title: Limited and defined truncation at the C terminus enhances receptor binding and degranulation activity of the neutrophil-activating peptide 2 (NAP-2). Comparison of native and recombinant NAP-2 variants.
abstract: We have previously described a C-terminally truncated variant of the chemokine neutrophil-activating peptide 2 (NAP-2) that exhibited higher neutrophil-stimulating capacity than the full-size polypeptide. To investigate the impact of the NAP-2 C terminus on biological activity and receptor binding, we have now purified the novel molecule to homogeneity. Furthermore, we have cloned, expressed in Escherichia coli, and purified full-size recombinant NAP-2 (rNAP-2-(1-70)) and a series of C-terminally deleted variants (rNAP-2-(1-69) to rNAP-2-(1-64)). Biochemical and immunochemical analyses revealed that the natural NAP-2 variant was structurally identical to the rNAP-2-(1-66) isoform. As compared with their respective native and recombinant full-size counterparts, both molecules exhibited approximately 3-4-fold enhanced potency in the induction of neutrophil degranulation as well as 3-fold enhanced binding affinity for specific receptors on these cells. All other variants were considerably less active. The natural occurrence of a NAP-2 variant truncated by exactly four residues at the C terminus suggests that limited and defined proteolysis at this site plays a role in the regulation of the biological function of the chemokine.
authors.affiliation: Department of Immunology and Cell Biology, Forschungsinstitut Borstel, Federal Republic of Germany.
authors.forename: J E
authors.initials: JE
authors.lastname: Ehlert
authors.forename: F
authors.initials: F
authors.lastname: Petersen
authors.forename: M H
authors.initials: MH
authors.lastname: Kubbutat
authors.forename: J
authors.initials: J
authors.lastname: Gerdes
authors.forename: H D
authors.initials: HD
authors.lastname: Flad
authors.forename: E
authors.initials: E
authors.lastname: Brandt
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D001792:Blood Platelets / Q000378:metabolism; D015153:Blotting, Western; D003001:Cloning, Molecular; D003238:Connective Tissue / Q000378:metabolism; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D014644:Genetic Variation; D006801:Humans; D008214:Lymphocytes / Q000378:metabolism*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009504:Neutrophils / Q000187:drug effects / Q000502:physiology*; D010455:Peptides / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology; D012333:RNA, Messenger / Q000097:blood / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D017384:Sequence Deletion; D001620:beta-Thromboglobulin
chemical_list: D017931:DNA Primers; C061615:PPBP protein, human; D010455:Peptides; D012333:RNA, Messenger; D011994:Recombinant Proteins; D001620:beta-Thromboglobulin; C009862:connective tissue-activating peptide