IDENTIFICATION OF A COMMON HYALURONAN-BINDING MOTIF IN THE HYALURONAN-BINDING PROTEINS RHAMM, CD44 AND LINK PROTEIN

We have previously identified two hyaluronan (HA) binding domains in t he HA receptor, RHAMM, that occur near the carboxyl-terminus of this p rotein. We show here that these two HA binding domains are the only HA binding regions in RHAMM, and that they contribute approximately equa lly to the HA binding ability of this receptor. Mutation of domain II using recombinant polypeptides of RHAMM demonstrates that K423 and R43 1, spaced seven amino acids apart, are critical for HA binding activit y. Domain I contains two sets of two basic amino acids, each spaced se ven residues apart, and mutation of these basic amino acids reduced th eir binding to HA - Sepharose. These results predict that two basic am ino acids flanking a seven amino acid stretch [hereafter called B(X(7) )B] are minimally required for HA binding activity. To assess whether this motif predicts HA binding in the intact RHAMM protein, we mutated all basic amino acids in domains I and II that form part of these mot ifs using site-directed mutagenesis and prepared fusion protein from t he mutated cDNA. The altered RHAMM protein did not bind HA, confirming that the basic amino acids and their spacing are critical for binding . A specific requirement for arginine or lysine residues was identifie d since mutation of K430, R431 and K432 to histidine residues abolishe d binding. Clustering of basic amino acids either within or at either end of the motif enhanced HA binding activity while the occurrence of acidic residues between the basic amino acids reduced binding. The B(X (7))B motif, in which B is either R or K and X(7) contains no acidic r esidues and at least one basic amino acid, was found in all HA binding proteins molecularly characterized to date. Recombinant techniques we re used to generate chimeric proteins containing either the B(X(7))B m otifs present in CD44 or link protein, with the amino-terminus of RHAM M (amino acids 1-238) that does not bind HA. All chimeric proteins con taining the motif bound HA in transblot analyses. Site-directed mutati ons of these motifs in CD44 sequences abolished HA binding. Collective ly, these results predict that the motif of B(X(7))B as a minimal bind ing requirement for HA in RHAMM, CD44 and link protein, and occurs in all HA binding proteins described to date.