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

doi:10.1074/jbc.272.3.1725

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.

Highlights

Recruitment and accumulation of circulating leukocytes at the site of inflammation are mediated in part by a family of small molecular weight proteins called chemokines [1,2,3]
Two receptors (IL-8R1 and IL-8R2) that bind CXC chemokines have been identified and characterized in neutrophils, and they belong to the superfamily of seven transmembrane domain-containing proteins that bind to G-proteins
Neutrophil Activation by Monomeric Chemokines observed in structures of neutrophil-activating peptide-2 (NAP-2) and melanoma growth-stimulatory activity (MGSA), monomeric analogs of NAP-2 and MGSA were synthesized using a similar strategy employed in the synthesis of the monomeric IL-8 [19]

Summary

Three well studied proteins of the CXC chemokine family are

Interleukin-8 (IL-8), melanoma growth-stimulatory activity (MGSA), and neutrophil-activating peptide-2 (NAP-2). We subsequently demonstrated that an analog in which Leu-25 was substituted with N-methylleucine (L25NMe) was monomeric and had similar functional properties as the native IL-8 [19] The structure of this synthetic IL-8 monomer was solved by 1H NMR spectroscopy and was shown to be largely similar to that of the monomeric unit in the NMR and x-ray structures of the native dimer [16]. Taken together, these observations suggested that dimerization of IL-8 is essential neither for structural integrity nor for functional activation. Functional data indicate that the residues at the dimer interface are not directly involved in receptor binding

Neutrophil Activation by Monomeric Chemokines

EXPERIMENTAL PROCEDURES

Experimental conditions

This study

RESULTS AND DISCUSSION