N-Acetylgalactosamine (GalNAc) Transfer to the Common Carbohydrate-Protein Linkage Region of Sulfated Glycosaminoglycans

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Background: EXTL2 is one of three EXT-like genes homologous to the tumor suppressor EXT gene family members and encodes an N-acetylhexosaminyltransferase. Results: EXTL2 terminated polymerization of glycosaminoglycan (GAG) chains by transferring an N-acetylglucosamine residue to the phosphorylated tetrasaccharide linkage region. Conclusion: EXTL2 controlled GAG biosynthesis in a xylose kinase-dependent manner. Significance: Lack of EXTL2 causes GAG overproduction associated with pathological processes.

Section: Embryonic Fibroblastsmentioning

confidence: 87%

EXTL2, a Member of the EXT Family of Tumor Suppressors, Controls Glycosaminoglycan Biosynthesis in a Xylose Kinase-dependent Manner

Nadanaka

Zhou

Kagiyama

et al. 2013

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“…Chondroitin GalNAcT-2 is a unique ␤1,4-GalNAc transferase that utilizes the tetrasaccharide serine, GlcUA␤1-3Gal␤1-3Gal␤1-4Xyl␤1-O-Ser, from the linkage region as an acceptor substrate. Previously, when this compound was tested as an acceptor together with a sugar donor, UDP-GalNAc, to search for such GalNAcTs using fetal bovine sera or mouse mastocytoma cell extracts as the enzyme source, only ␣-GalNAc transferase activity was detected, resulting in the exclusive production of an ␣-GalNAc-capped pentasaccharide, GalNAc␣1-4GlcUA␤1-3Gal␤1-3Gal␤1-4Xyl␤1-O-Ser (27,40,41). Therefore, high expression of ␣-GalNAc transferase might have interfered with the identification of a ␤1,4-GalNAc transferase, such as chondroitin GalNAcT-2.…”

Section: Discussionmentioning

confidence: 99%

“…GlcUA␤1-3Gal␤1-O-C 2 H 4 NHCbz was also synthesized. 2 Chondro-hexasaccharide (GlcUA␤1-3GalNAc) 3 was prepared from chondroitin as previously described (27 Construction of a Soluble Form of the Novel Chondroitin GalNAcT-A cDNA fragment of a truncated form of the novel chondroitin GalNAcT lacking the first 36 N-terminal amino acids was amplified by reverse transcription PCR with total RNA derived from G361 human melanoma cells (ATCC CRL-1424) as a template using a 5Ј-primer (5Ј-CGCGGATCCTTGTTAGGCAAATACACATTAATAAG-3Ј) containing an in-frame BamHI site and a 3Ј-primer (5Ј-CGCGGATCCGTTTT-GTGGTTCATACAGTAACGC-3Ј) containing a BamHI site located 37 bp downstream from the stop codon. PCR was carried out with Pfu polymerase (Stratagene, La Jolla, CA) for 30 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 120 s in 5% (v/v) dimethyl sulfoxide.…”

Section: Materials-udp-[u-mentioning

confidence: 99%

Molecular Cloning and Expression of a Second Chondroitin N-Acetylgalactosaminyltransferase Involved in the Initiation and Elongation of Chondroitin/Dermatan Sulfate

Uyama¹,

Kitagawa²,

Tanaka³

et al. 2003

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117

We identified a novel human chondroitin N-acetylgalactosaminyltransferase, designated chondroitin GalNAcT-2 after a BLAST analysis of the GenBank TM data base using the sequence of a previously described human chondroitin N-acetylgalactosaminyltransferase (chondroitin GalNAcT-1) as a probe. The new cDNA sequence contained an open reading frame encoding a protein of 542 amino acids with a type II transmembrane protein topology. The amino acid sequence displayed 60% identity to that of human chondroitin GalNAcT-1. Like chondroitin GalNAcT-1, the expression of a soluble form of the protein in COS-1 cells produced an active enzyme, which not only transferred ␤1,4-N-acetylgalactosamine (GalNAc) from UDP-[ 3 H]GalNAc to a polymer chondroitin representing growing chondroitin chains (␤-GalNAc transferase II activity) but also to GlcUA␤1-3Gal␤1-O-C 2 H 4 NHCbz, a synthetic substrate for ␤-GalNAc transferase I that transfers the first GalNAc to the core tetrasaccharide in the protein-linkage region of chondroitin sulfate. In contrast, the tetrasaccharide serine (GlcUA␤1-3Gal␤1-3Gal␤1-4Xyl␤1-O-Ser) derived from the linkage region, which is an inert acceptor substrate for chondroitin GalNAcT-1, served as an acceptor substrate. The coding region of this enzyme was divided into seven discrete exons, which is similar to the genomic organization of the chondroitin GalNAcT-1 gene, and was localized to chromosome 10q11.22. Northern blot analysis revealed that the chondroitin GalNAcT-2 gene exhibited a ubiquitous but differing expression in human tissues, and the expression pattern differed from that of chondroitin GalNAcT-1. Thus, we demonstrated redundancy in the chondroitin GalNAc transferases involved in the biosynthetic initiation and elongation of chondroitin sulfate, which is important for understanding the biosynthetic mechanisms leading to the selective chain assembly of chondroitin/dermatan sulfate on the linkage region tetrasaccharide common to various proteoglycans containing chondroitin/dermatan sulfate and heparin/heparan sulfate chains.

“…Asialo-ovine submaxillary mucin was obtained by treating ovine submaxillary mucin prepared according to Tettamanti and Pigman (18) with Arthrobacter ureafaciens sialidase (Nakarai Tesque, Kyoto, Japan). Chondro-hexasaccharide (GlcUA␤1-3GalNAc) 3 and chondro-octasaccharide (GlcUA␤1-3GalNAc) 4 were prepared from chondroitin as described previously (19). (GlcUA␤1-3GlcNAc) 5 was prepared from hyaluronan as described previously (20).…”

mentioning

confidence: 99%

Molecular Cloning and Expression of a Human Chondroitin Synthase

Kitagawa¹,

Uyama²,

Sugahara³

2001

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188

150

We have identified a human chondroitin synthase from the HUGE (human unidentified gene-encoded large proteins) protein data base by screening with two keywords: "one transmembrane domain" and "galactosyltransferase family." The identified protein consists of 802 amino acids with a type II transmembrane protein topology. The protein showed weak homology to the ␤1,3-galactosyltransferase family on the amino-terminal side and to the ␤1,4-galactosyltransferase family on the carboxyl-terminal side. The expression of a soluble recombinant form of the protein in COS-1 cells produced an active enzyme, which transferred not only the glucuronic acid (GlcUA) from UDP-[ 14 C]GlcUA but also Nacetylgalactosamine (GalNAc) from UDP-[ 3 H]GalNAc to the polymer chondroitin. Identification of the reaction products demonstrated that the enzyme was chondroitin synthase, with both ␤1,3-GlcUA transferase and ␤1,4-GalNAc transferase activities. The coding region of the chondroitin synthase was divided into three discrete exons and localized to chromosome 15. Northern blot analysis revealed that the chondroitin synthase gene exhibited ubiquitous but markedly differential expression in the human tissues examined. Thus, we demonstrated that analogous to human heparan sulfate polymerases, the single polypeptide chondroitin synthase possesses two glycosyltransferase activities required for chain polymerization.