Contribution of the putative heparan sulfate-binding motif BBXB of RANTES to transendothelial migration

Glycobiology. 2002 Sep;12(9):535-43. doi: 10.1093/glycob/cwf069.

Abstract

The chemokines are a family of small chemoattractant proteins that have a range of functions, including activation and promotion of vectorial migration of leukocytes. Regulation on activation, normal T cell expressed and secreted (RANTES; CCL5), a member of the CC-chemokine subfamily, has been implicated in a variety of immune responses. In addition to the interaction of CC-chemokines with their cognate cell-surface receptors, it is known that they also bind to glycosaminoglycans (GAGs), including heparan sulfate. This potential for binding to GAG components of proteoglycans on the cell surface or within the extracellular matrix might allow formation of the stable chemokine concentration gradients necessary for leukocyte chemotaxis. In this study, we created a panel of mutant RANTES molecules containing neutral amino acid substitutions within putative, basic GAG-binding domains. Despite showing reduced binding to GAGs, it was found that each mutant containing a single amino acid substitution induced a similar leukocyte chemotactic response within a concentration gradient generated by free solute diffusion. However, we found that the mutant K45A had a significantly reduced potential to stimulate chemotaxis across a monolayer of microvascular endothelial cells. Significantly, this mutant bound to the CCR5 receptor and showed a potential to mobilize Ca(2+) with an affinity similar to the wild-type protein. These results show that the interaction between RANTES and GAGs is not necessary for specific receptor engagement, signal transduction, or leukocyte migration. However, this interaction is required for the induction of efficient chemotaxis through the extracellular matrix between confluent endothelial cells.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Motifs
  • Amino Acid Sequence
  • Animals
  • Binding Sites
  • CHO Cells
  • Calcium / metabolism
  • Cell Line
  • Chemokine CCL5 / chemistry
  • Chemokine CCL5 / genetics
  • Chemokine CCL5 / metabolism*
  • Chemotaxis, Leukocyte
  • Cloning, Molecular
  • Cricetinae
  • Endothelium / cytology
  • Endothelium / metabolism*
  • Flow Cytometry
  • Heparitin Sulfate / metabolism*
  • Ion Transport
  • Radioligand Assay
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism

Substances

  • Chemokine CCL5
  • Recombinant Proteins
  • Heparitin Sulfate
  • Calcium