Tracheal antimicrobial peptide, a cysteine-rich peptide from mammalian tracheal mucosa: peptide isolation and cloning of a cDNA

Abstract

Extracts of the bovine tracheal mucosa have an abundant peptide with potent antimicrobial activity. The 38-amino acid peptide, which we have named tracheal antimicrobial peptide (TAP), was isolated by a sequential use of size-exclusion, ion-exchange, and reverse-phase chromatographic fractionations using antimicrobial activity as a functional assay. The yield was approximately 2 micrograms/g of wet mucosa. The complete peptide sequence was determined by a combination of peptide and cDNA analysis. The amino acid sequence of TAP is H-Asn-Pro-Val-Ser-Cys-Val-Arg-Asn-Lys-Gly-Ile-Cys-Val-Pro-Ile-Arg-Cys-Pr o- Gly-Ser-Met-Lys-Gln-Ile-Gly-Thr-Cys-Val-Gly-Arg-Ala-Val-Lys-Cys-Cys-Arg- Lys-Lys - OH. Mass spectral analysis of the isolated peptide was consistent with this sequence and indicated the participation of six cysteine residues in the formation of intramolecular disulfide bonds. The size, basic charge, and presence of three intramolecular disulfide bonds is similar to, but clearly distinct from, the defensins, a well-characterized class of antimicrobial peptides from mammalian circulating phagocytic cells. The putative TAP precursor is predicted to be relatively small (64 amino acids), and the mature peptide resides at the extreme carboxyl terminus and is bracketed by a short putative propeptide region and an inframe stop codon. The mRNA encoding this peptide is more abundant in the respiratory mucosa than in whole lung tissue. The purified peptide had antibacterial activity in vitro against Escherichia coli, Staphylococcus aureus, Klebsiella pneumonia, and Pseudomonas aeruginosa. In addition, the peptide was active against Candida albicans, indicating a broad spectrum of activity. This peptide appears to be, based on structure and activity, a member of a group of cysteine-rich, cationic, antimicrobial peptides found in animals, insects, and plants. The isolation of TAP from the mammalian respiratory mucosa may provide insight into our understanding of host defense of this vital tissue.