Salt-bridge interactions between acidic and basic amino acids contribute to the structural stability of proteins and to protein-protein interactions. A conserved salt-bridge is a canonical feature of the α-defensin antimicrobial peptide family, but the role of this common structural element has not been fully elucidated. We have investigated mouse Paneth cell α-defensin cryptdin-4 (Crp4) and peptide variants with mutations at Arg7 or Glu15 residue positions to disrupt the salt-bridge and assess the consequences on Crp4 structure, function, and stability. NMR analyses showed that both (R7G)-Crp4 and (E15G)-Crp4 adopt native-like structures, evidence of fold plasticity that allows peptides to reshuffle side chains and stabilize the structure in the absence of the salt-bridge. In contrast, introduction of a large hydrophobic side chain at position 15, as in (E15L)-Crp4 cannot be accommodated in the context of the Crp4 primary structure. Regardless of which side of the salt-bridge was mutated, salt-bridge variants retained bactericidal peptide activity with differential microbicidal effects against certain bacterial cell targets, confirmation that the salt-bridge does not determine bactericidal activity per se. The increased structural flexibility induced by salt-bridge disruption enhanced peptide sensitivity to proteolysis. Although sensitivity to proteolysis by MMP7 was unaffected by most Arg7 and Glu15 substitutions, every salt-bridge variant was degraded extensively by trypsin. Moreover, the salt-bridge facilitates adoption of the characteristic α-defensin fold as shown by the impaired in vitro refolding of (E15D)-proCrp4, the most conservative salt-bridge disrupting replacement. In Crp4, therefore, the canonical α-defensin salt-bridge facilitates adoption of the characteristic α-defensin fold, which decreases structural flexibility and confers resistance to degradation by proteinases.

In the small bowel, Paneth cells at the base of the crypts of Lieberkühn secrete ␣-defensins and additional antimicrobial peptides at high levels in response to cholinergic stimulation and when exposed to bacterial antigens (1-4). Paneth cell ␣-defensins show broad spectrum antimicrobial activities and constitute the majority of bactericidal peptide activity in Paneth cell secretions (2, 5-7). The release of Paneth cell products into the crypt lumen is inferred to protect mitotically active crypt cells from colonization by potential pathogens and to confer protection from enteric infection (2, 8 -10). The most compelling evidence for a Paneth cell role in enteric innate immunity is evident from studies of mice transgenic for a human Paneth cell ␣-defensin, HD-5, which are completely immune to infection and systemic disease from orally administered Salmonella enterica serovar typhimurium (11).The biosynthesis of ␣-defensins requires post-translational activation by lineage-specific proteinases (12, 13). Although the enzymes that mediate pro-␣-defensin processing in myeloid and epithelial cells differ, the overall processing schemes are the same. Both myeloid and Paneth cell ␣-defensins derive from ϳ10-kDa prepropeptides that contain canonical signal sequences, electronegative proregions, and a 3.5-4-kDa mature ␣-defensin peptide in the C-terminal portion of the precursor (13-16). Pro-␣-defensin processing in mouse Paneth cells is catalyzed by matrix metalloproteinase-7 (MMP-7) 3 and takes place intracellularly and prior to secretion (17,18). In mouse small intestinal epithelium, only Paneth cells express MMP-7 as components of dense core secretory granules (12), and the bactericidal activity of mouse Paneth cell ␣-defensins depends completely on activation of 8.4-kDa pro-Crps by MMP-7-catalyzed proteolysis (12, 18). MMP-7 gene disruption ablates pro-Crp processing such that mature, activated Crp peptides are absent from the small intestine, and innate immunity to oral bacterial infection is impaired in MMP-7-null mice (12).

Recombinant expression of alpha-defensins can be obtained at efficient levels in Escherichia coli. Amplified alpha-defensin or pro-alpha-defensin coding cDNA sequences are cloned directionally between EcoRI and SalI sites of the pET-28a expression vector and expressed in E. coli BL21 RIS cells. Cells growing exponentially in nutrient-rich liquid medium are induced to express the recombinant protein by addition of 50 mM isopropyl beta-D-1-thiogalactopyranoside for 3-6 h. After bacterial cells collected by centrifugation are lysed in 6 M guanidine-HCl under non-reducing conditions, the expressed defensin fused to its 6xHis-34 amino acid N-terminal fusion partner is purified by affinity chromatography on nickel-NTA columns. A Met codon introduced at the N terminus of expressed Met-free peptides provides a unique CNBr cleavage site, enabling release of the alpha-defensin free of ancillary residues by sequential C18 RP-HPLC. Molecular masses of C18 RP-HPLC purified peptides are confirmed by MALDI-TOF mass spectrometry, and peptide homogeneity is assessed using analytical RP-HPLC and acid-urea polyacrylamide gel electrophoresis. alpha-Defensins prepared in this manner are biochemically equivalent to the natural molecules.

Abstractα-Defensin biosynthesis requires the proteolytic conversion of inactive precursors to microbicidal forms. In mouse Paneth cell pro-α-defensin proCrp4 , anionic amino acids positioned near the proregion N-terminus inhibit proCrp4 activity by an apparent charge neutralization mechanism. Because most pro-α-defensins contain proregions of highly conserved chain length, we tested whether decreasing the distance between the inhibitory acidic residues of the proregion and the α-defensin component of the precursor would alter proCrp4 inhibition. Accordingly, two proCrp4 deletion variants, (Δ44-53)-proCrp4 and (Δ44-58)-proCrp4, truncated in a manner corresponding to deletions between MMP-7 cleavage sites, were prepared and assayed for bactericidal peptide activity. Consistent with the properties of full-length proCrp4 , (Δ44-53)-proCrp4 and (Δ44-58)-proCrp4 were processed effectively by MMP-7, lacked bactericidal activity at high peptide levels and over a 3 h exposure period, and failed to induce permeabilization of live E. coli in vitro. Thus, bringing the inhibitory proregion domain into greater proximity with the Crp4 component of the precursor did not alter the activity of this pro-α-defensin. Therefore, the conserved distance that separates inhibitory acidic proregion residues from the Crp4 peptide is not critical to maintaining proCrp4 in an inactive state.

Protein-expression profiling of serum is a common approach to the discovery of potential diagnostic and therapeutic markers of disease. Like any other proteome, the serum proteome is characterized by protein expression across a large dynamic range. This single facet requires the employment of fractionation procedures prior to detection of protein. The authors use a combination of conventional column chromatography with array-based chromatography to simplify the serum proteome into subproteomes, thus providing a greater representation of the serum proteome. Robotics is employed to increase the throughput of sample processing. These procedures result in large amounts of data that are analyzed through a series of preprocessing and postprocessing steps. A well-designed serum profiling project can therefore result in the discovery of statistically sound, clinically meaningful protein biomarkers.