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United States Department of Agriculture

Agricultural Research Service

Title: Anionic Antimicrobial Peptide May Originate As a Larger Precursor Protein (Poster Presentation for the 101st Asm Annual Meeting, May 20-24, 2001)

Authors
item HEIDARI, MOHAMMAD
item Brogden, Kim

Submitted to: American Society for Microbiology Annual Meeting
Publication Type: Abstract Only
Publication Acceptance Date: May 20, 2001
Publication Date: N/A

Technical Abstract: Three small antimicrobial, anionic peptides (AP) were originally isolated from ovine pulmonary surfactant and recently detected in human broncho- alveolar lavage (BAL) fluid. However, their origin in BAL fluid and tissues of the respiratory tract is unknown. In this study, we used affinity-purified mouse monoclonal antibodies to synthetic H-DDDDDDD-OH to detect AP containing molecules in respiratory tissues. In Western blots, monoclonal antibody 1G9-1C2 (5.0 ug/ml) detected 4 bands in solubilized turbinate and tracheal epithelial cells (53.7, 31.2, 28.0, and 25.7 kDa) and 5 bands in lung homogenates (53.5, 37.1, 32.2, 28.0, 25.7 kDa). Only a single band was seen in solubilized liver and small intestine homogenates, and no bands were seen in blots containing BAL fluid, albumin, kidney or spleen homogenates. As a first step in identifying candidate precursor AP gene(s), bands were excised and the N terminus was sequenced. All bands from ovine pulmonary tissues contained an N-terminus of VLTAAEKANV; and all bands from human pulmonary tissue contained an N- terminus of VLSTAEKANV, suggesting that these proteins are related and highly conserved between human and sheep. Degenerate oligonucleotides were then prepared and used as hybridization probes to screen both sheep and human genomic libraries. Large nucleotide fragments containing the N terminus sequences and the C terminus anionic peptide sequences were identified suggesting that AP may be part of a larger predicted protein. AP may originate as part of an intracellular precursor protein with multi- step processing, leading to the release of the heptapeptide into mucosal secretions. There it may interact with other pulmonary innate defenses to prevent microbial infection. (Research supported by Cystic Fibrosis Found.)

Last Modified: 9/10/2014
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