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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.

SUBMITTER: Diamond G

PROVIDER: S-EPMC51571 | biostudies-other | 1991 May

REPOSITORIES: biostudies-other

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

Diamond G G Zasloff M M Eck H H Brasseur M M Maloy W L WL Bevins C L CL

Proceedings of the National Academy of Sciences of the United States of America 19910501 9

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 ...[more]

PMID: 2023943

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Project description:The health threat caused by multiresistant bacteria has continuously increased and recently peaked with pathogens resistant to all current drugs. This has triggered intense research efforts to develop novel compounds to overcome the resistance mechanisms. Thus, antimicrobial peptides (AMPs) have been intensively studied, especially the family of proline-rich AMPs (PrAMPs) that was successfully tested very recently in murine infection models. PrAMPs enter bacteria and inhibit chaperone DnaK. Here, we studied the toxicity of intracellular PrAMPs in HeLa and SH-SY5Y cells. As PrAMPs cannot enter most mammalian cells, we coupled the PrAMPs with penetratin (residues 43 to 58 in the antennapedia homeodomain) via a C-terminally added cysteine utilizing a thioether bridge. The resulting construct could transport the covalently linked PrAMP into mammalian cells. Penetratin ligation reduced the MIC for Gram-negative Escherichia coli only slightly (1 to 8 ?mol/liter) but increased the activity against the Gram-positive Micrococcus luteus up to 32-fold (MIC ? 1 ?mol/liter), most likely due to more effective penetration through the bacterial membrane. In contrast to native PrAMPs, the penetratin-PrAMP constructs entered the mammalian cells, aligned around the nucleus, and associated with the Golgi apparatus. At higher concentrations, the constructs reduced the cell viability (50% inhibitory concentration [IC(50)] ? 40 ?mol/liter) and changed the morphology of the cells. No toxic effects or morphological changes were observed at concentrations of 10 ?mol/liter or below. Thus, the IC(50) values were around 5 to 40 times higher than the MIC values. In conclusion, PrAMPs are in general not toxic to mammalian cells, as they do not pass through the membrane. When shuttled into mammalian cells, however, PrAMPs are only slightly cross-reactive to mammalian chaperones or other intracellular mammalian proteins, providing a second layer of safety for in vivo applications, even if they can enter some human cells.

Project description:To date the complete sequence of only one mammalian mucin cDNA, MUC1, has been reported, although several mucin proteins have been partially characterized. Here we report the nucleotide sequence of a canine tracheal mucin cDNA containing two potential translation initiation codons, one translation termination codon and a poly(A) tail. A lambda gt11 cDNA library prepared from canine tracheal epithelial cells was screened with polyclonal anti-apo-canine tracheal mucin antibodies with the aim of obtaining the deduced amino acid sequence of the mucin core protein. Antibody-positive clones containing overlapping inserts of various lengths were purified and used for nucleotide sequencing. Based on the sequencing data, synthetic oligonucleotide primers were constructed and both ends (5' and 3') of the cDNA were determined. The complete sequence was 3.7 kb and included an open reading frame with coding capacity for 1118 aa, two translation initiation ATG codons in context with Kozak consensus sequences, one polyadenylylation site, and a poly(A) stretch. The protein was rich in Thr, Pro, Ser, Gly, and Ala and poor in Tyr, Phe, and Trp. Although tandem repeats of amino acids were absent in the deduced canine tracheal mucin sequence, motifs TPTPTP and TTTTPV appeared 13 and 19 times, respectively. The C-terminal region contained a Cys-rich domain (although a few Cys residues were also present in the middle of the protein) as has been reported for bovine submaxillary mucin, porcine submaxillary mucin, rat intestinal mucin, human intestinal mucin, and frog skin mucin. This suggested that a broad group of mucins contain such a Cys-rich domain whose functional significance is yet to be understood. Three potential N-glycosylation sites were present in canine tracheal mucin and the amino acid sequence showed homology with both human tracheal and intestinal mucins. The N-terminal domain showed more flexibility (probably due to a high number of Pro residues in this region) when analyzed by the University of Wisconsin Genetics Computer Group program package to determine the predicted secondary structure. Evaluation of the transcripts using the canine mucin cDNA as a probe indicated a polydisperse message with total RNA.

Dataset Information

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

Publications

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

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