Premium
An analytical mass spectrometry method of determining binding constants between chemokine, posttranslationally modified chemokine receptor N‐terminal peptides, and glycosaminoglycan
Author(s) -
Jen Connie H.,
Leary Julie A.
Publication year - 2010
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.24.1_supplement.lb156
Subject(s) - ccl7 , chemistry , chemokine receptor , ccr2 , sulfation , ccr1 , biochemistry , receptor , chemokine
Chemokines play roles in inducing chemotaxis, extravasation, and activation of leukocytes. Tyrosine sulfation of the chemokine receptor is crucial for binding to its ligands and signaling. A robust analytical method for measuring the contribution of this posttranslational modification to the receptor to binding of its ligand is lacking. Using Nano‐electrospray time‐of‐flight mass spectrometry, we determined the association constant of chemokine, CCL7 to variably sulfated CCR2 peptides. Association constants between CCL7‐CCR2, CCL7‐monosulfated CCR2, and CCL7‐ disulfated CCR2 were determined to be 1.1 x 10 4 M −1 , 3.9 x 10 4 M −1 , and 4.0 x 10 5 M −1 , respectively. Chemokines also bind to glycosaminoglycans (GAGs). Mutation studies suggest a partial overlap between the GAG and receptor binding domains, but it has been debatable whether a ternary complex forms. Using mass spectrometry we did not observe significant ternary complex formation. Thus, competitive binding experiments between Arixtra and disulfated CCR2 were conducted. As the concentration of disulfated CCR2 is increased from 0μM to 10μM two observations are made suggesting that CCR2 competes with GAG for CCL7 binding: 1. more noncovalent complexes between CCL7 and CCR2; 2. Less noncovalent interactions between CCL7 and Arixtra.