Activation of Paneth Cell α-Defensins in Mouse Small Intestine

Abstract

The release of endogenous antimicrobial peptides by mammalian epithelial cells contributes to innate mucosal immunity (1, 2). The crypts of Lieberkü hn in the small intestine of most mammals contain Paneth cells that secrete ␣-defensins (cryptdins), lysozyme, secretory phospholipase A 2 , xanthine oxidase, CD95 ligand, CD15, and tumor necrosis factor-␣ as components of apically oriented secretory granules (3-10). Although certain Paneth cell ␣-defensins have been detected in mouse skin and testis (11,12) and in human oropharyngeal and urogenital mucosa (13,14), in the small intestine, ␣-defensins are specific to Paneth cells (9). Exposure of Paneth cells to cholinergic agonists or bacterial stimuli elicits granule discharge into the crypt lumen (15), and carbamylcholine mediates secretion via increased cytosolic Ca 2ϩ (16). Regardless of how mouse Paneth cell secretion is stimulated, cryptdins constitute ϳ70% of the released bactericidal activity, and the concentration of cryptdins is estimated to be 25 mM at the point of secretion in the crypt lumen (15).␣-Defensins are processed from inactive proforms by specific proteolytic cleavage steps. Both neutrophil and Paneth cell ␣-defensins derive from ϳ10-kDa prepropeptides that contain canonical signal sequences, acidic proregions, and an ϳ3.5-kDa mature ␣-defensin peptide in the C-terminal portion of the precursor. For example, maturation of myeloid pro-␣-defensins appears to involve two primary cleavage steps, and most ␣-defensins in mature phagocytic leukocytes are completely processed (17)(18)(19)(20). In a heterologously expressed human neutrophil pro-␣-defensin, deletions in the prosegment adjacent to the proregion-defensin junction impairs post-translational processing in 32DCL3 cells (19).In mouse Paneth cells, matrix metalloproteinase-7 (MMP-7 1 ; matrilysin) mediates the processing and activation of ␣-defensins from 8.4-kDa proforms (21). MMP-7 gene disruption ablates procryptdin processing, resulting in accumulation of cryptdin precursors and the absence of activated mature cryptdin peptides in the small intestine (21). Lacking functional cryptdin peptides, MMP-7-null mice have a defect in clearance of intestinal infections, and they succumb more rapidly and to lower doses of virulent Salmonella typhimurium compared with control mice (21). Thus, the cryptdin deficiency resulting from defective procryptdin activation is associated with a measurable deficit in mucosal immunity and increased risk of systemic disease.In this study, cryptdin biosynthesis was investigated by characterizing details of intracellular procryptdin processing in

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

“…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.…”

Interactions of Mouse Paneth Cell α-Defensins and α-Defensin Precursors with Membranes

“…For commercially obtained peptides, melittin was purchased from Sigma-Aldrich, and the Crp1 proregion consisting of the following primary structure: DPIQNTDEET KTEEQPGEDD QAVSVSFGDP EGTSLQEES was synthesized by Quality Controlled Biochemicals, Inc. (Hopkinton, MA). The properties of the synthetic prosegment have been reported previously (10).…”

“…1C), pro-Crp4 did not interact with lipid/PDA model membranes. Mouse cryptdins are processed from inactive pro-Crp precursors by MMP-7-mediated proteolysis (10), which is essential for the activation of bactericidal mature ␣-defensin peptides (10). 3 To determine whether the lack of pro-Crp4 bactericidal activity correlates with an inability of the precursor to perturb membranes, interactions between pro-Crp4 and Crp4 with lipid/PDA vesicles were compared using DMPG/DMPC/PDA vesicles and found to be markedly different (Fig.…”

“…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).…”

“…In particular, this colorimetric assay has provided insights into mechanisms of membrane permeation, including the degree of interfacial disruption by peptides, the depth of peptide penetration into the lipid bilayer, and changes in membrane fluidity in response to peptide exposure (16,17). 2 Crp4 was chosen for this study, because this secreted peptide functions in the extracellular compartment (20,21) and is the most bactericidal mouse Paneth cell ␣-defensin (4,5,22) and because the molecular and cellular details of Crp4 activation from inactive pro-Crp4 by MMP-7 proteolysis are known (10). 3 Furthermore, Crp4 has a unique structure among the known ␣-defensins in that its polypeptide backbone contains a threeresidue deletion in the loop formed by the Cys 3 -Cys 5 disulfide bond (4,5,22).…”