Mouse Paneth cell defensins: primary structures and antibacterial activities of numerous cryptdin isoforms

Abstract

Cryptdins are antimicrobial peptides of the defensin family that are produced by intestinal Paneth cells. mRNAs encoding 17 cryptdin isoforms have been characterized from a cDNA library generated from a single jejunal crypt. Six cryptdin cDNAs correspond to known peptides, and the remainder predict 11 novel Paneth cell defensins. Most cryptdin cDNAs have > or = 93% nucleotide sequence identity overall, except for cryptdin 4 and 5 cDNAs, whose respective mature peptide-encoding regions are only 74 and 78% identical to that of cryptdin 1. Cryptdin cDNAs differ at a small number of nucleotide positions: frequent substitutions were found in codons 38 and 52 of the propiece and in codons 68, 73, 76, 87, and 89 of the deduced peptides; cDNA clones with changes in codons 74, 83, and 88 were found, but there were fewer of these. The antimicrobial activities of cryptdins 1 to 6 were tested against Escherichia coli ML35 in two assays. In an agar diffusion assay, the potencies of cryptdins 1 to 3, 5, and 6 were approximately equivalent to that of rabbit neutrophil defensin NP-1 but cryptdin 4 was 30 times more active than NP-1. In a bactericidal assay system, cryptdins 1 and 3 to 6 were equally active at 10 micrograms/ml but cryptdin 2 and rabbit NP-1 were not active at this concentration. Since cryptdins 2 and 3 differ only at residue 10 (Thr and Lys, respectively), this amino acid appears to function in bactericidal interaction with E. coli. The demonstration that Paneth cells express a diverse population of microbicidal defensins further implicates cryptdins in restricting colonization or invasion of small intestinal epithelium by bacteria.