Structure and function of the glycosaminoglycan binding site of chemokine macrophage-inflammatory protein-1 beta.
The ability of chemokines to bind to glycosaminoglycans (GAGs) on cell surfaces and in the extracellular matrix is thought to play a crucial role in chemokine function. We investigated the structural basis for chemokine binding to GAGs by using in vitro mutagenesis to identify amino acids of chemokine macrophage-inflammatory protein-1 beta (MIP-1 beta) that contribute to its interaction with the model GAG heparin. Among six basic residues that are organized into a single basic domain in the folded MIP-1 beta monomer, three (R18, K45, and R46) were found to contribute significantly to heparin binding. Of these, R46 was found to play a dominant role, and proved essential for the interaction of MIP-1 beta with both heparin and heparan sulfate in physiological salt. The results of this mutational analysis have implications for the structure of the MIP-1 beta-heparin complex, and a comparison of these results with those obtained by mutational analysis of the MIP-1 alpha-heparin interaction suggests a possible structural difference between the MIP-1 beta-heparin and MIP-1 alpha-heparin complexes. To determine whether GAG binding plays an important role in receptor binding and cellular activation by MIP-1 beta, the activities of wild-type MIP-1 beta and R46-substituted MIP-1 beta were compared in assays of T lymphocyte chemotaxis. The two proteins proved equipotent in this assay, arguing that interaction of MIP-1 beta with GAGs is not intrinsically required for functional interaction of MIP-1 beta with its receptor.
Koopmann
W
W
Department of Immunology, Duke University Medical Center, Durham, NC 27710, USA.
Ediriwickrema
C
C
Krangel
M S
MS
D000409:Alanine / Q000235:genetics; D019943:Amino Acid Substitution / Q000235:genetics; D001120:Arginine / Q000235:genetics; D001665:Binding Sites / Q000235:genetics / Q000276:immunology; D002460:Cell Line; D002474:Cell-Free System / Q000276:immunology; D054407:Chemokine CCL4; D002634:Chemotaxis, Leukocyte; D002846:Chromatography, Affinity; D002847:Chromatography, Agarose; D006025:Glycosaminoglycans / Q000378:metabolism*; D006493:Heparin / Q000378:metabolism; D006801:Humans; D019402:Macrophage Inflammatory Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008407:Mast Cells / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D013329:Structure-Activity Relationship
D054407:Chemokine CCL4; D006025:Glycosaminoglycans; D019402:Macrophage Inflammatory Proteins; D006493:Heparin; D001120:Arginine; D000409:Alanine