytic activation of precursors by matrix metalloproteinase-7 (MMP-7), prompting an analysis of the relative sensitivities of native and mutant Crp4 and pro-Crp4 molecules to degradation by MMP-7. Although native Crp4 and the α-defensin moiety of proCrp4 resisted proteolysis completely, all disulfide variants were degraded extensively by MMP-7. Crp4 bactericidal activity was eliminated by MMP-7 cleavage. Thus, rather than determining α-defensin bactericidal activity, the Crp4 disulfide arrangement confers essential protection from degradation by this critical activating proteinase.

The mammalian defensins comprise the ␣-, ␤-, and -defensin families of cationic, Cys-rich antimicrobial peptides, and each subfamily is characterized by a distinctive tridisulfide array (1). ␣-Defensins are cationic, amphipathic, 3-4-kDa peptides with a ␤-sheet polypeptide backbone and broad spectrum antimicrobial activities (1). The consensus ␣-defensin tertiary structure is established by six cysteines that are spaced in a pattern that facilitates the formation of invariant disulfide bonds between Cys I -Cys VI , Cys II -Cys IV , and Cys III -Cys V (2) (Fig. 1). These conserved ␣-defensin disulfide pairings have been inferred to have a role in determining, perhaps critically, the bactericidal activity of these peptides.
Paneth cell ␣-defensins confer enteric immunity (3) and, thus, knowledge of determinants of peptide activity and biosynthetic regulation will improve the understanding of the role of these ␣-defensins in mucosal immunity. For example, mouse Paneth cell ␣-defensins, termed cryptdins (Crps), 1 are secreted into the lumen of small intestinal crypts at concentrations of 25-100 mg/ml, four orders of magnitude greater than their minimum bactericidal concentrations (4). In mice, Paneth cell ␣-defensin precursors (proCrps) are processed to their biologically active forms by specific proteolytic cleavage events catalyzed by matrix metalloproteinase-7 (MMP-7, matrilysin). Disruption of the MMP-7 gene abrogates proCrp activation, eliminating the accumulation of functional mature Crp peptides from the small intestine (5). Consequently, MMP-7-null mice have impaired enteric innate immunity in response to oral bacterial infection (5). Also, in mice transgenic for the human Paneth cell ␣-defensin HD5, the minitransgene is expressed specifically in Paneth cells, and the mice are immune to oral infection by virulent strains of Salmonella enterica serovar Typhimurium (serovar Typhimurium) (3).
Here, we report on the role of the disulfide array in the mouse Paneth cell ␣-defensin cryptdin-4 (Crp4) (6,7). Paired Ala for Cys amino acid substitutions in Crp4 were tested for effects on bactericidal activity and resistance to the activating proteinase MMP-7. Mutations that disrupted disulfide bonds did not inactivate peptide bactericidal activity regardless of position. However, Crp4 and proCrp4 molecules with disrupted disulfides were proteolyzed extensively by MMP-7, disclosing a critical protective role for the disulfide array in peptide biosynthesis.

Preparation of Recombinant Crp4 Peptide Variants-Recombinant
Crp4 peptides were expressed in Escherichia coli as N-terminal His 6tagged fusion proteins from the EcoRI and SalI sites of the pET28a expression vector (Novagen, Inc., Madison, WI) as described (8,9). The Crp4-coding cDNA sequences were amplified using the forward primer ER1-Met-C4-F (5Ј-GCGCGAATTCATCGAGGGAAGGATGGGTTTGT-TATGCTATTGT-3Ј) paired with the reverse primer pMALCrp4-R (5Ј-ATATATGTCGACTCAGCGACAGCAGAGCGTGTACAATAAATG-3Ј) as reported previously (9). For proCrp4, the forward primer pETPCr4-F (5Ј-GCGCGAATTCATGGATCCTATCCAA AACACA-3Ј) was paired with the reverse primer SLpMALCrp4R (5Ј-ATATATGTCGACTGT-TCAGCGGCGGGGGCAGCAGTACAA-3Ј), corresponding to nucleotides 104 -119 and 301-327 in preproCrp4 cDNA (8). The underlined codons in the forward primers denote Met codons introduced upstream of each peptide N terminus to provide a CNBr cleavage site (8,9). In all instances, reactions were performed using the GeneAmp PCR Core Reagents (Applied Biosystems, Foster City, CA) by incubating the reaction mixture at 94°C for 5 min followed by successive cycles at 94°C for 30 s, 60°C for 30 s, and 72°C for 30 s for 30 cycles and then a final extension reaction at 72°C for 7 min.
Mutagenesis at Cys Residue Positions-Mutations were introduced into Crp4 by PCR as described previously (8) in the order described below. In the first round of mutagenesis the Crp4 construct in pET-28a (9) was used as template. In PCR reaction number 1, a mutant forward primer, e.g. Crp4-C11A-F, containing the mutation for peptide residue position 11 flanked by three natural codons was paired with the reverse primer T7 terminator (Invitrogen), a downstream sequencing primer in the pET-28a vector. In PCR reaction number 2, the mutant reverse primer Crp4-C11A-R, the reverse complement of the mutant forward primer, was paired with the T7 promoter forward primer, again from the pET-28a. After amplification at 94°C for 5 min followed by successive cycles at 94°C for 30 s, 60°C for 30 s, and 72°C for 30 s for 30 cycles and then a final extension reaction at 72°C for 7 min, samples of purified products from reactions number 1 and number 2 were com- bined as templates in PCR reaction number 3 using the T7 promoter and terminator primers as amplimers. All mutated Crp4 templates were cloned in pCR-2.1 TOPO, verified by DNA sequencing, excised with SalI and EcoRI, subcloned into pET28a plasmid DNA (Novagen, Inc.), and transformed into E. coli BL21(DE3)-CodonPlus-RIL cells (Stratagene) for recombinant expression. The underlined codons in the forward primers denote Met codons introduced upstream of each peptide N terminus to provide a CNBr cleavage site (8,9).
Purification of Recombinant Crp4 Proteins-Recombinant proteins were expressed and purified as His-tagged Crp4 fusion peptides as described (8). Briefly, recombinant proteins were expressed at 37°C in Terrific Broth medium by induction with 0.1 mM isopropyl-␤-D-1-thiogalactopyranoside for 6 h at 37°C, cells were lysed by sonication in 6 M guanidine-HCl in 100 mM Tris-Cl (pH 8.1), and the soluble protein fraction was clarified by centrifugation (8 -10). His-tagged Crp4 fusion peptides were purified using nickel-nitrilotriacetic acid (Qiagen) resin affinity chromatography (8). After CNBr cleavage, Crp4 peptides were purified by C18 reverse-phase high performance liquid chromatography (RP-HPLC) and quantitated by bicinchoninic acid (Pierce), and the molecular masses of the purified peptides were determined using matrix-assisted laser desorption ionization mode mass spectrometry (Voyager-DE MALDI-TOF, PE-Biosystems, Foster City, CA) in the Mass Spectroscopy Facility, Department of Chemistry, University of California, Irvine, CA.
NMR Spectroscopy-Samples of Crp4 and the mutants used for NMR analysis contained 2 mg of Crp4, 0.6 mg of (C6A/C21A)-Crp4, and Ͻ0.3 mg of the other mutants dissolved in 0.5 ml of 95% H 2 O/5% D 2 O at pH 4. One-dimensional and two-dimensional total correlation spectroscopy with a MLEV17 mixing time of 80 ms and two-dimensional nuclear Overhauser effect spectroscopy spectra with a mixing time of 200 ms were recorded for all analogues on a Bruker DMX 750 MHz spectrometer at 298 K. In all experiments, the carrier frequency was set at the center of the spectrum on the solvent signal, and all spectra were recorded in phase-sensitive mode using the time-proportional phase increment method. Solvent suppression was achieved by a modified WATERGATE sequence. Two-dimensional spectra collected with Ͼ4000 data points in the f2 dimension and 512 increments on the f1 dimension over a spectral width corresponding to 12 ppm. Resonance assignments were achieved by standard sequential assignment strategies (11).
Cleavage of Crp4 and proCrp4 Disulfide Variants with MMP-7 in Vitro-Recombinant Crp4, proCrp4, and variants with site-directed mutations in the disulfide array were digested with MMP-7 and analyzed for proteolysis by AU-PAGE, and samples of the proteolytic digests were tested in bactericidal peptide assays and analyzed by Nterminal sequencing by Edman degradation as described previously (8). Samples (11 g) of proCrp4 and all proCrp4 variants, as well as 5-g samples of Crp4 and variants, were incubated with an activated recombinant human MMP-7 (0.3ϳ1.0 g) catalytic domain (Calbiochem, La Jolla, CA) in buffer containing 10 mM HEPES (pH 7.4), 150 mM NaCl, and 5 mM CaCl 2 for 18 -24 h at 37°C (8). Equimolar samples of all digests were analyzed by AU-PAGE, and 3-g quantities of complete digests were subjected to five or more cycles of Edman degradation in the University of California, Irvine Biomedical Protein and Mass Spectrometry Resource Facility.
The biological effects of MMP-7-mediated proteolysis of Crp4 molecules with mutations in the disulfide array was assayed by conducting bactericidal peptide assays as above. Bacterial target cells consisting of exponentially growing bacteria (ϳ1 ϫ 10 6 CFU/ml) were incubated with equimolar quantities (0 to 20 g/ml) of Crp4 or pro-Crp4 peptide variants that had been incubated overnight at 37°C with or without MMP-7.

Mutagenesis of the Crp4 Disulfide Array-Recombinant
Crp4 variants with site-directed mutations in the tridisulfide array ( Fig. 1A) were prepared by expression in E. coli using the pET-28 vector system (8). As shown in Fig. 1, Crp4 variants included molecules null for the following: (a) individual Cys I -Cys VI , Cys II -Cys IV , or Cys III -Cys V disulfides; (b) both the Cys I -Cys VI and Cys III -Cys V bonds; and (c) a Crp4 peptide with all Cys residues converted to Ala and, thus, disulfide-null. All variant Crp4 peptides were purified to homogeneity by RP-HPLC as verified by analytical RP-HPLC (not shown) and AU-PAGE analyses in which the peptides migrated as expected relative to native Crp4 (Fig. 1C) (8). Alkylation of ␣-defensins disrupts ␤-sheet structure, linearizing the molecule and reducing its mobility in AU-PAGE (13). Similarly, the mobility of these variant Crp4 molecules was diminished with increased numbers of disrupted disulfides (Fig. 1C).
A series of one-dimensional and two-dimensional total correlation spectroscopy and nuclear Overhauser effect spectroscopy NMR spectra was recorded to assess the structural integrity of recombinant Crp4 and the disulfide-deficient variants. The two-dimensional spectra were sequentially assigned and used to derive chemical shifts for the backbone protons, which are a sensitive monitor of structure as summarized in Fig. 2. Native Crp4 and the C6A/C21A variant have widely dispersed amide signals characteristic of well folded peptides ( Fig. 2A). On the other hand, the amide signals for the C4A/C29A, C11A/ C28A, and C4A/C11A/C28A/C29A variants have a narrow amide dispersion typical of random coil conformations. Confirmation that the native peptide and the C6A/C21A variant are well folded and that the other variants are not is evident from the ␣H secondary shifts (Fig. 2B), i.e. the differences between the observed chemical shifts of a given amino acid and those for the corresponding residue in a random coil peptide. The presence of several consecutive residues with positive ␣H secondary shifts of magnitude Ͼ0.1 ppm provides a strong indication of ␤-strand structure. Such regions are seen in Crp4 and in the C6A/C21A mutant and correspond to the regions comprising a triplestranded ␤-sheet typically seen in other ␣-defensins (Fig. 2B,  arrows). By contrast, the C4A/C29A, C11A/C28A, and C4A/ C11A/C28A/C29A Crp4 mutants have small secondary shifts characteristic of random coil peptides. These trends correspond well with the gel migration data in Fig. 1C, where the C6A/ C21A variant migrates similarly to the native peptide, but the others have diminished mobility as would be expected for disordered peptides.
The ␣-Defensin Disulfide Array Does Not Determine Crp4 Bactericidal Activity in Vitro-To investigate the role of the Crp4 disulfide array, we assayed the in vitro bactericidal activities of Crp4 disulfide mutants against several bacterial species in relation to native Crp4 (Fig. 3). The overall bactericidal activities of Crp4 and Cys 3 Ala Crp4 variants were similar, although not identical, with all of the peptides reducing bacterial cell survival by at least 1000-fold at concentrations at or below 25 g/ml (Figs. 3 and 4 and data not shown). Because differences in peptide bactericidal activities become more apparent in assays against species with inherently lower antimicrobial peptide sensitivities, the peptides were tested against strains of wild-type serovar Typhimurium, which has low ␣-defensin susceptibility relative to other species of bacteria (14 -17). Three disulfide mutants, the C6A/C21A, C4A/C11A/C28A/C29A, and C4A/C6A/C11A/ C21A/C28A/C29A variants of Crp4, were consistently more active than native Crp4 against wild-type serovar Typhimurium (Fig. 4). Therefore, Crp4 bactericidal activity is independent of disulfide mutagenesis with molecules lacking more than one disulfide bond showing enhanced microbicidal activities, although the dose-response curves of certain peptides varied modestly (Figs. 3 and 4). Because mutagenesis at Crp4 disulfides did not induce loss of function, we considered alternative roles for the disulfide array, including protection of the peptide from degradation by the activating proteinase.
Disulfide Bonds Protect Crp4 from Proteolysis by MMP-7-Production of functional mouse Paneth cell ␣-defensins requires that MMP-7 mediate proteolytic cleavage of inactive proCrps (5). To test whether the disulfide array protects the Crp4 moiety from MMP-7 proteolysis during activation, we first assayed for cleavage products of native and mutant Crp4 molecules exposed to MMP-7 (Fig. 5). As reported previously (8), native Crp4 was completely resistant to MMP-7 in vitro, but all Crp4 peptides with disrupted cystine pairings were degraded extensively (Fig. 5A). As expected, MMP-7 activated native proCrp4 as shown by AU-PAGE analyses (Fig. 5A) and in functional assays (Fig. 6A, and see below). Consistent with peptide structures, the major degradation products detected on the gels (Fig. 5A) all have increased mobilities relative to the uncleaved disulfide-deficient peptide, except for (C6A/C21A)-Crp4. Its high intrinsic mobility is due to the loss of native-like globular structure on proteolysis, whereas the degradation products of the other, random coil peptide variants increased in mobility.

DISCUSSION
The tridisulfide array is a universal and defining feature of the ␣-defensins (1,2,18,19), but Crp4 bactericidal activity does not require that the array be intact (Figs. 3 and 4). Similarly unanticipated were results showing that Crp4 variants lacking two or three disulfide bonds were more bactericidal against serovar Typhimurium than the parent molecule (Fig. 4). All Crp4 and proCrp4 disulfide mutants were degraded by MMP-7 at several internal positions as determined by N-terminal peptide sequence analysis (Figs. 5B and 7B), from which we conclude that the disulfide array protects the Crp4 ␣-defensin moiety during activating proteolysis. Although these studies have focused on the mouse Paneth cell pro-␣-defensin processing enzyme (5,20), similar findings have been observed for corresponding mutations in RMAD-4 and RED-4, myeloid and Paneth cell ␣-defensins, respectively (21,22), from rhesus macaque (not shown). We speculate that the disulfide array also may protect ␣-defensins from degradation in phagolysosomes, after release into the small intestinal lumen or in the extracellular environment at sites of inflammation.
Of the one-, two-, and three-disulfide Crp4 mutants, C6A/ C21A adopts the most native-like structure and is the variant most resistant to MMP-7 induced degradation. If C6A/C21A so resembles native structure, why is it susceptible to proteolysis at all when Crp4 is completely resistant? The answer appears to be due to the enhanced molecular flexibility of this mutant relative to Crp4. Evidence for this possibility may be seen in the significantly broadened NMR signals for all of the amide protons in the C6A/C21A mutant relative the native peptide (compare the upper two traces in Fig. 2A) and in the reduced size of ␣H secondary shifts (compare the upper two traces in Fig. 2B). Signal broadening is particularly acute at residues 6 -8 and 25-26, and the ␣H signals for these residues are broadened beyond detection in (C6A/C21A)-Crp4. The enhanced mobility near Cys 6 reflects the removal of a crosslinking disulfide bond and potential disruption of the first strand of the triple-stranded ␤-sheet, whereas the broadening at Phe 25 -Leu 26 is associated with an extended hairpin turn between the second and third ␤-strands. This turn appears to be one of the major sites for proteolytic degradation of the mutant peptide with three cleavages occurring nearby, including one directly at the Phe 25 -Leu 26 peptide bond. In the structure of the rabbit kidney ␣-defensin RK-1 (23), this hairpin turn is relatively solvent-exposed and, by homology, is predicted to be exposed similarly in native Crp4 and more so in (C6A/C21A)-Crp4, where a disulfide bond that tethers this region to the molecular core is absent. Overall, RK-1 and Crp4 have similarly folded structures (not shown), even though their primary structures are quite different. Relative to Crp4, RK-1 contains two additional residues between Cys IV and Cys V , i.e. between strands 2 and 3. Possibly, the structure of the turn between these two strands would be less extended in Crp4 than in RK-1, but residues in the turn still would be solvent-accessible and a major site of proteolytic degradation. The enhanced flexibility of the (C6A/C21A)-Crp4 mutant presumably facilitates access to the enzyme active site, thus increasing the degree of proteolysis.
The disulfide connectivities of Crp4 variants with just a single disrupted disulfide, C4A/29A, C6A/21A, and C11A/28A, were analyzed by MALDI-TOF MS after digestion with MMP-7 (see Fig. 5). For (C11A/C28A)-Crp4, the only disulfide connectivities consistent with the detected peptide masses of 2250.6, 878.0, 3110.4, 1868.2, 3310.9, and 2837.3 atomic mass units are the predicted Cys I -Cys VI and Cys II -Cys IV bonds, confirming the correct pairings for this peptide. On the basis of similar findings, we could exclude the possibility of Cys I -Cys III and Cys V -Cys VI disulfide pairings in (C6A/C21A)-Crp4 as well as Cys II -Cys III and Cys IV -Cys V disulfide bonds in (C4A/C29A)-Crp4, because no peptide masses consistent with those respective bonding patterns were detected. However, MALDI-TOF MS analysis of (C6A/C21A)-Crp4 MMP-7 digests was unable to distinguish correct Cys I -Cys VI and Cys III -Cys V bond pairings from a possible Cys I -C V and Cys III -Cys VI folded variant. Similarly, we could not differentiate between correct Cys II -Cys IV and Cys III -Cys V connectivities in (C4A/C29A)-Crp4 from a possible Cys II -Cys V and Cys III -Cys IV misfolded variant. Thus, in the case of these two mutants, the relation of peptide tertiary structure to activity is uncertain. Nevertheless, the disulfide pairings of (C11A/C28A)-Crp4, (C4A/C11A/C28A/C29A)-Crp4 with a solitary Cys II -Cys IV bond, and disulfide-null (C4A/C6A/ C11A/C21A/C28A/C29A)-Crp4 are unambiguous.
Alterations in the tridisulfide array of ␤-defensin hBD-3 also have little effect on its microbicidal activity (24). Although ␣and ␤-defensins both have six Cys residues that form specific and invariant disulfide bond pairings (2,25), the spacing of ␣and ␤-defensin cysteines and their Cys-Cys pairings differ, and they have markedly different precursor structures. The ␣-defensin cystine connectivities are Cys I -Cys VI , Cys II -Cys IV , and Cys III -Cys V , and the pairings of ␤-defensins are Cys I -Cys V , Cys II -Cys IV , and Cys III -Cys VI , yet the peptides have similar folded conformations (26 -31). Of the six hBD-3 variants with mispaired Cys connectivities analyzed (32), the microbicidal activities of native hBD-3, mispaired variants, and disulfide-null hBD-3 were the same (24,32). Similarly, the bactericidal activity of bovine ␤-defensin BNBD-12 against E. coli was also independent of the disulfide array (33). The possible role of ␤-defensin disulfide connectivities in conferring resistance to proteolysis is unknown to our knowledge, perhaps because the mechanisms of ␤-defensin posttranslational processing remain obscure.
Studies with model membranes support the view that Paneth cell and myeloid ␣-defensins kill their targets by permeabilizing the cell envelope, thus leading to dissipation of electrochemical gradients, although the mechanisms of individual peptides often differ (19,34). Mouse Crp4 induces graded leakage from quenched fluorophore-loaded large unilamellar vesicles (9,10,35,36), and preliminary results show that the Crp4 disulfide variants described here induce large unilamellar vesicle leakage by the same mechanism and at levels corresponding to their relative bactericidal activities. 2 Although the disulfide array has been thought to facilitate peptide-membrane interactions by maintaining a constrained amphipathic ␤-sheet structure, those interactions clearly are independent of disulfide bonding. Perhaps the disordered, random coil structures of the disulfide variants in aqueous solution (Fig. 2) assume the ␤-sheet structure of disulfide-stabilized Crp4 when in hydrophobic environments that mimic the lipid-water interface at the membrane surface. Alternatively, in the absence of constraints imposed by the disulfide array, the Crp4 molecule may adopt an unrelated configuration that retains amphipathicity and membrane-disruptive behavior.
Transcripts coding for ␣-defensins with mutations at disulfide bonds accumulate in mouse small bowel. For example, C57BL/6 mouse small intestine expresses at least 12 ␣-defensin genes with mutations at varied Cys residue positions. For example, certain mutations are predicted to disrupt the Cys I -Cys VI disulfide bond, including (C6Y)-Crp (GenBank TM accession number AV070313) and two different (C6F)-Crps (AV-064537 and AV061023). An additional (C6F)-Crp mutant also has an Arg 35 to Cys substitution that could enable the formation of an alternative Cys I -Cys VI bond (AV070855). Double mutants of the Cys I -Cys VI linkage also exist as exemplified by (C1W/C6F)-Crp (AV067626) and (C1S/C6F)-Crp (AV067447). Three different (C5W)-Crp mutant peptides would disrupt the Cys III -Cys V bond (AV064900, AV070633, and AV066474), and a (C4F)-Crp peptide would lack the Cys II -Cys IV disulfide (AV-065642). In addition to these predicted single disulfide bond disruptions, (C1S/C2S/C3F/C6F)-Crp (AV066139) and (C3F/C-4F/C5S/C6F)-Crp (AV070606) mutants would lack all disulfides typical of ␣-defensins. Although the actual disulfide connectivities in these deduced Crp mutants are unknown, especially in vivo, our findings predict that the ␣-defensin component of these expressed proforms would be degraded during MMP-7-mediated activation. Given the demonstrated impact of Paneth cell ␣-defensins on enteric immunity (3), a loss of function caused by MMP-7-or trypsin-mediated proteolysis of naturally occurring ␣-defensin disulfide mutants could have adverse consequences for innate immunity in the small intestine.