Core protein dependence of epimerization of glucuronosyl residues in galactosaminoglycans

Abstract

Chondroitin sulfate and dermatan sulfate proteoglycans are distinguished by differences in their proportion of D-glucuronosyl and L-iduronosyl residues, the latter being formed by chondroitin-glucuronate 5-epiomerase during or after glycosaminoglycan chain polymerization. To investigate the influence of the core protein on the extent of epimerization, we expressed chimeric proteins in 293 HEK cells constructed from intact or modified Met1-Gln153 of decorin (DCN), which normally has a single dermatan sulfate chain at Ser34, in combination with intact or modified Leu241-Ser353 of CSF-1, which has a chondroitin sulfate attachment site at Ser309. Transfected DCNM1-Q153, like full-length DCN, contained ∼20% L-iduronate. Conversely, transfected CSF-1L241-S353, attached C-terminally on the DCN prepropeptide, contained almost exclusively D-glucuronate. Transfected intact chimeric DCNM1-Q153-CSF-1L241-S353, with two glycosaminoglycan chains, also contained almost exclusively D-glucuronate in chains at both sites, as did chimeras in which alanine was substituted for serine at either of the glycosaminoglycan attachment sites. Nevertheless, undersulfated intact chimeric proteoglycan was an effective substrate for epimerization of glucuronate to iduronate residues when incubated with microsomal proteins and 3′-phosphoadenylylphosphosulfate. C-terminal truncation constructs were prepared from the full-length chimera with an alanine substitution at the CSF-1 glycosaminoglycan attachment site. Transfected truncations retaining the alanine-blocked site contained chains with essentially only glucuronate, whereas those further truncated by 49 or more amino acids and missing the modified attachment site contained chains with ∼15% iduronate. This 49-amino acid region contains a 7-amino acid motif that appears to be conserved in several chondroitin sulfate proteoglycans. The results are consistent with a model in which the core protein, possibly via this motif, is responsible for routing to subcellular compartments with or without sufficient access to chondroitin-glucuronate 5-epimerase for the addition of chains with or without iduronate residues, respectively.