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The bactericidal activity of mouse ␣-defensins (cryptdins) requires proteolytic activation of inactive precursors by matrix metalloproteinase-7 (matrilysin, EC 3.4.24.23, MMP-7 a ). To investigate mechanisms of cryptdin-4 (Crp4) peptide interactions with membrane bilayers and to determine whether MMP-7-mediated proteolysis activates the membrane disruptive activity of Crp4, associations of Crp4 and melittin with biomimetic lipid/ polydiacetylene chromatic vesicles were characterized. The peptides differ in their sensitivity to vesicle lipid composition and their depth of bilayer penetration. Crp4 undergoes strong interfacial binding onto lipid bilayers with disruption of the bilayer head group region, unlike melittin, which inserts more deeply into the hydrophobic core of the bilayer. Colorimetric and tryptophan fluorescence studies showed that Crp4 insertion is favored by negatively charged phospholipids and that zwitterionic and Escherichia coli phospholipids promote stronger interfacial binding; melittin-membrane interactions were independent of either variable. In contrast to the membrane disruptive activity of Crp4, pro-Crp4 did not perturb vesicular membranes, consistent with the lack of bactericidal activity of the precursor, and incubation of Crp4 with prosegment in trans blocked Crp4 and G1W-Crp4 membrane interactions at concentrations that inhibit Crp4 bactericidal activity. CD measurements showed that Crp4 has an expected ␤-sheet structure that is not evident in the pro-Crp4 CD trace or when Crp4 is incubated with prosegment, indicating that the ␤-sheet signal is attenuated by proregion interactions or possibly disrupted by the prosegment. Collectively, the results suggest that the prosegment inhibits Crp4 bactericidal activity by blocking peptidemediated perturbation of target cell membranes, a constraint that is relieved when MMP-7 cleaves the prosegment.Antimicrobial peptides have been identified as components of innate immunity in every living organism investigated (1), and the mammalian ␣-defensins were among the first antimicrobial peptide families to be recognized and characterized (2, 3). ␣-Defensins are major constituents of both azurophilic granules in mammalian phagocytic leukocytes and secretory granules of mammalian Paneth cells, where they are termed cryptdins (Crps) 1 in mice (4, 5). In contrast to ␣-defensins in cells of myeloid origin, which provide a nonoxidative means for killing microorganisms after phagocytosis (6), Paneth cell ␣-defensins are secreted to function in the extracellular compartment (7,8). These cationic peptides with molecular masses of 3-4 kDa contain six cysteine residues that form a distinctive tridisulfide array to produce an amphipathic peptide, a feature that is essential for its microbicidal activity (6). In mouse Paneth cells, the production of mature bactericidal ␣-defensins requires proteolytic activation by matrix metalloproteinase-7 (9), a process that precedes secretion (10).Human and rabbit neutrophil ␣-defensins are structurally and functionally disti...

␣-Defensins are mediators of mammalian innate immunity, and knowledge of their structure-function relationships is essential for understanding their mechanisms of action. We report here the NMR solution structures of the mouse Paneth cell ␣-defensin cryptdin-4 (Crp4) and a mutant (E15D)-Crp4 peptide, in which a conserved Glu 15 residue was replaced by Asp. Structural analysis of the two peptides confirms the involvement of this Glu in a conserved salt bridge that is removed in the mutant because of the shortened side chain. Despite disruption of this structural feature, the peptide variant retains a well defined native fold because of a rearrangement of side chains, which result in compensating favorable interactions. Furthermore, salt bridge-deficient Crp4 mutants were tested for bactericidal effects and resistance to proteolytic degradation, and all of the variants had similar bactericidal activities and stability to proteolysis. These findings support the conclusion that the function of the conserved salt bridge in Crp4 is not linked to bactericidal activity or proteolytic stability of the mature peptide.

We have established an assay to measure indirectly the titres of anti-Dsg3 serum antibodies against the Ca(2+) -dependent epitopes, based on the differences between EDTA-untreated and EDTA-treated ELISA index values, as a routine laboratory test to reflect the pathogenic anti-Dsg3 serum antibody titres more accurately.

Defensins are cationic antimicrobial peptides with a broad spectrum. Recently human beta-defensin 2 (hBD-2) has been isolated from psoriatic skin; however, its exact localization and fate have not been fully understood. We studied the distribution pattern of hBD-2 in skin tissues of psoriasis and other inflammatory skin diseases. In the upper spinous and granular layer of psoriasis vulgaris hBD-2 was present in the cytoplasm. In the horny layer the positive signals were in a basket-weave pattern, indicating possible accumulation of hBD-2 in the intercellular space. The similar pattern of hBD-2 distribution was observed in the lesions of nummular eczema and atopic dermatitis. hBD-2 was not detected in the section of normal elbow and knee skin. When isolated psoriatic scales were stained, hBD-2 was detected in a wrapping paper-like distribution pattern surrounding the corneocytes. In horny layer of psoriatic skin hBD-2 was closely associated or colocalized with elafin, which is known to be in extracellular space, as demonstrated by double staining. Western blot analysis using cultured human keratinocytes detected hBD-2 with an expected size in the conditioned medium and in the cell lysates when stimulated with 5% FCS or IL-alpha. These results indicate that hBD-2 was synthesized and remained in cytoplasm in the upper spinous and granular layer, and then secreted into intercellular space in the horny layer. This dynamic change in hBD-2 distribution in epidermis is certainly relevant to function as an innate host defense mechanism against invading micro-organisms.