Premium
Determinants of Mouse Alpha‐Defensin Bactericidal Activity
Author(s) -
Weeks Colby Sanford,
Tanabe Hiroki,
Crampton Steve P.,
Cocco Melanie J.,
Ouellette Andre J.
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.4.a649-b
Subject(s) - recombinant dna , proteolysis , peptide , in vitro , defensin , chemistry , biochemistry , protein precursor , cleavage (geology) , bacteria , microbiology and biotechnology , biology , enzyme , gene , genetics , paleontology , fracture (geology)
Mouse Paneth cell cryptdins are expressed as inactive propeptides and are activated upon proteolysis by MMP‐7. MMP‐7 cleavage sites Ser 43 ↓ Ile 44 , Ala 53 ↓ Leu 54 , and Ser 58 ↓ Leu 59 are conserved in the cryptdin family. All three recombinant processing intermediates, proCrp4 (44‐92) , proCrp4 (54‐92) , and proCrp4 (59‐92) , exhibit bactericidal activity, revealing that the activating cleavage step occurs at Ser 43 ↓ Ile 44 . A recombinant proCrp4 molecule with all acidic residues upstream of Ser 43 ↓ Ile 44 mutated to Gly showed activity comparable to Crp4 in in vitro bactericidal assays. The conversion of acidic residues in the prosegment of proCrp4 to Gly shows an intramolecular interaction between acidic residues in the prosegment and cationic residues in the defensin domain is required for peptide inactivity. In addition, an examination of the α‐defensin family reveals a ratio of 9:1 in favor of Arg compared to Lys. To investigate the prevalence of Arg in α‐defensins, a Crp4 variant was produced recombinantly with all the Arg mutated to Lys. (R→K)‐Crp4 was consistently less active than native Crp4 against a panel of bacteria tested in in vitro bactericidal assays. This is more apparent when (R→K)‐Crp4 was assayed against bacteria with less susceptibility to α‐defensins. This evidence supports the conclusion that the selection of Arg in α‐defensins is to maintain optimal activity against pathogens.