Elevated Expression of Paneth Cell CRS4C in Ileitis-prone SAMP1/YitFc Mice: REGIONAL DISTRIBUTION, SUBCELLULAR LOCALIZATION, AND MECHANISM OF ACTION

Michael T Shanahan,Alda Vidrich,Yoshinori Shirafuji,Claire L Dubois,Agnes Henschen-Edman,Susan J Hagen,Steven M Cohn,André J Ouellette

doi:10.1074/jbc.m109.083220

Abstract

Paneth cells at the base of small intestinal crypts of Lieberkühn secrete host defense peptides and proteins, including alpha-defensins, as mediators of innate immunity. Mouse Paneth cells also express alpha-defensin-related Defcr-rs genes that code for cysteine-rich sequence 4C (CRS4C) peptides that have a unique CPX triplet repeat motif. In ileitis-prone SAMP1/YitFc mice, Paneth cell levels of CRS4C mRNAs and peptides are induced more than a 1000-fold relative to non-prone strains as early as 4 weeks of age, with the mRNA and peptide levels highest in distal ileum and below detection in duodenum. CRS4C-1 peptides are found exclusively in Paneth cells where they occur only in dense core granules and thus are secreted to function in the intestinal lumen. CRS4C bactericidal peptide activity is membrane-disruptive in that it permeabilizes Escherichia coli and induces rapid microbial cell K(+) efflux, but in a manner different from mouse alpha-defensin cryptdin-4. In in vitro studies, inactive pro-CRS4C-1 is converted to bactericidal CRS4C-1 peptide by matrix metalloproteinase-7 (MMP-7) proteolysis of the precursor proregion at the same residue positions that MMP-7 activates mouse pro-alpha-defensins. The absence of processed CRS4C in protein extracts of MMP-7-null mouse ileum demonstrates the in vivo requirement for intracellular MMP-7 in pro-CRS4C processing.

Highlights

Cessed cysteine-rich sequence 4C (CRS4C) in protein extracts of matrix metalloproteinase-7 (MMP-7)-null mouse ileum demonstrates the in vivo requirement for intracellular MMP-7 in pro-CRS4C processing
At 4 weeks of age, transcript levels of the Defcrrs10 gene, which codes for a CRS4C-4 peptide isoform [32, 33], were already elevated ϳ1000-fold in SAMP1/YitFc ileum relative to age-matched AKR and C57BL/6 mice, non-ileitis-prone reference strains in which levels are inherently low (Fig. 1A)
Defcr-rs genes relative to the Crp (Defcr)-rs10 gene expression was specific for the ileum, with CRS4C mRNAs occurring at barely detectable levels in jejunum

Summary

The abbreviations used are

Cryptdin; MMP-7, matrix metalloproteinase-7; CRS4C, cysteine-rich sequence 4C; pro-Crp, pro-cryptdin; AU-PAGE, acid-urea PAGE; HPLC, high performance liquid chromatography; MALDITOF, matrix-assisted laser desorption ionization time-of-flight; PIPES, 1,4piperazinediethanesulfonic acid; ONPG, 2-ortho-nitrophenyl ␤-D-galactopyranoside; ONP, o-nitrophenol. Defcr-rs gene second exons code for Cys-rich, cationic CRS4C peptides that are not ␣-defensin paralogs. Instead, they are characterized by seven repeats of a CPX triplet motif that is unique to this defensin peptide subfamily and found only in the mouse [32, 34] (see supplemental Fig. S1). Native CRS4C peptides purified from mouse small intestine exist as disulfide-stabilized homodimers and heterodimers that are antibacterial [33]. Details of their expression patterns, post-translational processing, and mechanisms of action remain obscure. MMP-7 can activate proCRS4C-1 in vitro by a mechanism similar to mouse pro-␣-defensin processing, converting inactive pro-CRS4C molecules to bactericidal, membrane-disruptive peptides

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION