GLYCOSYLATION PROVIDES BOTH STIMULATORY AND INHIBITORY EFFECTS ON CELL-SURFACE AND SOLUBLE CD44 BINDING TO HYALURONAN

Glycosylation has been implicated in the regulation of CD44-mediated c ell binding of hyaluronan (HA). However, neither the relative contribu tion of N- and O-linked glycans nor the oligosaccharide structures tha t alter CD44 affinity for HA have been elucidated, To determine the ef fect of selective alteration of CD44 oligosaccharide composition on th e affinity of CD44 for HA, we developed a novel strategy based on the use of affinity capillary electrophoresis (ACE). Soluble recombinant C D44-immunoglobulin fusion proteins were overproduced in the mutant CHO cell line ldl-D, which has reversible defects in both N- and O-linked oligosaccharide synthesis, Using this cell line, a panel of recombina nt glycosidases, and metabolic glycosidase inhibitors, CD44 glycoforms with defined oligosaccharide structures were generated and tested for HA affinity by ACE. Because ldl-D cells express endogenous cell surfa ce CD44, the effect of any given glycosylation change on the ability o f cell surface and soluble CD44 to bind HA could be compared. Four dis tinct oligosaccharide structures were found to effect CD44-mediated HA binding: (a) the terminal alpha 2,3-linked sialic acid on N-linked ol igosaccharides inhibited binding; (b) the first N-linked N-acetylgluco samine residue enhanced binding; (c) O-linked glycans on N-deglycosyla ted CD44 enhanced binding; and (d) N-acetylgalactosamine incorporation into non-N-linked glycans augmented HA binding by cell surface CD44. The first three structures induced up to a 30-fold alteration in the i ntrinsic CD44 affinity for HA (K-d = 5 to > 150 mu M). The fourth augm ented CD44-mediated cellular HA avidity without changing the intrinsic HA affinity of soluble CD44.