Identification of the glycosaminoglycan-attachment site of mouse invariant-chain proteoglycan core protein by site-directed mutagenesis.

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Abstract
The invariant chain (Ii), a nonpolymorphic glycoprotein that associates with the immunoregulatory Ia proteins encoded by the major histocompatibility complex, has a proteoglycan form (Ii-CS) that bears a chondroitin sulfate glycosaminoglycan. In this proteoglycan form, Ii may remain associated with Ia at the cell surface. Inhibitors that prevent the addition of glycosaminoglycan to Ii have been found to depress antigen-presenting function. Ii does not have multiple candidate glycosaminoglycan-attachment sites, and we used site-directed mutagenesis to replace a candidate serine glycosaminoglycan-acceptor site with alanine at position 201 in the murine Ii protein. Transfection of the normal or altered gene into Ii-negative COS-7 cells showed that equivalent amounts of core Ii protein and its acidic, terminally glycosylated forms were synthesized, but the Ala-201 mutant Ii did not give rise to Ii-CS. The mutant protein had apparently normal transport through the Golgi compartment and associated stably with Ia molecules. Thus, this mutation directly identifies the site of glycosaminoglycan addition and shows that it can be eliminated without adversely affecting the overall biosynthesis of Ii.