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-epimerase 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 Met(1)-Gln(153) of decorin (DCN), which normally has a single dermatan sulfate chain at Ser(34), in combination with intact or modified Leu(241)-Ser(353) of CSF-1, which has a chondroitin sulfate attachment site at Ser(309). Transfected DCN(M1-Q153), like full-length DCN, contained approximately 20% l-iduronate. Conversely, transfected CSF-1(L241-S353), attached C-terminally on the DCN prepropeptide, contained almost exclusively d-glucuronate. Transfected intact chimeric DCN(M1-Q153)-CSF-1(L241-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 approximately 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.
Highlights
The assembly of glycosaminoglycan chains on core proteins is a multistep process that begins most often with the formation of a specific carbohydrate-protein linkage region and continues with the alternating addition of D-GlcUA and either D-GlcNAc or D-GalNAc followed by sulfation and additional polymer modifications
That DCN is always linked with a glycosaminoglycan chain, whereas CSF-1 has to be considered as a part-time proteoglycan [25, 26]
Digestion of [35S]methionine-labeled proteoglycan preparations resulted in the appearance of core proteins of 29 kDa in accordance with the prediction derived from the cDNA of the constructs used for transfection
Summary
Construction of Chimeric cDNA—First, the cDNA for DCNM1-Q153, including 120 bp of the 5Ј-untranslated sequence and a stop codon, was amplified by PCR from full-length human cDNA [28] in pGem3Z by using the primer pair 5Ј-TAATACGACTCACTATAG-3Ј (T7-forward primer) and 5Ј-CTGAAGAGTTTTGGGCATTTTTTC-3Ј (reverse and complement primer). Fluorophore-assisted carbohydrate electrophoresis [36] after chondroitin ABC, ACII, and B lyase digestion, respectively, was used to determine the ratio of 4- and 6-sulfated N-acetylgalactosamine-containing disaccharides in the chondroitin sulfate- and dermatan sulfatecontaining domains of the glycosaminoglycan chains of the various chimeric proteins. The medium was digested with 300 units of hyaluronate lyase from Streptomyces hyalurolyticus (Sigma) for 2 h at 37 °C and subjected to ammonium sulfate precipitation and chromatography on 0.5 ml of DEAE-Trisacryl M (Serva) column as described except that the starting buffer contained only 50 mM NaCl. The column was washed with buffer containing 100 mM NaCl, and proteoglycans were desorbed from the resin with 1.0 ml of 300 mM NaCl in 20 mM Tris/HCl, pH 7.2. Other Methods—Polyacrylamide gel electrophoresis in the presence of SDS followed by fluorography was performed as previously described [30]
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