A competitive binding study of chemokine, sulfated receptor, and glycosaminoglycan interactions by nano-electrospray ionization mass spectrometry.
Chemokines are secreted proteins that play roles in inducing chemotaxis, extravasation, and activation of leukocytes associated with inflammatory or homeostatic processes. Tyrosine sulfation of the chemokine receptor has been shown to be important for binding and signaling. We have applied a mass spectrometry method to measure the contribution of this posttranslational modification to binding of its ligand chemokine. Using nano-electrospray time-of-flight mass spectrometry (nano-ESI TOF MS), we determined the association constants of C-C motif chemokine 7 (CCL7) with C-C chemokine receptor type 2 (CCR2), monosulfated CCR2, and disulfated CCR2 peptides to be 1.1×10(4)M(-1), 3.9×10(4)M(-1), and 4.0×10(5)M(-1), respectively. To our knowledge, this is the first reported association constant measurement between a protein and sulfated peptide using MS. Furthermore, nano-ESI MS was used to characterize noncovalent binding interactions among CCL7, Arixtra (a pentasaccharide glycosaminoglycan [GAG] analog), and disulfated CCR2 peptide. A lack of observable ternary complex formation prompted investigation of competitive binding. Results of this study suggest that CCR2 competes partially with GAG for CCL7 binding and that disulfated CCR2 peptide has a higher binding affinity than Arixtra, which correlates with data from association constant measurements for CCL7-disulfated CCR2 and CCL7-Arixtra.
Jen
Connie H
CH
Department of Molecular and Cellular Biology, University of California, Davis, Davis, CA 95616, USA.
Leary
Julie A
JA
D001667:Binding, Competitive; D054410:Chemokine CCL7 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000077425:Fondaparinux; D006801:Humans; D007700:Kinetics; D036103:Nanotechnology; D011134:Polysaccharides / Q000737:chemistry*; D011485:Protein Binding; D054390:Receptors, CCR2 / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D013431:Sulfates / Q000737:chemistry
D054410:Chemokine CCL7; D011134:Polysaccharides; D054390:Receptors, CCR2; D011994:Recombinant Proteins; D013431:Sulfates; D000077425:Fondaparinux