Intramolecular inhibition of human defensin HNP-1 by its propiece.
We examined mechanisms that protect host defense cells from their cytotoxic effector molecules. Human neutrophil peptides (HNP) 1-3 are microbicidal and cytotoxic defensins, initially synthesized as 94-amino acid preproHNP(1-94), cotranslationally proteolyzed to proHNP(20-94), then converted by removal of the anionic propiece to mature HNP(65-94)(HNP-1 and -3) and HNP(66-94) (HNP-2). We hypothesized that during synthesis and subcellular sorting the anionic propiece inhibits the cytotoxicity of the cationic defensin. We expressed preproHNP-1 cDNA in recombinant baculovirus-infected insect cells that secreted the normally transient proHNP-1(20-94) into the medium. Cyanogen bromide cleaved proHNP-1(20-94) at the fortuitously located Met64 to yield mature recombinant HNP-1(65-94) and unlinked propiece. Recombinant and native HNP-1 purified from PMN were identical as judged by mass spectrometry, retention time in reverse-phase high performance liquid chromatography, migration on acid-urea polyacrylamide gels, and reaction with a conformation-specific antibody. Recombinant and native HNP-1 had comparable microbicidal activity towards Listeria monocytogenes and were similarly potent in permeabilizing K562 leukemia cells, but proHNP-1(20-94) was virtually inactive in both assays. Addition of unlinked propiece (proHNP-1(20-64) with Met64-->homoserine) inhibited the bactericidal and cell-permeabilizing activity of mature HNP-1 in a dose-dependent manner. Linked, and to a lesser extent unlinked, propiece interfered with the binding of HNP-1 to target cells. The propiece thus acts as an efficient intramolecular inhibitor of defensin HNP-1 cytotoxicity.
Valore
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Will Rogers Institute Pulmonary Research Laboratory, Department of Medicine, UCLA School of Medicine, Los Angeles, California, 90095-1736, USA.
Martin
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Harwig
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SS
Ganz
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T
D000595:Amino Acid Sequence; D000818:Animals; D001770:Blood Bactericidal Activity; D001798:Blood Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D003602:Cytotoxicity, Immunologic; D018076:DNA, Complementary / Q000235:genetics; D023082:Defensins; D006801:Humans; D008089:Listeria monocytogenes / Q000187:drug effects; D008969:Molecular Sequence Data; D009504:Neutrophils / Q000276:immunology / Q000378:metabolism; D017924:Nucleopolyhedroviruses / Q000235:genetics; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D018411:Spodoptera; D014407:Tumor Cells, Cultured; D023084:alpha-Defensins
D001798:Blood Proteins; D018076:DNA, Complementary; D023082:Defensins; D011498:Protein Precursors; D011994:Recombinant Proteins; D023084:alpha-Defensins; C406413:human neutrophil peptide 1; C406416:human neutrophil peptide 2