REGULATION OF CD44 BINDING TO HYALURONAN BY GLYCOSYLATION OF VARIABLYSPLICED EXONS

The hyaluronan (HA)-binding function (lectin function) of the leukocyt e homing receptor, CD44, is tightly regulated. Herein we address possi ble mechanisms that regulate CD44 isoform-specific Hii binding. Bindin g studies with melanoma transfectants expressing CD44H, CD44E, or with soluble immunoglobulin fusions of CD44H and CD44E (CD44H-Rg, CD44E-Rg ) showed that although both CD44 isoforms can bind HA, CD44H binds HA more efficiently than CD44E. Using CD44-Rg fusion proteins we show tha t the variably spliced exons in CD44E, V8-V10, specifically reduce the lectin function of CD44, while replacement of V8-V10 by an ICAM-1 imm unoglobulin domain restores binding to a level comparable to that of C D44H. Conversely, CD44 bound HA very weakly when exons V8-V10 were rep laced with a CD34 mucin domain, which is heavily modified by O-linked glycans. Production of CD44E-Rg or incubation of CD44E-expressing tran sfectants in the presence of an O-linked glycosylation inhibitor resto red HA binding to CD44H-Rg and to cell surface CD44H levels, respectiv ely. We conclude that differential splicing provides a regulatory mech anism for CD44 lectin function and that this effect is due in part to O-linked carbohydrate moieties which are added to the Ser/Thr rich reg ions encoded by the variably spliced CD44 exons. Alternative splicing resulting in changes in protein glycosylation provide a novel mechanis m for the regulation of lectin activity.