A biophysical insight into the RANTES-glycosaminoglycan interaction.
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 Kd 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.
Rek
Angelika
A
ProtAffin Biotechnologie AG, Reininghausstrasse 13a, A-8020 Graz, Austria.
Brandner
Barbara
B
Geretti
Elena
E
Kungl
Andreas J
AJ
D045744:Cell Line, Tumor; D018946:Chemokine CCL5 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002633:Chemotaxis; D002942:Circular Dichroism; D004926:Escherichia coli / Q000235:genetics; D005454:Fluorescence Polarization; D006497:Heparan Sulfate / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D011485:Protein Binding; D055503:Protein Multimerization; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012965:Sodium Chloride / Q000737:chemistry; D020349:Surface Plasmon Resonance
D018946:Chemokine CCL5; D006497:Heparan Sulfate; D011994:Recombinant Proteins; D012965:Sodium Chloride