Biosynthesis of decorin and glypican

Chondroitin sulfate (CS) and dermatan sulfate (DS) have been implicated in the processes of neural development in the brain. In this study, we characterized developmentally regulated brain CS/DS chains using a single chain antibody, GD3G7, produced by the phage display technique. Evaluation of the specificity of GD3G7 toward various glycosaminoglycan preparations showed that this antibody specifically reacted with squid CS-E (rich in the GlcUA␤1-3GalNAc(4,6-O-sulfate) disaccharide unit E), hagfish CS-H (rich in the IdoUA␣1-3GalNAc(4,6-Osulfate) unit iE), and shark skin DS (rich in both E and iE units). In situ hybridization for the expression of N-acetylgalactosamine-4-sulfate 6-O-sulfotransferase in the postnatal mouse brain, which is involved in the biosynthesis of CS/DS-E, showed a widespread expression of the transcript in the developing brain except at postnatal day 7, where strong expression was observed in the external granule cell layer in the cerebellum. The expression switched from the external to internal granule cell layer with development. Immunohistochemical localization of GD3G7 in the mouse brain showed that the epitope was relatively abundant in the cerebellum, hippocampus, and olfactory bulb. GD3G7 suppressed the growth of neurites in embryonic hippocampal neurons mediated by CS-E, suggesting that the epitope is embedded in the neurite outgrowth-promoting motif of CS-E. In addition, a CS-E decasaccharide fraction was found to be the critical minimal structure needed for recognition by GD3G7. Four discrete decasaccharide epitopic sequences were identified. The antibody GD3G7 has broad applications in investigations of CS/DS chains during the central nervous system's development and under various pathological conditions.

Section: Identified the Antibody Gd3g7 Has Broad Applications In Invmentioning

confidence: 99%

Functions of Chondroitin Sulfate/Dermatan Sulfate Chains in Brain Development

Purushothaman

Fukuda

Mizumoto

et al. 2007

“…Since we showed that phosphorylated xyloside cannot serve as a substrate for GalT-I, it can be suggested that phosphorylation may arrest the biosynthesis of some GAG chains and thus may represent a regulatory mechanism in the biosynthesis rate of PGs. In a similar way, ␣-GalNAc capping of the core tetrasaccharide primer (36) and 4,6-disulfatation of GalNAc residues of CS chains (37) have been postulated to represent chain termination mechanisms. However, this issue awaits further investigation.…”

Section: Table II Kinetic Analysis Of Glcat-i Towards Gal␤1-3gal␤1-o-mentioning

confidence: 99%

Phosphorylation and Sulfation of Oligosaccharide Substrates Critically Influence the Activity of Human β1,4-Galactosyltransferase 7 (GalT-I) and β1,3-Glucuronosyltransferase I (GlcAT-I) Involved in the Biosynthesis of the Glycosaminoglycan-Protein Linkage Region of Proteoglycans

Gulberti

Lattard

Fondeur

et al. 2005

We determined whether the two major structural modifications, i.e. phosphorylation and sulfation of the glycosaminoglycan-protein linkage region (GlcA␤1-3Gal␤1-3Gal␤1-4Xyl␤1), govern the specificity of the glycosyltransferases responsible for the biosynthesis of the tetrasaccharide primer. We analyzed the influence of C-2 phosphorylation of Xyl residue on human ␤1,4-galactosyltransferase 7 (GalT-I), which catalyzes the transfer of Gal onto Xyl, and we evaluated the consequences of C-4/C-6 sulfation of Gal␤1-3Gal (Gal2-Gal1) on the activity and specificity of ␤1,3-glucuronosyltransferase I (GlcAT-I) responsible for the completion of the glycosaminoglycan primer sequence. For this purpose, a series of phosphorylated xylosides and sulfated C-4 and C-6 analogs of Gal␤1-3Gal was synthesized and tested as potential substrates for the recombinant enzymes. Our results revealed that the phosphorylation of Xyl on the C-2 position prevents GalT-I activity, suggesting that this modification may occur once Gal is attached to the Xyl residue of the nascent oligosaccharide linkage. On the other hand, we showed that sulfation on C-6 position of Gal1 of the Gal␤1-3Gal analog markedly enhanced GlcAT-I catalytic efficiency and we demonstrated the importance of Trp 243 and Lys 317 residues of Gal1 binding site for enzyme activity. In contrast, we found that GlcAT-I was unable to use digalactosides as acceptor substrates when Gal1 was sulfated on C-4 position or when Gal2 was sulfated on both C-4 and C-6 positions. Altogether, we demonstrated that oligosaccharide modifications of the linkage region control the specificity of the glycosyltransferases, a process that may regulate maturation and processing of glycosaminoglycan chains.

“…1 The abbreviations used are: DS, dermatan sulfate; CS, chondroitin sulfate; C4ST, chondroitin 4-O-sulfotransferase; HNK-1ST, HNK-1 sulfotransferase; GalNAc4ST, N-acetylgalactosamine 4-O-sulfotransferase; PAPS, 3Ј-phosphoadenosine 5Ј-phosphosulfate; ⌬HexUA, 4,5-unsaturated hexuronic acid or 4-deoxy-␣-L-threo-hex-4-ene-pyranosyluronic acid; ⌬Di-0S, ⌬ 4,5 HexUA␣1-3GalNAc; ⌬Di-4S, ⌬ 4,5 HexUA␣1-3GalNAc(4-O-sulfate); ⌬Di-6S, ⌬ 4,5 HexUA␣1-3GalNAc(6-O-sulfate); ⌬Di-diS D , ⌬ see Refs. [25][26][27]). 4-O-Sulfation of GalNAc residues is a typical modification found in CS/DS at higher frequency and was once postulated to be a prerequisite for the adjacent IdoUA formation by glucuronyl C5-epimerase, and GalNAc 4-O-sulfotransferase(s) had been suggested to be a critical and rate-limiting factor during DS biosynthesis (28).…”

mentioning

confidence: 99%

Specificities of Three Distinct Human Chondroitin/Dermatan N-Acetylgalactosamine 4-O-Sulfotransferases Demonstrated Using Partially Desulfated Dermatan Sulfate as an Acceptor

Mikami

Mizumoto

Kago

et al. 2003

111

4-O-Sulfation of GalNAc is a high frequency modification of chondroitin sulfate and dermatan sulfate (DS), and three major GalNAc 4-O-sulfotransferases including dermatan 4-O-sulfotransferase-1 (D4ST-1) and chondroitin 4-O-sulfotransferases-1 and -2 (C4ST-1 and -2) have been identified. 4-O-Sulfation of GalNAc during DS biosynthesis had long been postulated to be a prerequisite for iduronic acid (IdoUA) formation by C5-epimerization of GlcUA. This hypothesis has recently been argued based on enzymological studies using microsomes that C5-epimerization precedes 4-O-sulfation, which was further supported by the specificity of the cloned D4ST-1 with predominant preference for IdoUA-GalNAc flanked by GlcUA-GalNAc over IdoUA-GalNAc flanked by IdoUA-GalNAc in exhaustively desulfated dermatan. Whereas the counterproposal explains the initial reactions, apparently it cannot rationalize the synthetic mechanism of IdoUA-GalNAc(4-O-sulfate)-rich clusters typical of mature DS chains. In this study, we examined detailed specificities of the three recombinant human 4-O-sulfotransferases using partially desulfated DS as an acceptor. Enzymatic analysis of the transferase reaction products showed that D4ST-1 far more efficiently transferred sulfate to GalNAc residues in -IdoUA-GalNAc-IdoUA-than in -GlcUA-GalNAc-GlcUA-sequences. In contrast, C4ST-1 showed the opposite preference, and C4ST-2 used GalNAc residues in both sequences to comparable degrees, being consistent with its phylogenetic relations to D4ST-1 and C4ST-1. Structural analysis of the oligosaccharides, which were isolated after chondroitinase AC-I digestion of the 35 S-labeled transferase reaction products, revealed for the first time that D4ST-1, as compared with C4ST-1 and C4ST-2, most efficiently utilized GalNAc residues located not only in the sequence -IdoUA-GalNAc-IdoUA-but also in -GlcUA-GalNAc-IdoUA-and -IdoUA-GalNAc-GlcUA-. The isolated oligosaccharide structures also suggest that 4-O-sulfation promotes subsequent 4-O-sulfation of GalNAc in the neighboring disaccharide unit.