Differential Roles of TwoN-Acetylgalactosaminyltransferases, CSGalNAcT-1, and a Novel Enzyme, CSGalNAcT-2

Abstract

By a tblastn search with ␤1,4-galactosyltransferases as query sequences, we found an expressed sequence tag that showed similarity in ␤1,4-glycosyltransferase motifs. The full-length complementary DNA was obtained by a method of 5-rapid amplification of complementary DNA ends. The predicted open reading frame encodes a typical type II membrane protein comprising 543 amino acids, the sequence of which was highly homologous to chondroitin sulfate N-acetylgalactosaminyltransferase (CSGalNAcT-1), and we designated this novel enzyme CSGalNAcT-2. CSGalNAcT-2 showed much stronger Nacetylgalactosaminyltransferase activity toward glucuronic acid of chondroitin poly-and oligosaccharides, and chondroitin sulfate poly-and oligosaccharides with a ␤1-4 linkage, i.e. elongation activity for chondroitin and chondroitin sulfate, but showed much weaker activity toward a tetrasaccharide of the glycosaminoglycan linkage structure (GlcA-Gal-Gal-Xyl-O-methoxyphenyl), i.e. initiation activity, than CSGalNAcT-1. Transfection of the CSGalNAcT-1 gene into Chinese hamster ovary cells yielded a change of glycosaminoglycan composition, i.e. the replacement of heparan sulfate on a syndecan-4/fibroblast growth factor-1 chimera protein by chondroitin sulfate, however, transfection of the CSGalNAcT-2 gene did not. The above results indicated that CSGalNAcT-1 is involved in the initiation of chondroitin sulfate synthesis, whereas CSGalNAcT-2 participates mainly in the elongation, not initiation. Quantitative real-time PCR analysis revealed that CSGalNAcT-2 tran-

Chondroitin Sulfate N-Acetylgalactosaminyltransferase-1 Plays a Critical Role in Chondroitin Sulfate Synthesis in Cartilage

“…4B). In tissues other than cartilage, CSGalNacT-1 is expressed at the highest level in the thyroid gland and placenta, which exhibit expression only up to 1.5-5-fold that of CSGalNAcT-2 (20). In addition, various cell lines show relatively low levels of CSGalNAcT-1 expression compared with other enzymes (18).…”

“…Syndecan-4 in CSGalNAcT-1-overexpressing COS7 cells contains a large amount of CS (20). CSGalNAcT-1 overexpression in Balb/3T3 cells, a cell line established from normal mouse fibroblast cells, achieves ϳ5-fold CS synthesis.…”

“…The amplified fragment was subcloned into pcDNA/Hygro (Invitrogen) using the HindIII and XbaI sites, as previously described (20). The CSGalNAcT-1/pcDNA3.1(ϩ), CSS-1/pcDNA3.1(ϩ), CSS-2/pcDNA3.1(ϩ), and CSGlcAT/pcDNA3.1(ϩ) vectors were constructed by subcloning the full-length cDNA into pcDNA3.1 (Invitrogen).…”

Genistein-mediated inhibition of glycosaminoglycan synthesis as a basis for gene expression-targeted isoflavone therapy for mucopolysaccharidoses

“…However, in the process leading to synthesis of GAGs, the monomers are carbohydrates or their derivatives that are also involved in many other metabolic pathways. 10 Therefore, a putative competitor that could block activity of one of enzymes taking part in GAG synthesis, would most probably interfere with many other metabolic pathways, by blocking other biochemical reactions, thus giving potentially serious side effects. Therefore, to avoid this problem we aimed to develop SRT based on regulation of expression of genes coding for specific GAG synthetases.…”

Molecular Cloning and Characterization of a Novel Human β1,4-N-Acetylgalactosaminyltransferase, β4GalNAc-T3, Responsible for the Synthesis of N,N′-Diacetyllactosediamine, GalNAcβ1–4GlcNAc

“…In the case of CSSs, the ␤4GalNAc-T domain exists in the C-terminal region. Chondroitin sulfate Nacetylgalactosaminyltransferase I and II (CSGalNAc-T1 and -T2) both act as ␤4GalNAc-Ts in the initiation and elongation of chondroitin sulfate synthesis (13)(14)(15)(16).…”