Biosynthesis of the Proteoglycan Decorin

Abstract

Biosynthesis of decorin was investigated by incubating a rat fibroblast cell line with various radiolabelled protein and galactosaminoglycan precursors. The following cell-associated and distinct intermediates were isolated and identified: a pool of non-glycosylated core protein, two pools of decorin with incomplete chains, one with three sulphated disaccharide repeats and another with five or more sulphated disaccharide repeats, as well as decorin with mature chains. Results of pulsekhase experiments indicated that these pools represented discrete stages in chain growth. Treatment with brefeldin A, which blocks transport from the endoplasmic reticulum to the Golgi, resulted in accumulation of decorin with an incomplete chain containing six or seven largely unsulphated disaccharide repeats. During recovery from drug treatment, 4-sulfation reappeared earlier than 6-sulfation. The results suggest that the galactosaminoglycan assembly-line consists of separate multienzyme complexes that build only a limited section of the chain. Most connective tissues contain small leucine-rich proteoglycans substituted with dermatan sulphate or chondroitin sulfate depending on the source (Ciister and Fransson, 1981 ; Coster et al., 1984;Heineglrd et al., 1985; Heinegird and Oldberg, 1993;Cheng et al., 1994). One of these proteoglycans is named decorin because it decorates collagen fibrils in a characteristic manner (Krusius and Ruoslahti, 1986; Scott et al., 1981). The single galactosaminoglycan chain is attached to a serine residue located to the extreme N-terminal portion. In decorin isolated from extracellular matrices the N-terminal sequence is DEASGIlP in the rat (Kokenyesi and Woessner, 1990; Abramson and Woessner, 1992), and DEASGIGP in bovine and human decorin (Brennan et al., 1984), both of which conform with the proposed recognition consensus sequence for glycosaminoglycan attachment (Bourdon et al., 1987). Comparisons between the N-terminal sequence of extracellular decorin and the sequences deduced from cDNA indicate that decorin is synthesized as a precursor with a 14-amino-acid-long propeptide. We have studied expression and secretion of decorin in COS and CHO cells transformed with vectors encoding both wild-type rat decorin and variants with deletions in the N-terminal region (Oldberg et al., 1996). Cells expressing deletion variants synthesized proteoglycans with a shorter galactosaminoglycan chain compared with those derived from wild-type decorin with an intact N-terminal domain.

Biosynthesis of the Proteoglycan Decorin Transient 2‐Phosphorylation of Xylose during Formation of the Trisaccharide Linkage Region

“…The cell line, anti-(rat decorin), glycosaminoglycan, oligosaccharide and disaccharide standards, decorin proteoglycan and core protein, chondroitin lyases, prepacked columns and media, brefeldin A and proteinase inhibitors will be described in a forthcoming report (Moses et al, 1997). A linkage-region preparation from dermatan sulfate was prepared as described (Fransson, 1968).…”

“…In work described in a report to be published shortly (Moses et al, 1997), we studied early steps in galactosaminoglycan assembly in a rat fibroblast cell line that synthesizes and secretes decorin. By using an antiserum directed against the core protein we detected three pools of intermediates substituted with the linkage region plus either three or five or more sulfated repeats or with a mature chain.…”

Determination of the Glycosaminoglycan-Protein Linkage Region Oligosaccharide Structures of Proteoglycans from Drosophila melanogaster and Caenorhabditis elegans

“…Recent simultaneous analysis of both CS and HS chains of the hybrid type proteoglycan syndecan-1 molecule has suggested the Xyl phosphorylation of both CS and HS chains (34). Structural analysis of the biosynthetic intermediate oligosaccharides formed in the cultured rat fibroblasts has suggested that dephosphorylation takes place soon after the transfer reaction of GlcUA to the trisaccharide (57)(58)(59). In vitro enzyme assays using crystallized human glucuronyltransferase I as enzyme (60) and Gal-Gal-Xyl(2-O-phosphate)-Ser as acceptor have demonstrated the higher enzyme activity with the phosphorylated acceptor substrate.…”

Comparative Analysis of Proteoglycans Synthesized by Chick Corneal Stromal Cells in Cell Culture and Organ Culture.

“…These differences in molecular size appear to derive primarily from the differences in the sulfation and length of glycosaminoglycan chains of PGs in each fraction. Although the length of glycosaminoglycan chains was not determined in this study, the undersulfated and nonsulfated glycosaminoglycan chains of PGs in Fractions 1 and 2 appear to be shorter than those in Fraction 3, because the differences between the elution positions on chromatographies are too great to be accounted for only by the extent of sulfation; furthermore, it has been reported that the sulfation of the glycosaminoglycan chain occurs during its polymerization 28,29) and that the glycosyltransferases involved in the chain elongation have higher activity toward the sulfated oligosaccharides than toward the nonsulfated ones 30,31) (the sulfation could be a rate-limiting step of the chain elongation). The results described above are consistent with those reported by Funderburgh et al 21) and Schrecengost et al 22) The chromatographies in this study also show, however, that PGs with short, highly sulfated and long, nonsulfated glycosaminoglycan chains were present in both organ and cell cultures.…”