Bh. Yang et al., IDENTIFICATION OF A COMMON HYALURONAN-BINDING MOTIF IN THE HYALURONAN-BINDING PROTEINS RHAMM, CD44 AND LINK PROTEIN, EMBO journal, 13(2), 1994, pp. 286-296
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.