Lysine 58 and Histidine 66 at the C-terminal α-Helix of Monocyte Chemoattractant Protein-1 Are Essential for Glycosaminoglycan Binding

Monocytes rolling on the endothelial cell layer interact with monocyte chemoattractant protein-1 (MCP-1) that is tethered to the proteoglycans on the luminal side of the endothelial cells and consequently initiate adhesion of monocytes in the early phase of immune response. The amino acid residues in MCP-1 involved in tethering to the proteoglycans have not been elucidated. MCP-1 showed binding to [3H]heparin with aK D of 1.5 μm. We substituted lysine or histidine residues at the C-terminal end of MCP-1 with alanine residues and tested these mutants for their ability to bind heparin, heparan sulfate, hyaluronic acid, and chondroitin sulfate-C. Substitution of Lys-58 or His-66 drastically reduced glycosaminoglycan binding. Substitution of Lys-56 or deletion of the five amino acid residues at the C terminus, including Lys-75, did not alter the heparin binding ability, suggesting that the other lysine residues at the C terminus are not involved in glycosaminoglycan binding. MCP-1 and its mutants did not bind hyaluronic acid as strongly as the other subunits of the GAGs. Substitution of Lys-58 or His-66 by alanine that prevented glycosaminoglycan binding did not affect Ca2+ influx, receptor binding, or chemotactic activity elicited by the chemokine on monocytic THP-1 cells. Therefore, we conclude that the Lys-58 and His-66 residues in the C-terminal α-helix of MCP-1 are essential for glycosaminoglycan binding and probably for the binding to the endothelial surface proteoglycans. Monocytes rolling on the endothelial cell layer interact with monocyte chemoattractant protein-1 (MCP-1) that is tethered to the proteoglycans on the luminal side of the endothelial cells and consequently initiate adhesion of monocytes in the early phase of immune response. The amino acid residues in MCP-1 involved in tethering to the proteoglycans have not been elucidated. MCP-1 showed binding to [3H]heparin with aK D of 1.5 μm. We substituted lysine or histidine residues at the C-terminal end of MCP-1 with alanine residues and tested these mutants for their ability to bind heparin, heparan sulfate, hyaluronic acid, and chondroitin sulfate-C. Substitution of Lys-58 or His-66 drastically reduced glycosaminoglycan binding. Substitution of Lys-56 or deletion of the five amino acid residues at the C terminus, including Lys-75, did not alter the heparin binding ability, suggesting that the other lysine residues at the C terminus are not involved in glycosaminoglycan binding. MCP-1 and its mutants did not bind hyaluronic acid as strongly as the other subunits of the GAGs. Substitution of Lys-58 or His-66 by alanine that prevented glycosaminoglycan binding did not affect Ca2+ influx, receptor binding, or chemotactic activity elicited by the chemokine on monocytic THP-1 cells. Therefore, we conclude that the Lys-58 and His-66 residues in the C-terminal α-helix of MCP-1 are essential for glycosaminoglycan binding and probably for the binding to the endothelial surface proteoglycans. monocyte chemoattractant protein macrophage inflammatory protein glycosaminoglycan phosphate-buffered saline polyacrylamide gel electrophoresis. Chemokines are small chemotactic proteins, which mediate directional migration of leukocytes from the blood to the site of injury. Usually the chemokines, produced at the site of injury, cause leukocyte migration (1Baggiolini M. Nature. 1998; 392: 565-568Crossref PubMed Scopus (2385) Google Scholar, 2Aukrust P. Ueland T. Muller F. Andreassen A.K. Nordoy I. Aas H. Kjekshus 1998; Scopus Google Scholar, F. PubMed Scopus Google Scholar, T. T. PubMed Scopus Google Scholar, PubMed Google chemoattractant protein-1 of the chemokine of monocytes and in to and in inflammatory 1998; PubMed Google Scholar, P. 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The amino at the C-terminal α-helix of MCP-1 that probably in the binding of MCP-1 with GAGs. The of Lys-58 and His-66 with alanine the heparin binding ability of of lysine with alanine did not affect binding. deletion of five amino including from the C-terminal α-helix of MCP-1 did not affect the heparin binding The D of heparin binding to MCP-1 and its mutants at 1.5 μm. the the MCP-1 or to chondroitin and heparan strongly to hyaluronic the chemokine and is on The of on the binding of to have the involved in binding. chemokine the C terminus is and in the of that have been to involved in of heparin binding by of to residues from the C-terminal α-helix of M. PubMed Scopus Google of chemokine of of at and at with and in the α-helix heparin binding Google in of the with the the of the lysine residues in binding the that of and with heparin binding that by of and with M. 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I. PubMed Scopus Google from the of of chemokine is small in of the binding The of the the of the chemokine and the binding to the the F. I. 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Usually the chemokines, produced at the site of injury, cause leukocyte migration (1Baggiolini M. Nature. 1998; 392: 565-568Crossref PubMed Scopus (2385) Google Scholar, 2Aukrust P. Ueland T. Muller F. Andreassen A.K. Nordoy I. Aas H. Kjekshus 1998; Scopus Google Scholar, F. PubMed Scopus Google Scholar, T. T. PubMed Scopus Google Scholar, PubMed Google chemoattractant protein-1 of the chemokine of monocytes and in to and in inflammatory 1998; PubMed Google Scholar, P. PubMed Scopus Google Scholar, PubMed Scopus Google of Google Scholar, M. T. T. H. PubMed Scopus Google Scholar, T. H. PubMed Scopus Google Scholar, T. Google Scholar, F. P. PubMed Scopus Google PubMed Scopus Google 1998; PubMed Scopus Google and M. 1998; PubMed Google The of MCP-1 of and by α-helix at the C-terminal end PubMed Scopus Google cells and MCP-1 in to and M. Google Scholar, T. H. H. PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, M. Google Scholar, PubMed Scopus Google migration to the site of is by chemokine T. T. PubMed Scopus Google Scholar, PubMed Google The receptor of on the monocytes rolling on the endothelial MCP-1 tethered to on the luminal surface of the endothelial cells Scopus Google Scholar, H. Nature. PubMed Scopus Google Scholar, F. I. PubMed Scopus Google receptor binding the monocyte adhesion to the endothelial by and migration of the monocytes the MCP-1 to the of of MCP-1 at the site of or are that are by the of glycosaminoglycan side at The are heparin, chondroitin sulfate, heparan sulfate, sulfate, and hyaluronic acid by the PubMed Scopus Google side are to MCP-1 from endothelial cells. residues in the C-terminal α-helix of of chemokine to for heparin binding Google C-terminal of the α-helix of heparin binding M. PubMed Scopus Google acid residues involved in the heparin binding of the of chemokine are not that residues involved in binding heparin as as the receptor PubMed Scopus Google that residues for binding with receptor PubMed Scopus Google involved in the binding of MCP-1 have not been elucidated. we that of Lys-58 and His-66 with alanine in the C-terminal α-helix of not the other residues at the C-terminal binding receptor binding, Ca2+ influx, or chemotactic residues in the C terminus, from the to for binding the of of on binding for deletion of five amino including and of residues and at the C terminus of MCP-1 to for the of on binding. of the mutants in the cell showed that and in the not of the showed at with MCP-1 showed that with the protein in not of MCP-1 with [3H]heparin We the by and that heparin to MCP-1 with D of 1.5 and mutants of MCP-1 showed drastically reduced binding with that of the MCP-1 protein and the other and mutants [3H]heparin as as the MCP-1 with D and and binding and Ca2+ by MCP-1 and its binding of Ca2+ of binding and are of heparin, chondroitin heparan sulfate, and hyaluronic acid, not did not bind the to The for at in the binding of the MCP-1 tested with as heparan sulfate, chondroitin and hyaluronic on the in the [3H]heparin binding chemokine for heparan sulfate, and heparin with to bind The D for heparan sulfate, chondroitin and heparin to other of the D for the in I. of MCP-1 for hyaluronic acid with the for heparin, heparan sulfate, and chondroitin heparin and other for binding MCP-1 and its of MCP-1 or with [3H]heparin or with heparin chondroitin heparan or hyaluronic acid and [3H]heparin to the protein as and and mutants to [3H]heparin and The D for and mutants μm. from and D of and that the or deletion of residues did not alter the binding of the to binding of or mutants reduced the heparin, or heparan with [3H]heparin with for binding the and to the binding of the for hyaluronic acid with the for the of the GAGs. The for mutants and not tested to their heparin binding of histidine in binding is as and are the involved residues in other for the of histidine in binding of of heparin binding in binding with showed the of with of strongly the of histidine of MCP-1 in heparin binding. D for heparin binding with from at to at and to at of heparin binding to are the as in that of the of amino the of the chemokine and affect its binding to and we tested the in the C-terminal the receptor binding, of Ca2+ influx, and the monocytic cell which receptor of binding tested in cells in of of The mutants the receptor with the D as the the in the C terminus did not affect receptor of with of of the MCP-1 and mutants with THP-1 cells and of the the of the by of by the mutants of MCP-1 and the to that at the that the amino acid at residues and did not affect the as as Ca2+ of by MCP-1 and its mutants in THP-1 cells. by of MCP-1 or its mutants in THP-1 cells with of by MCP-1 and which with of THP-1 cells and the The is as of the elicited by the cells with of monocytes tested at of MCP-1 and and The mutants showed chemotactic activity to that with of chemotactic activity at of the chemokine and at and of MCP-1 and its mutants in THP-1 cells by MCP-1 and its in THP-1 cells with MCP-1 and the by we the as the of the migration to MCP-1 at in The the from the residues in the C terminus, from the to for binding the of of on binding for deletion of five amino including and of residues and at the C terminus of MCP-1 to for the of on binding. of the mutants in the cell showed that and in the not of the showed at with MCP-1 showed that with the protein in not of MCP-1 with [3H]heparin We the by and that heparin to MCP-1 with D of 1.5 and mutants of MCP-1 showed drastically reduced binding with that of the MCP-1 protein and the other and mutants [3H]heparin as as the MCP-1 with D and and and are of heparin, chondroitin heparan sulfate, and hyaluronic acid, not did not bind the to The for at the binding of the MCP-1 tested with as heparan sulfate, chondroitin and hyaluronic on the in the [3H]heparin binding chemokine for heparan sulfate, and heparin with to bind The D for heparan sulfate, chondroitin and heparin to other of the D for the in I. of MCP-1 for hyaluronic acid with the for heparin, heparan sulfate, and chondroitin and mutants to [3H]heparin and The D for and mutants μm. from and D of and that the or deletion of residues did not alter the binding of the to binding of or mutants reduced the heparin, or heparan with [3H]heparin with for binding the and to the binding of the for hyaluronic acid with the for the of the GAGs. The for mutants and not tested to their heparin binding The of histidine in binding is as and are the involved residues in other for the of histidine in binding of of heparin binding in binding with showed the of with of strongly the of histidine of MCP-1 in heparin binding. D for heparin binding with from at to at and to at of amino the of the chemokine and affect its binding to and we tested the in the C-terminal the receptor binding, of Ca2+ influx, and the monocytic cell which receptor of binding tested in cells in of of The mutants the receptor with the D as the the in the C terminus did not affect receptor binding. of with of of the MCP-1 and mutants with THP-1 cells and of the the of the by of by the mutants of MCP-1 and the to that at the that the amino acid at residues and did not affect the as as Ca2+ of monocytes tested at of MCP-1 and and The mutants showed chemotactic activity to that with of chemotactic activity at of the chemokine and at and of MCP-1 and its mutants proteoglycans on the luminal surface of endothelial cells are to MCP-1 to to monocytes by the blood the residues of MCP-1 involved in binding, we tested the of of residues at the C terminus of MCP-1 with alanine on binding. The amino at the C-terminal α-helix of MCP-1 that probably in the binding of MCP-1 with GAGs. The of Lys-58 and His-66 with alanine the heparin binding ability of of lysine with alanine did not affect binding. deletion of five amino including from the C-terminal α-helix of MCP-1 did not affect the heparin binding The D of heparin binding to MCP-1 and its mutants at 1.5 μm. the the MCP-1 or to chondroitin and heparan strongly to hyaluronic the chemokine and is on The of on the binding of to have the involved in binding. chemokine the C terminus is and in the of that have been to involved in of heparin binding by of to residues from the C-terminal α-helix of M. PubMed Scopus Google of chemokine of of at and at with and in the α-helix heparin binding Google in of the with the the of the lysine residues in binding the that of and with heparin binding that by of and with M. PubMed Scopus Google of the chemokine as on cell and and the residues at and are to involved in heparin binding. that these residues for to bind heparin PubMed Scopus Google Scholar, PubMed Scopus Google on the of on chemokine binding to heparin, the to the for heparin binding PubMed Google been M. PubMed Scopus Google of other M. M. PubMed Scopus Google Scholar, H. PubMed Google Scholar, PubMed Scopus Google strongly that of amino in the probably the heparin binding to the to the binding of and the on the amino acid residues of MCP-1 involved in binding, is that the chemokine involved in binding. the for Lys-58 and His-66 in the C-terminal α-helix of MCP-1 heparin binding. these residues are in to in heparin binding. The of the and that are to involved in binding, strongly by the of binding that showed for with of from the of histidine in heparin binding, D from at to at Substitution at or deletion of residues from the C terminus did not affect heparin binding, suggesting that the residues at and in binding. to the these residues not the α-helix PubMed Scopus Google the binding site of the for and the receptor is not of amino acid residues to involved in binding to receptor binding PubMed Scopus Google in the amino acid residues that binding site not to for receptor binding and by in PubMed Scopus Google in which the amino at and with amino did bind the and showed chemotactic activity P. in Substitution of the amino acid residues in the binding affect the receptor binding and not have the of in the for showed PubMed Scopus Google Scholar, P. in that the binding not with the receptor binding. the of that in the α-helix that drastically heparin binding on receptor binding, and receptor binding of MCP-1 from binding and strongly that the α-helix is involved in the MCP-1 to the binding cause of F. I. PubMed Scopus Google from the of of chemokine is small in of the binding The of the the of the chemokine and the binding to the the F. I. PubMed Scopus Google that the residues in the C-terminal α-helix are involved in the tethering of the chemokine is with for the MCP-1 to the the C-terminal α-helix is to the chemokine to the luminal side of the endothelial cells of the blood the the amino acid residues and with the proteoglycans. the and the of for binding to the receptor on the monocyte rolling on the endothelial on the been that the residues and at the and and at the of MCP-1 interact with its receptor PubMed Scopus Google Scholar, PubMed Google Scholar, PubMed Google Lys-58 and His-66 residues at the C-terminal α-helix of MCP-1 bind proteoglycans of endothelial layer with the receptor binding. the of of the receptor with the of the chemokine that the chemokine of the as by that the of in receptor PubMed Google Scholar, PubMed Google and by the binding of to the protein receptor Scopus Google Scholar, PubMed Scopus Google The of the chemokine interact with the and the The proteoglycans on the luminal surface of endothelial cells are to MCP-1 to to monocytes by the blood the residues of MCP-1 involved in binding, we tested the of of residues at the C terminus of MCP-1 with alanine on binding. The amino at the C-terminal α-helix of MCP-1 that probably in the binding of MCP-1 with GAGs. The of Lys-58 and His-66 with alanine the heparin binding ability of of lysine with alanine did not affect binding. deletion of five amino including from the C-terminal α-helix of MCP-1 did not affect the heparin binding The D of heparin binding to MCP-1 and its mutants at 1.5 μm. the the MCP-1 or to chondroitin and heparan strongly to hyaluronic the chemokine and is on The of on the binding of to have the involved in binding. chemokine the C terminus is and in the of that have been to involved in of heparin binding by of to residues from the C-terminal α-helix of M. PubMed Scopus Google of chemokine of of at and at with and in the α-helix heparin binding Google in of the with the the of the lysine residues in binding the that of and with heparin binding that by of and with M. PubMed Scopus Google of the chemokine as on cell and and the residues at and are to involved in heparin binding. that these residues for to bind heparin PubMed Scopus Google Scholar, PubMed Scopus Google on the of on chemokine binding to heparin, the to the for heparin binding PubMed Google been M. PubMed Scopus Google of other M. M. PubMed Scopus Google Scholar, H. PubMed Google Scholar, PubMed Scopus Google strongly that of amino in the probably the heparin binding to the to the binding of and the on the amino acid residues of MCP-1 involved in binding, is that the chemokine involved in binding. the for Lys-58 and His-66 in the C-terminal α-helix of MCP-1 heparin binding. these residues are in to in heparin binding. The of the and that are to involved in binding, strongly by the of binding that showed for with of from the of histidine in heparin binding, D from at to at Substitution at or deletion of residues from the C terminus did not affect heparin binding, suggesting that the residues at and in binding. to the these residues not the α-helix PubMed Scopus Google The the binding site of the for and the receptor is not of amino acid residues to involved in binding to receptor binding PubMed Scopus Google in the amino acid residues that binding site not to for receptor binding and by in PubMed Scopus Google in which the amino at and with amino did bind the and showed chemotactic activity P. in Substitution of the amino acid residues in the binding affect the receptor binding and not have the of in the for showed PubMed Scopus Google Scholar, P. in that the binding not with the receptor binding. the of that in the α-helix that drastically heparin binding on receptor binding, and receptor binding of MCP-1 from binding and strongly that the α-helix is involved in the MCP-1 to the binding cause of F. I. PubMed Scopus Google from the of of chemokine is small in of the binding The of the the of the chemokine and the binding to the the F. I. PubMed Scopus Google The that the residues in the C-terminal α-helix are involved in the tethering of the chemokine is with for the MCP-1 to the the C-terminal α-helix is to the chemokine to the luminal side of the endothelial cells of the blood the the amino acid residues and with the proteoglycans. the and the of for binding to the receptor on the monocyte rolling on the endothelial on the been that the residues and at the and and at the of MCP-1 interact with its receptor PubMed Scopus Google Scholar, PubMed Google Scholar, PubMed Google Lys-58 and His-66 residues at the C-terminal α-helix of MCP-1 bind proteoglycans of endothelial layer with the receptor binding. the of of the receptor with the of the chemokine that the chemokine of the as by that the of in receptor PubMed Google Scholar, PubMed Google and by the binding of to the protein receptor Scopus Google Scholar, PubMed Scopus Google The of the chemokine interact with the and the We for [3H]heparin and and for