Neutrophil-activating Peptide-2 and Melanoma Growth-stimulatory Activity Are Functional as Monomers for Neutrophil Activation

Krishna Rajarathnam, Cyril M. Kay, Beatrice Dewald, Marlene Wolf, Marco Baggiolini, Ian Clark-Lewis, Brian D. Sykes
1997 Journal of Biological Chemistry  
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
more » ... in MGSA, was substituted with the bulky methyl group (NH 3 NCH 3 ). 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 Ca 2؉ 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 M. Similarly, MGSA showed a monomer-dimer equilibrium with a K d of ϳ5 M. 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. 2 The numbering corresponds to the sequence in IL-8. The corresponding positions are 2, 3 and 4 in NAP-2 and 6, 7 and 8 in MGSA.
doi:10.1074/jbc.272.3.1725 pmid:8999852 fatcat:jizhajvlhfhujezsnxl3bbekz4