A biophysical insight into the RANTES-glycosaminoglycan interaction

作者：Rek Angelika; Brandner Barbara; Geretti Elena; Kungl Andreas J^*

来源：Biochimica et Biophysica Acta-Proteins and Proteomics, 2009, 1794(4): 577-582.

DOI：10.1016/j.bbapap.2009.01.001

• Heparin
• Heparan sulfate
• Chemokine
• Fluorescence spectroscopy
• Circular dichroism

Binding of chemokines to glycosaminoglycans (GAGs) represents a crucial step in leukocyte attraction and activation. Since chemokine oligomerisation was shown to be important for GAG binding, the apparent oligomerisation constant of RANTES was determined to be 225 nM using fluorescence anisotropy. In the presence of heparan sulfate, chemokine oligomerisation was found to be promoted by the glycosaminoglycan as expressed in the increase in cooperativity and a shift towards higher melting temperatures in thermal unfolding experiments. In surface plasmon resonance investigations of RANTES-GAG binding kinetics using a heparan sulfate-coated chip, GAG-induced oligomerisation led to a bell-shaped (bi-phasic) Scatchard plot referring to cooperativity in the chemokine-GAG interaction. This was absent in the oligomerisation-deficient RANTES mutants N46R and Q48K. We have further investigated the dependence of RANTES-GAG dissociation constants on oligosaccharide chain length by performing isothermal fluorescence titrations with size-defined heparin and heparan sulfate oligosaccharides as chemokine ligands. Heparin dp18 and heparan sulfate dp14 yielded the highest affinities with K(d) values of 31.7 nM and 42.9 nM, respectively. Far-UV CD spectroscopy revealed a significant conformational change of RANTES upon heparan sulfate binding which is suggested to be a pre-requisite for oligomerisation and thus for optimal GPCR activation in vivo. This was shown by the impaired chemotactic activity of the RANTES N46R and Q48K mutants.

• 出版日期2009-4