Molecular And Biochemical Analysis Of SPACRCAN Binding To Hyaluronan And Other Glycosaminoglycans

Abstract

Abstract: : Purpose: Photoreceptors project from the outer retina into an organized IPM containing hyaluronan (HA) and two novel proteoglycans (SPACR and SPACR CAN). How these molecules interact to organize this matrix is not known. HA is present in this matrix and is a candidate for involvement in IPM organization. Because RHAMM–type HA–binding motifs are present in the deduced sequence of SPACR and SPACR CAN, these proteins may be involved in this binding. In this study we define the interactions between HA and putative RHAMM–type HA–binding motifs present in the deduced amino acid sequence of SPACRCAN. Since SPACRCAN is a chondroitin sulfate proteoglycan and because heparan sulfate proteoglycans are also expressed by photoreceptors, we evaluated the possibility that these RHAMM–type HA–binding motifs may also bind chondroitin and heparin GAGs. Methods:We sub–cloned three fragments of mouse SPACRCAN that contain the putative RHAMM–type HA–binding motifs into the pGEX–2TK vector and have generated several mutations in the HA–binding motifs in the respective constructs. The constructs were then expressed in E. coli BL21. To determine the binding properties of the proteins to HA, chondroitin sulfates and heparin, proteins purified from the cell extracts, were subjected to CPC precipitation analysis and ELISA on plates pre–coated with either HA or heparin. Results:We found that each recombinant protein binds HA. Binding decreased when residues in the RHAMM–type HA–binding motifs were mutated. We also evaluated the binding of these recombinant proteins to heparin and chondroitin sulfates. We found that each of the recombinant proteins binds to both heparin and chondroitin sulfate GAGs. Binding to chondroitin sulfates involved these HA–binding motifs, since mutagenesis of these motifs reduced binding. Heparin binding was probably not mediated through these hyaluronan binding motifs since heparin binding persisted after mutagenesis. Conclusions:This provides the first direct evidence that the RHAMM–type hyaluronan binding motifs are involved in HA binding in SPACRCAN. These studies provide the first evidence defining the molecular interaction of specific molecules comprising the IPM.

Keywords: extracellular matrix • proteoglycans/glycosaminoglycans • protein structure/function