Importance of Basic Residues and Quaternary Structure in the Function of MIP-1: CCR5 Binding and Cell Surface Sugar I

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• 作者：Jennifer S. Laurence ; Cé ; dric Blanpain ; Anne De Leener ; Marc Parmentier ; and Patricia J. LiWang
• 刊名：Biochemistry
• 出版年：2001
• 出版时间：April 24, 2001
• 年：2001
• 卷：40
• 期：16
• 页码：4990 - 4999
• 全文大小：115K
• 年卷期：v.40,no.16(April 24, 2001)
• ISSN：1520-4995

文摘

Chemokines direct immune cells toward sites of infection by establishing a gradient acrossthe extracellular matrix of the tissue. This gradient is thought to be stabilized by ligation of chemokinesto sulfated polysaccharides known as glycosaminoglycans (GAGs) that are found on the surface ofendothelial and other cells as well as in the tissue matrix. GAGs interact with chemokines and in somecases cause them to aggregate. The interaction between cell surface GAGs and chemokines has also beenpostulated to play a role in the anti-HIV activity of some chemokines, including MIP-1mages/gifchars/beta2.gif" BORDER=0 ALIGN="middle">. Since manyproteins interact with GAGs by utilizing basic residues, we mutated R18, K45, R46, and K48 in MIP-1mages/gifchars/beta2.gif" BORDER=0 ALIGN="middle">to investigate the role of these residues in GAG binding and CCR5 function. We find that no singleamino acid substitution alone has a dramatic effect on heparin binding, although change at R46 has amoderate effect. However, binding to heparin is completely abrogated in a mutant (K45A/R46A/K48A)in which the entire "40's loop" has been neutralized. A functional study of these mutants reveals that thecharged residues in this 40's loop, particularly K48 and R46, are critical mediators of MIP-1mages/gifchars/beta2.gif" BORDER=0 ALIGN="middle"> binding toits receptor CCR5. However, despite the partially overlapping function of the residues in the 40's loop inbinding to both CCR5 and heparin, the presence of cell surface sugars does not appear to be necessary forthe ability of MIP-1mages/gifchars/beta2.gif" BORDER=0 ALIGN="middle"> to function on its receptor CCR5, as enzymatic removal of GAGs from cells resultsin little effect on MIP-1mages/gifchars/beta2.gif" BORDER=0 ALIGN="middle"> activity. Because the means by which the chemokine gradient transmitsinformation to the recruited cells is not well defined, we also mutated the basic residues in MIP(9), atruncated form of MIP-1mages/gifchars/beta2.gif" BORDER=0 ALIGN="middle"> that is impaired in its ability to dimerize, to probe whether the quaternarystructure of this chemokine influences its ability to bind heparin. None of the truncated variants bound aswell as the full-length proteins containing the same mutation, suggesting that the MIP-1mages/gifchars/beta2.gif" BORDER=0 ALIGN="middle"> dimer participatesin heparin binding.