AMINO-ACID DETERMINANTS THAT DRIVE HEPARAN-SULFATE ASSEMBLY IN A PROTEOGLYCAN

To study how cells regulate the composition of glycosaminoglycan chain s on proteoglycans, we have examined the assembly of chains on chimeri c proteoglycans containing segments of betaglycan (transforming growth factor-beta Type III receptors) fused to protein A. Transient express ion of the chimeras in Chinese hamster ovary cells revealed that only two glycosaminoglycan attachment sites exist. One site at Ser(535) sup ported both chondroitin sulfate and heparan sulfate synthesis, whereas the site at Ser(546) supported only chondroitin sulfate. The composit ions of the chimeras were the same in CHO-K1, CHOP-C4, BHK-21, and HeL a S3 cells and in chimeras containing polyhistidine fused to the C ter minus, Deletion experiments showed that the assembly of heparan sulfat e chains on the chimeras required a peptide segment of less than or eq ual to 16 amino acids (SPGDSS(535)-GWPDGYEDLE) and the first 5 amino a cids were not essential, Truncation of the acidic cluster (EDLE), site -directed mutation of the acidic residues in the cluster or deletion o f the sequence between the cluster and the Ser attachment site decreas ed heparan sulfate assembly. Mutation of Trp(537) adjacent to the site also decreased heparan sulfate assembly. More importantly, introducin g tryptophan next to three different Ser-Gly dipeptides in betaglycan and syndecan-1 chimeras stimulated assembly of heparan sulfate. Thus, one type of heparan sulfate attachment site consists of a Ser-Gly dipe ptide and a flanking cluster of acidic residues. An adjacent tryptopha n residue can augment the proportion of heparan sulfate.