The BBXB Motif of RANTES Is the Principal Site for Heparin Binding and Controls Receptor Selectivity

Abstract

The chemokine RANTES (regulated on activation normal T cell expressed and secreted; CCL5) binds selectively to glycosaminoglycans (GAGs) such as heparin, chondroitin sulfate, and dermatan sulfate. The primary sequence of RANTES contains two clusters of basic residues, 44RKNR47 and55KKWVR59. The first is a BBXB motif common in heparin-binding proteins, and the second is located in the loop directly preceding the C-terminal helix. We have mutated these residues to alanine, both as point mutations as well as triple mutations of the 40s and 50s clusters. Using a binding assay to heparin beads with radiolabeled proteins, the44AANA47 mutant demonstrated an 80% reduction in its capacity to bind heparin, whereas the55AAWVA59 mutant retained full binding capacity. Mutation of the 44RKNR47 site reduced the selectivity of RANTES binding to different GAGs. The mutants were tested for their integrity by receptor binding assays on CCR1 and CCR5 as well as their ability to induce chemotaxis in vitro. In all assays the single point mutations and the triple 50s cluster mutation caused no significant difference in activity compared with the wild type sequence. However, the triple 40s mutant showed a 80-fold reduction in affinity for CCR1, despite normal binding to CCR5. It was only able to induce monocyte chemotaxis at micromolar concentrations. The triple 40s mutant was also able to inhibit HIV-1 infectivity, but consistent with its abrogated GAG binding capacity, it no longer induced enhanced infectivity at high concentrations. The chemokine RANTES (regulated on activation normal T cell expressed and secreted; CCL5) binds selectively to glycosaminoglycans (GAGs) such as heparin, chondroitin sulfate, and dermatan sulfate. The primary sequence of RANTES contains two clusters of basic residues, 44RKNR47 and55KKWVR59. The first is a BBXB motif common in heparin-binding proteins, and the second is located in the loop directly preceding the C-terminal helix. We have mutated these residues to alanine, both as point mutations as well as triple mutations of the 40s and 50s clusters. Using a binding assay to heparin beads with radiolabeled proteins, the44AANA47 mutant demonstrated an 80% reduction in its capacity to bind heparin, whereas the55AAWVA59 mutant retained full binding capacity. Mutation of the 44RKNR47 site reduced the selectivity of RANTES binding to different GAGs. The mutants were tested for their integrity by receptor binding assays on CCR1 and CCR5 as well as their ability to induce chemotaxis in vitro. In all assays the single point mutations and the triple 50s cluster mutation caused no significant difference in activity compared with the wild type sequence. However, the triple 40s mutant showed a 80-fold reduction in affinity for CCR1, despite normal binding to CCR5. It was only able to induce monocyte chemotaxis at micromolar concentrations. The triple 40s mutant was also able to inhibit HIV-1 infectivity, but consistent with its abrogated GAG binding capacity, it no longer induced enhanced infectivity at high concentrations. glycosaminoglycan interleukin regulated on activation normal T cell expressed and secreted Chinese hamster ovary high pressure liquid chromatography heteronuclear single quantum coherence human immunodeficiency virus, type 1 wild type Chemokines selectively recruit and activate leukocyte populations, both during routine immunosurveillance and also during inflammation. The migration of cells is believed to require immobilization of the chemokines on proteoglycans in the extracellular matrix and on the endothelial cell (2Rot A. Immunol. Today. 1992; 13: 291-294Abstract Full Text PDF PubMed Scopus (410) Google Scholar, 3Springer T.A. Cell. 1994; 76: 301-314Abstract Full Text PDF PubMed Scopus (6374) Google Scholar). The glycosaminoglycan (GAG)1 moiety of the proteoglycan shows a wide range of structures, with heparin, heparan sulfate, chondroitin sulfate, and dermatan sulfate being important members of the family. Changes in the type of intensity of proteoglycan expression are known to happen in a wide variety of inflammatory diseases. It has been suggested that these changes in glycosaminoglycan expression play a role in the localization of the inflammatory response, by localizing inflammatory cytokines and chemokines (4Roberts C.R. Wight T.N. Hascall V.C. Crystal R.G. West J.B. Weibel E. Barnes P.J. The Lung. Lippincott-Raven, Philadelphia2000Google Scholar, 5Jackson D.G. Biochem. Soc. Trans. 1997; 25: 220-224Crossref PubMed Scopus (21) Google Scholar, 6Wasty F. Alavi M.Z. Moore S. Diabetologia. 1993; 36: 316-322Crossref PubMed Scopus (109) Google Scholar, 7Marquezini M.V. Strunz C.M. Dallan L.A. Toledo O.M. Cardiology. 1995; 86: 143-146Crossref PubMed Scopus (19) Google Scholar).Chemokines are a large family of small proteins with a remarkably highly conserved three-dimensional monomeric structure (Ref. 8Wells T.C. Proudfoot A.I. Inflamm. Res. 1999; 48: 353-362Crossref PubMed Scopus (37) Google Scholar and Fig.1). This conserved structure is mediated by the formation of two disulfide bridges imposed by the conserved 4-cysteine motif rather than identity at the level of primary sequence, which can be as low as 20%. The majority of chemokines (MIP-1α and -1β being the exceptions) 2The following chemokines with the new standardized nomenclature (1Zlotnik A. Yoshie O. Immunity. 2000; 12: 121-127Abstract Full Text Full Text PDF PubMed Scopus (3248) Google Scholar) are cited: IL-8, CXCL8; SDF-1, CXCL12; MIP-1α, CCL3; MIP-1β, CCL4; RANTES, CCL5; and MCP-1, CCL2. 2The following chemokines with the new standardized nomenclature (1Zlotnik A. Yoshie O. Immunity. 2000; 12: 121-127Abstract Full Text Full Text PDF PubMed Scopus (3248) Google Scholar) are cited: IL-8, CXCL8; SDF-1, CXCL12; MIP-1α, CCL3; MIP-1β, CCL4; RANTES, CCL5; and MCP-1, CCL2. are highly basic proteins with an isoelectric point around pH 9.0. All chemokines are able to bind heparin, although with varying affinities. We have previously that selectivity for the for chemokines IL-8, RANTES, MIP-1α, and F. Proudfoot T.N. 1999; PubMed Scopus Google Scholar). RANTES was to have the range of selectivity with an affinity of for heparin than for chondroitin sulfate. its isoelectric is able to bind to that the is a of basic chemokines with The of chemokines with glycosaminoglycans the binding of the chemokines to their in F. Proudfoot T.N. 1999; PubMed Scopus Google Scholar). However, the has also been to the activity of chemokines in A. S. A. 1993; PubMed Scopus Google Scholar, PubMed Scopus Google a basic have been to be a common heparin-binding motif for proteins PubMed Google Scholar, PubMed Scopus Google Scholar). of the RANTES sequence showed that it two clusters of basic a BBXB cluster on the 40s loop and cluster of basic residues the C-terminal on the 50s loop The C-terminal has been to be in GAG binding for chemokines such as E. Biochem. 1995; PubMed Scopus Google Proudfoot PubMed Scopus Google and S. Full Text Full Text PDF PubMed Scopus Google Scholar) but BBXB We have mutated the in these as point mutations as well as triple to a role for single but that the triple mutation of the 40s cluster 80% of the heparin binding capacity, whereas mutation of the 50s cluster has no that these basic residues in the 40s loop are also important for CCR1 but for CCR5 an on RANTES for CCR1 and GAG are in the activation and of cells in and routine the migration of it has been suggested (2Rot A. Immunol. Today. 1992; 13: 291-294Abstract Full Text PDF PubMed Scopus (410) Google Scholar) that cells of that are by the of the chemokine with matrix and the expression of glycosaminoglycans is regulated at the site of be of the that is all chemokines with glycosaminoglycans and heparin, is selectivity in F. Proudfoot T.N. 1999; PubMed Scopus Google Scholar, 1994; Full Text Full Text PDF PubMed Scopus Google Scholar). It that RANTES shows the selectivity for the different GAG its for heparin and chondroitin sulfate by of F. Proudfoot T.N. 1999; PubMed Scopus Google Scholar). This a level of selectivity that is the by in binding and activation assays that have in the chemokine being as rather was for that the of chemokines was located in the and in the loop J.B. Full Text PDF PubMed Google Scholar, Full Text PDF PubMed Google Scholar, 1997; 36: PubMed Scopus Google whereas GAG binding was located in the C-terminal of these proteins Proudfoot PubMed Scopus Google Scholar, 1992; PubMed Scopus Google Scholar). However, as the for chemokines are it that is a in GAG binding are in the loop in two and SDF-1, although the are on different of the a The 40s loop is to be the site of GAG for the chemokines and whereas the C-terminal is in and GAG binding a and to two clusters of basic residues on RANTES was in of both of chemokines binding to heparin have been by a variety of to is The and mutants no longer to 1997; Full Text Full Text PDF PubMed Scopus Google and the mutant no longer bind the chromatography was in the of at Immunol. 1999; Google Scholar). However, Proudfoot PubMed Scopus Google A. O. A. A. F. F. 1999; Full Text Full Text PDF PubMed Scopus Google and the mutants of RANTES that have were all able to bind to a but were at a of than the However, a with that only the mutants in the 40s to have for The role of the 40s was by the of and on the binding of a RANTES, although the binding of the to than that for Proudfoot PubMed Scopus Google Scholar). The assay with to heparin that the 40s has a to the capacity of RANTES to bind to glycosaminoglycan whereas the cluster of basic residues in the 50s loop preceding the C-terminal no role in heparin be that the GAG binding are to the BBXB The binding of of the chemokines in have SDF-1, and the the residues in IL-8, and Proudfoot PubMed Scopus Google as well in MCP-1, and S. Full Text Full Text PDF PubMed Scopus Google are has been that cell GAG expression is for the activity of chemokines in although a role in chemokine S. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). of the GAG binding has no on receptor binding and activation in the of IL-8, SDF-1, and has a as has been for binding to CCR1 S. A. PubMed Scopus Google Scholar). We have both for the mutation of has no significant on its binding to and activation of CCR5 but its with This is by the of of CCR5 activation by the mutation of in Immunol. 1999; Google whereas mutations in the of CCR1 binding but have been for CCR5. of the of the extracellular of these two an these are CCR1 has and residues, only which basic and and which whereas CCR5 has and basic and and it that the of CCR1 is to be whereas in CCR5 it be This that of the of CCR1 play a role in the receptor but of CCR5 and its that are in This is by the important in the of RANTES was the for CCR1 whereas for CCR5 binding the residues in RANTES are and 1997; 36: PubMed Scopus Google Scholar) and in were P.J. 2000; PubMed Scopus Google Scholar). However, in these the role of the residues in the 40s loop in to receptor binding was The of the in affinity for CCR1 by the RANTES mutant is by its in ability to induce chemotaxis of which CCR1 as the RANTES receptor F. S. T.N. 1999; Full Text Full Text PDF PubMed Scopus Google was to that the triple 40s mutant that to the heparin beads retained the affinity for heparin as for the This that although is the GAG binding be residues that also can be of the three-dimensional is to be and play a role in binding We are We have previously that RANTES a significant of selectivity for different GAG F. Proudfoot T.N. 1999; PubMed Scopus Google Scholar). the selectivity previously for the GAG by RANTES to be mediated to a by the BBXB motif on the 40s of the in HIV-1 infectivity has been by the that at high RANTES A. E. Proudfoot Moore 1999; PubMed Google Scholar, Proudfoot Moore A. 1999; PubMed Google Scholar). have been to the of RANTES to on heparin F. Proudfoot T.N. 1999; PubMed Scopus Google Scholar, A. E. Proudfoot Moore 1999; PubMed Google which that the with an important RANTES mutants that are able to A. E. Proudfoot Moore 1999; PubMed Google Scholar). that the 80% reduction of the ability of the RANTES to bind to heparin the enhanced However, the ability of RANTES to inhibit HIV-1 infectivity on its ability to bind to the with its The triple 40s mutant CCR5 binding activity and is able to inhibit HIV-1 infectivity of HIV-1 has the heparin binding of RANTES but has that to be which are the residues that are for the heparin binding the residues in that play a role in GAG binding in CCR1 and the of is to the that it with the the of RANTES with activation a that such mutants to the important of the of GAG binding to in which has been for been and the role of GAG binding in inflammation. Chemokines selectively recruit and activate leukocyte populations, both during routine immunosurveillance and also during inflammation. The migration of cells is believed to require immobilization of the chemokines on proteoglycans in the extracellular matrix and on the endothelial cell (2Rot A. Immunol. Today. 1992; 13: 291-294Abstract Full Text PDF PubMed Scopus (410) Google Scholar, 3Springer T.A. Cell. 1994; 76: 301-314Abstract Full Text PDF PubMed Scopus (6374) Google Scholar). The glycosaminoglycan (GAG)1 moiety of the proteoglycan shows a wide range of structures, with heparin, heparan sulfate, chondroitin sulfate, and dermatan sulfate being important members of the family. Changes in the type of intensity of proteoglycan expression are known to happen in a wide variety of inflammatory diseases. It has been suggested that these changes in glycosaminoglycan expression play a role in the localization of the inflammatory response, by localizing inflammatory cytokines and chemokines (4Roberts C.R. Wight T.N. Hascall V.C. Crystal R.G. West J.B. Weibel E. Barnes P.J. The Lung. Lippincott-Raven, Philadelphia2000Google Scholar, 5Jackson D.G. Biochem. Soc. Trans. 1997; 25: 220-224Crossref PubMed Scopus (21) Google Scholar, 6Wasty F. Alavi M.Z. Moore S. Diabetologia. 1993; 36: 316-322Crossref PubMed Scopus (109) Google Scholar, 7Marquezini M.V. Strunz C.M. Dallan L.A. Toledo O.M. Cardiology. 1995; 86: 143-146Crossref PubMed Scopus (19) Google Scholar). Chemokines are a large family of small proteins with a remarkably highly conserved three-dimensional monomeric structure (Ref. 8Wells T.C. Proudfoot A.I. Inflamm. Res. 1999; 48: 353-362Crossref PubMed Scopus (37) Google Scholar and Fig.1). This conserved structure is mediated by the formation of two disulfide bridges imposed by the conserved 4-cysteine motif rather than identity at the level of primary sequence, which can be as low as 20%. The majority of chemokines (MIP-1α and -1β being the exceptions) 2The following chemokines with the new standardized nomenclature (1Zlotnik A. Yoshie O. Immunity. 2000; 12: 121-127Abstract Full Text Full Text PDF PubMed Scopus (3248) Google Scholar) are cited: IL-8, CXCL8; SDF-1, CXCL12; MIP-1α, CCL3; MIP-1β, CCL4; RANTES, CCL5; and MCP-1, CCL2. 2The following chemokines with the new standardized nomenclature (1Zlotnik A. Yoshie O. Immunity. 2000; 12: 121-127Abstract Full Text Full Text PDF PubMed Scopus (3248) Google Scholar) are cited: IL-8, CXCL8; SDF-1, CXCL12; MIP-1α, CCL3; MIP-1β, CCL4; RANTES, CCL5; and MCP-1, CCL2. are highly basic proteins with an isoelectric point around pH 9.0. All chemokines are able to bind heparin, although with varying affinities. We have previously that selectivity for the for chemokines IL-8, RANTES, MIP-1α, and F. Proudfoot T.N. 1999; PubMed Scopus Google Scholar). RANTES was to have the range of selectivity with an affinity of for heparin than for chondroitin sulfate. its isoelectric is able to bind to that the is a of basic chemokines with The of chemokines with glycosaminoglycans the binding of the chemokines to their in F. Proudfoot T.N. 1999; PubMed Scopus Google Scholar). However, the has also been to the activity of chemokines in A. S. A. 1993; PubMed Scopus Google Scholar, PubMed Scopus Google Scholar). The a basic have been to be a common heparin-binding motif for proteins PubMed Google Scholar, PubMed Scopus Google Scholar). of the RANTES sequence showed that it two clusters of basic a BBXB cluster on the 40s loop and cluster of basic residues the C-terminal on the 50s loop The C-terminal has been to be in GAG binding for chemokines such as E. Biochem. 1995; PubMed Scopus Google Proudfoot PubMed Scopus Google and S. Full Text Full Text PDF PubMed Scopus Google Scholar) but BBXB We have mutated the in these as point mutations as well as triple to a role for single but that the triple mutation of the 40s cluster 80% of the heparin binding capacity, whereas mutation of the 50s cluster has no that these basic residues in the 40s loop are also important for CCR1 but for CCR5 an on RANTES for CCR1 and GAG are in the activation and of cells in and routine the migration of it has been suggested (2Rot A. Immunol. Today. 1992; 13: 291-294Abstract Full Text PDF PubMed Scopus (410) Google Scholar) that cells of that are by the of the chemokine with matrix and the expression of glycosaminoglycans is regulated at the site of be of the that is all chemokines with glycosaminoglycans and heparin, is selectivity in F. Proudfoot T.N. 1999; PubMed Scopus Google Scholar, 1994; Full Text Full Text PDF PubMed Scopus Google Scholar). It that RANTES shows the selectivity for the different GAG its for heparin and chondroitin sulfate by of F. Proudfoot T.N. 1999; PubMed Scopus Google Scholar). This a level of selectivity that is the by in binding and activation assays that have in the chemokine being as rather was for that the of chemokines was located in the and in the loop J.B. Full Text PDF PubMed Google Scholar, Full Text PDF PubMed Google Scholar, 1997; 36: PubMed Scopus Google whereas GAG binding was located in the C-terminal of these proteins Proudfoot PubMed Scopus Google Scholar, 1992; PubMed Scopus Google Scholar). However, as the for chemokines are it that is a in GAG binding are in the loop in two and SDF-1, although the are on different of the a The 40s loop is to be the site of GAG for the chemokines and whereas the C-terminal is in and GAG binding a and to two clusters of basic residues on RANTES was in of both of chemokines binding to heparin have been by a variety of to is The and mutants no longer to 1997; Full Text Full Text PDF PubMed Scopus Google and the mutant no longer bind the chromatography was in the of at Immunol. 1999; Google Scholar). However, Proudfoot PubMed Scopus Google A. O. A. A. F. F. 1999; Full Text Full Text PDF PubMed Scopus Google and the mutants of RANTES that have were all able to bind to a but were at a of than the However, a with that only the mutants in the 40s to have for The role of the 40s was by the of and on the binding of a RANTES, although the binding of the to than that for Proudfoot PubMed Scopus Google Scholar). The assay with to heparin that the 40s has a to the capacity of RANTES to bind to glycosaminoglycan whereas the cluster of basic residues in the 50s loop preceding the C-terminal no role in heparin be that the GAG binding are to the BBXB The binding of of the chemokines in have SDF-1, and the the residues in IL-8, and Proudfoot PubMed Scopus Google as well in MCP-1, and S. Full Text Full Text PDF PubMed Scopus Google are has been that cell GAG expression is for the activity of chemokines in although a role in chemokine S. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). of the GAG binding has no on receptor binding and activation in the of IL-8, SDF-1, and has a as has been for binding to CCR1 S. A. PubMed Scopus Google Scholar). We have both for the mutation of has no significant on its binding to and activation of CCR5 but its with This is by the of of CCR5 activation by the mutation of in Immunol. 1999; Google whereas mutations in the of CCR1 binding but have been for CCR5. of the of the extracellular of these two an these are CCR1 has and residues, only which basic and and which whereas CCR5 has and basic and and it that the of CCR1 is to be whereas in CCR5 it be This that of the of CCR1 play a role in the receptor but of CCR5 and its that are in This is by the important in the of RANTES was the for CCR1 whereas for CCR5 binding the residues in RANTES are and 1997; 36: PubMed Scopus Google Scholar) and in were P.J. 2000; PubMed Scopus Google Scholar). However, in these the role of the residues in the 40s loop in to receptor binding was The of the in affinity for CCR1 by the RANTES mutant is by its in ability to induce chemotaxis of which CCR1 as the RANTES receptor F. S. T.N. 1999; Full Text Full Text PDF PubMed Scopus Google was to that the triple 40s mutant that to the heparin beads retained the affinity for heparin as for the This that although is the GAG binding be residues that also can be of the three-dimensional is to be and play a role in binding We are We have previously that RANTES a significant of selectivity for different GAG F. Proudfoot T.N. 1999; PubMed Scopus Google Scholar). the selectivity previously for the GAG by RANTES to be mediated to a by the BBXB motif on the 40s of the in HIV-1 infectivity has been by the that at high RANTES A. E. Proudfoot Moore 1999; PubMed Google Scholar, Proudfoot Moore A. 1999; PubMed Google Scholar). have been to the of RANTES to on heparin F. Proudfoot T.N. 1999; PubMed Scopus Google Scholar, A. E. Proudfoot Moore 1999; PubMed Google which that the with an important RANTES mutants that are able to A. E. Proudfoot Moore 1999; PubMed Google Scholar). that the 80% reduction of the ability of the RANTES to bind to heparin the enhanced However, the ability of RANTES to inhibit HIV-1 infectivity on its ability to bind to the with its The triple 40s mutant CCR5 binding activity and is able to inhibit HIV-1 infectivity of HIV-1 has the heparin binding of RANTES but has that to be which are the residues that are for the heparin binding the residues in that play a role in GAG binding in CCR1 and the of is to the that it with the the of RANTES with activation a that such mutants to the important of the of GAG binding to in which has been for been and the role of GAG binding in inflammation. Chemokines are in the activation and of cells in and routine the migration of it has been suggested (2Rot A. Immunol. Today. 1992; 13: 291-294Abstract Full Text PDF PubMed Scopus (410) Google Scholar) that cells of that are by the of the chemokine with matrix and the expression of glycosaminoglycans is regulated at the site of be of the that is all chemokines with glycosaminoglycans and heparin, is selectivity in F. Proudfoot T.N. 1999; PubMed Scopus Google Scholar, 1994; Full Text Full Text PDF PubMed Scopus Google Scholar). It that RANTES shows the selectivity for the different GAG its for heparin and chondroitin sulfate by of F. Proudfoot T.N. 1999; PubMed Scopus Google Scholar). This a level of selectivity that is the by in binding and activation assays that have in the chemokine being as rather It was for that the of chemokines was located in the and in the loop J.B. Full Text PDF PubMed Google Scholar, Full Text PDF PubMed Google Scholar, 1997; 36: PubMed Scopus Google whereas GAG binding was located in the C-terminal of these proteins Proudfoot PubMed Scopus Google Scholar, 1992; PubMed Scopus Google Scholar). However, as the for chemokines are it that is a in GAG binding are in the loop in two and SDF-1, although the are on different of the a The 40s loop is to be the site of GAG for the chemokines and whereas the C-terminal is in and GAG binding a and to two clusters of basic residues on RANTES was in of both The of chemokines binding to heparin have been by a variety of to is The and mutants no longer to 1997; Full Text Full Text PDF PubMed Scopus Google and the mutant no longer bind the chromatography was in the of at Immunol. 1999; Google Scholar). However, Proudfoot PubMed Scopus Google A. O. A. A. F. F. 1999; Full Text Full Text PDF PubMed Scopus Google and the mutants of RANTES that have were all able to bind to a but were at a of than the However, a with that only the mutants in the 40s to have for The role of the 40s was by the of and on the binding of a RANTES, although the binding of the to than that for Proudfoot PubMed Scopus Google Scholar). The assay with to heparin that the 40s has a to the capacity of RANTES to bind to glycosaminoglycan whereas the cluster of basic residues in the 50s loop preceding the C-terminal no role in heparin It be that the GAG binding are to the BBXB The binding of of the chemokines in have SDF-1, and the the residues in IL-8, and Proudfoot PubMed Scopus Google as well in MCP-1, and S. Full Text Full Text PDF PubMed Scopus Google are It has been that cell GAG expression is for the activity of chemokines in although a role in chemokine S. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). of the GAG binding has no on receptor binding and activation in the of IL-8, SDF-1, and has a as has been for binding to CCR1 S. A. PubMed Scopus Google Scholar). We have both for the mutation of has no significant on its binding to and activation of CCR5 but its with This is by the of of CCR5 activation by the mutation of in Immunol. 1999; Google whereas mutations in the of CCR1 binding but have been for CCR5. of the of the extracellular of these two an these are CCR1 has and residues, only which basic and and which whereas CCR5 has and basic and and it that the of CCR1 is to be whereas in CCR5 it be This that of the of CCR1 play a role in the receptor but of CCR5 and its that are in This is by the important in the of RANTES was the for CCR1 whereas for CCR5 binding the residues in RANTES are and 1997; 36: PubMed Scopus Google Scholar) and in were P.J. 2000; PubMed Scopus Google Scholar). However, in these the role of the residues in the 40s loop in to receptor binding was The of the in affinity for CCR1 by the RANTES mutant is by its in ability to induce chemotaxis of which CCR1 as the RANTES receptor F. S. T.N. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). It was to that the triple 40s mutant that to the heparin beads retained the affinity for heparin as for the This that although is the GAG binding be residues that also can be of the three-dimensional is to be and play a role in binding We are We have previously that RANTES a significant of selectivity for different GAG F. Proudfoot T.N. 1999; PubMed Scopus Google Scholar). the selectivity previously for the GAG by RANTES to be mediated to a by the BBXB motif on the 40s The of the in HIV-1 infectivity has been by the that at high RANTES A. E. Proudfoot Moore 1999; PubMed Google Scholar, Proudfoot Moore A. 1999; PubMed Google Scholar). have been to the of RANTES to on heparin F. Proudfoot T.N. 1999; PubMed Scopus Google Scholar, A. E. Proudfoot Moore 1999; PubMed Google which that the with an important RANTES mutants that are able to A. E. Proudfoot Moore 1999; PubMed Google Scholar). that the 80% reduction of the ability of the RANTES to bind to heparin the enhanced However, the ability of RANTES to inhibit HIV-1 infectivity on its ability to bind to the with its The triple 40s mutant CCR5 binding activity and is able to inhibit HIV-1 infectivity of HIV-1 This has the heparin binding of RANTES but has that to be which are the residues that are for the heparin binding the residues in that play a role in GAG binding in CCR1 and the of is to the that it with the the of RANTES with activation a that such mutants to the important of the of GAG binding to in which has been for been and the role of GAG binding in inflammation. We for of the