title: Neutrophil-activating peptide-2 and melanoma growth-stimulatory activity are functional as monomers for neutrophil activation.
abstract: Neutrophil-activating peptide-2 (NAP-2) and melanoma growth-stimulatory activity (MGSA) are members of the chemokine family of inflammatory proteins. The structures of NAP-2, determined by x-ray crystallography, and MGSA, elucidated by NMR spectroscopy, revealed a tetramer and dimer, respectively. In order to address the relevance of multimeric species to their activities on neutrophils, analogs of NAP-2 and MGSA were synthesized in which the backbone amide proton of Leu-22 in NAP-2, and Val-26 in MGSA, was substituted with the bulky methyl group (NH --> NCH3). These analogs were shown to be monomeric by sedimentation equilibrium ultracentrifugation studies and were similar to the corresponding native protein in assays for neutrophil elastase release and Ca2+ mobilization from IL-8R1 and IL-8R2 transformed cells. Sedimentation equilibrium studies of the native NAP-2 and MGSA were also carried out to address the association behavior. For NAP-2, there was no evidence for the tetramer, but an equilibrium between monomers and dimers and the dissociation constant was calculated to be 50-100 microM. Similarly, MGSA showed a monomer-dimer equilibrium with a Kd of approximately 5 microM. The data from the monomeric analogs and also the calculation of dissociation constants indicate that NAP-2 and MGSA have a tendency to associate above the concentrations required for maximal activity or for receptor activation, but at functional concentrations they are predominantly monomers.
authors.affiliation: Protein Engineering Network of Centres of Excellence (PENCE) and the Department of Biochemistry, University of Alberta, Edmonton, Alberta T6G 2S2, Canada. kkr@rigel.biochem.ualberta.ca
authors.forename: K
authors.initials: K
authors.lastname: Rajarathnam
authors.forename: C M
authors.initials: CM
authors.lastname: Kay
authors.forename: B
authors.initials: B
authors.lastname: Dewald
authors.forename: M
authors.initials: M
authors.lastname: Wolf
authors.forename: M
authors.initials: M
authors.lastname: Baggiolini
authors.forename: I
authors.initials: I
authors.lastname: Clark-Lewis
authors.forename: B D
authors.initials: BD
authors.lastname: Sykes
mesh_terms: D054360:Chemokine CXCL1; D019743:Chemokines, CXC; D002630:Chemotactic Factors / Q000737:chemistry / Q000378:metabolism*; D018360:Crystallography, X-Ray; D006133:Growth Substances / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D036341:Intercellular Signaling Peptides and Proteins; D019169:Jurkat Cells; D009682:Magnetic Resonance Spectroscopy; D018375:Neutrophil Activation; D010455:Peptides / Q000737:chemistry / Q000378:metabolism*; D013329:Structure-Activity Relationship; D001620:beta-Thromboglobulin
chemical_list: C475829:CXCL1 protein, human; D054360:Chemokine CXCL1; D019743:Chemokines, CXC; D002630:Chemotactic Factors; D006133:Growth Substances; D036341:Intercellular Signaling Peptides and Proteins; C061615:PPBP protein, human; D010455:Peptides; D001620:beta-Thromboglobulin; C009862:connective tissue-activating peptide