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

Background:The enzyme responsible for the dephosphorylation of xylose in glycosaminoglycans remains unknown. Results: A protein that removed the phosphate from the phosphorylated linkage trisaccharide and localized in the Golgi was identified. Inhibition of this protein phosphatase resulted in decreased glycosaminoglycan levels in cells. Conclusion:We identified the phosphoxylose phosphatase that regulates glycosaminoglycan synthesis. Significance: Transient phosphorylation of xylose residues controls glycosaminoglycan synthesis.

mentioning

confidence: 67%

Identification of Phosphatase That Dephosphorylates Xylose in the Glycosaminoglycan-Protein Linkage Region of Proteoglycans

Koike

Izumikawa

Sato

et al. 2014

“…These modifications show a rather wide distribution in various tissues of many different vertebrate species. 2-O-Phosphorylation of Xyl has been found for CS from shark cartilage (13), rat (40) and mouse (12) tumors, and for CS/DS from rat fibroblast cell line (41). It was also found for HS/Hep from bovine lung (10, 42).…”

Section: Discussionmentioning

confidence: 88%

“…Fransson and his co-workers (41,53,54) have reported that the phosphorylation of Xyl is a transient phenomenon and is not inhibited by brefeldin A, which interferes with progression from the endoplasmic reticulum to Golgi. They have also found in the early steps of the CS chain biosynthesis of decorin that the degree of phosphorylation increases to ϳ90% until the linkage region grows into the Gal-Gal-Xyl trisaccharide, and then dephosphorylation takes places extensively accompanied by glucuronidation.…”

Section: Discussionmentioning

confidence: 99%

“…In view of the possible modifications by sulfation and phosphorylation of the linkage region and their specific recognition by GlcUA transferase I as described above, the linkage region synthesis may be regulated variably during multiple modification and glycosylation steps and may yield an unglycanated part-time PG when inhibited during any one of these steps of the chain elongation during maturation of the linkage region tetrasaccharide sequence, which would provide multiple regulatory points during the conversion of a nonglycanated to glycanated PG. Vigorous examinations are required for detecting possible small biosynthetic intermediate oligosaccharides (41,53), which may be generated during the maturation process of the linkage region of the syndecan-1 GAG chains.…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Structural Characterization of Heparan Sulfate and Chondroitin Sulfate of Syndecan-1 Purified from Normal Murine Mammary Gland Epithelial Cells

Ueno¹,

Yamada²,

Zako³

et al. 2001

Syndecan-1, present on the surfaces of normal murine mammary gland epithelial cells, is a transmembrane hybrid proteoglycan, which bears glycosaminoglycan (GAG) side chains of heparan sulfate (HS) and chondroitin sulfate (CS). Purified syndecan-1 ectodomains were analyzed for disaccharide composition and the GAG-protein linkage region after digestion with bacterial lyases. The HS chains contained predominantly a nonsulfated unit with smaller proportions of two monosulfated, two disulfated, and a trisulfated unit, whereas CS chains were demonstrated for the first time to bear GlcUA-GalNAc(4-O-sulfate) as a major component as well as GlcUA-GalNAc, GlcUA-GalNAc(6-O-sulfate), and an E disaccharide unit GlcUA-GalNAc(4,6-O-disulfate) as minor yet appreciable components. Two kinds of linkage region tetrasaccharides, GlcUA-Gal-Gal-Xyl and GlcUA-Gal-Gal-Xyl(2-O-phosphate), were found for the HS chains in a molar ratio of 55:45. In marked contrast, an additional sulfated tetrasaccharide, GlcUA-Gal(4-Osulfate)-Gal-Xyl, was demonstrated only for the CS chains, and the unmodified phosphorylated and sulfated components were present at a molar ratio of 55:26: 19. The present study thus provided conclusive evidence for the hypothesis that 4-O-sulfation of Gal is peculiar to CS chains in contrast to the phosphorylation of Xyl, which is common to both HS and CS chains. These modifications may be required for biosynthetic maturation of the linkage region tetrasaccharide sequence, which is a prerequisite for creating the repeating disaccharide region of GAG chains and/or biosynthetic selective chain assembly of CS and HS chains.

“…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.…”

Section: Disaccharidesmentioning

confidence: 99%

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

Yamada

Okada

Ueno

et al. 2002

Caenorhabditis elegans and Drosophila melanogaster are relevant models for studying the roles of glycosaminoglycans (GAG) during the development of multicellular organisms. The genome projects of these organisms have revealed the existence of multiple genes related to GAG-synthesizing enzymes. Although the putative genes encoding the enzymes that synthesize the GAG-protein linkage region have also been identified, there is no direct evidence that the GAG chains bind covalently to core proteins. This study aimed to clarify whether GAG chains in these organisms are linked to core proteins through the conventional linkage region tetrasaccharide sequence found in vertebrates and whether modifications by phosphorylation and sulfation reported for vertebrates are present also in invertebrates. The linkage region oligosaccharides were isolated from C. elegans chondroitin in addition to D. melanogaster heparan and chondroitin sulfate after digestion with the respective bacterial eliminases and were then derivatized with a fluorophore 2-aminobenzamide. Their structures were characterized by gel filtration and anion-exchange high performance liquid chromatography in conjunction with enzymatic digestion and matrix-assisted laser desorption ionization time-of-flight spectrometry, which demonstrated a uniform linkage tetrasaccharide structure of -GlcUA-Gal-Gal-Xyl-or -GlcUA-Gal-Gal-Xyl(2-O-phosphate)-for C. elegans chondroitin and D. melanogaster CS, respectively. In contrast, the unmodified and phosphorylated counterparts were demonstrated in heparan sulfate of adult flies at a molar ratio of 73:27, and in that of the immortalized D. melanogaster S2 cell line at a molar ratio of 7:93, which suggests that the linkage region in the fruit fly first becomes phosphorylated uniformly on the Xyl residue and then dephosphorylated. It has been established here that GAG chains in both C. elegans and D. melanogaster are synthesized on the core protein through the ubiquitous linkage region tetrasaccharide sequence, suggesting that indispensable functions of the linkage region in the GAG synthesis have been well conserved during evolution. Mutations affecting the genes encoding putative proteins related to GAG biosynthetic enzymes have also been described for these organisms. Mutations in the tout velu (ttv) gene of D. melanogaster cause defects in Hedgehog movement in mosaic wing discs (1, 6). The ttv gene is a putative ortholog of vertebrate EXT1, which encodes a heparan polymerase and is associated with the hereditary multiple exostoses syndrome in humans (7). The pipe gene, which affects dorsal-ventral patterning of D. melanogaster development, encodes a homolog of vertebrate HS 2-O-sulfotransferase (2, 8). The sugarless and sulfateless genes, both of which affect the fibroblast growth factor signaling during the D. melanogaster development, en-