title: A novel molecular variant of the neutrophil-activating peptide NAP-2 with enhanced biological activity is truncated at the C-terminus: identification by antibodies with defined epitope specificity.
abstract: The human neutrophil-activating peptide 2 (NAP-2) belongs to the so-called beta-thromboglobulin/interleukin 8-family of chemotactic and reparative host defense cytokines. NAP-2 represents one of several N-terminally truncated cleavage products that originate from platelet-derived precursor molecules through proteolytic processing. Among these homologous isoforms that are comprised as beta-thromboglobulin antigen (beta-TG Ag), NAP-2 is recognized as the major component, having the highest potential for the activation of polymorphonuclear neutrophils (PMN). We now present evidence that there exists a second molecular form of NAP-2 with even higher biological activity. This novel isoform was detected in concentrates of culture supernatants from peripheral blood mononuclear cells, and could be separated from authentic NAP-2 by several steps of column chromatography. It had an N-terminus identical to that of NAP-2 but was biochemically different as indicated by its slightly lower molecular weight and a higher isoelectric point. To examine our hypothesis that the polypeptide represented a C-terminally truncated variant of NAP-2, we prepared synthetic peptides that were used for the induction and characterization of two rabbit antibody fractions, directed against different and defined epitopes within the C-terminal alpha-helix of the NAP-2 molecule. Comparison of reactivity patterns of these antibodies in Western blots as well as in a NAP-2 biological assay (PMN degranulation assay) confirmed that the variant NAP-2 was truncated at its C-terminus by at least one and by maximally three residues. The specific activity of the truncated polypeptide was estimated to be about four-fold higher than that of authentic NAP-2, as determined in the PMN degranulation assay. Thus, proteolytic modification at the C-terminus appears to play a role in the regulation of NAP-2-biological activity.
authors.affiliation: Forschungsinstitut Borstel, Department of Immunology and Cell Biology, Germany.
authors.forename: E
authors.initials: E
authors.lastname: Brandt
authors.forename: F
authors.initials: F
authors.lastname: Petersen
authors.forename: H D
authors.initials: HD
authors.lastname: Flad
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000939:Epitopes / Q000032:analysis; D006801:Humans; D008969:Molecular Sequence Data; D010455:Peptides / Q000032:analysis / Q000737:chemistry* / Q000276:immunology / Q000494:pharmacology; D011817:Rabbits; D013329:Structure-Activity Relationship; D001620:beta-Thromboglobulin / Q000032:analysis
chemical_list: D000911:Antibodies, Monoclonal; D000939:Epitopes; C061615:PPBP protein, human; D010455:Peptides; D001620:beta-Thromboglobulin; C009862:connective tissue-activating peptide