Dataset Information

GlcUA?1-3Gal?1-3Gal?1-4Xyl(2-O-phosphate) is the preferred substrate for chondroitin N-acetylgalactosaminyltransferase-1.

ABSTRACT: A deficiency in chondroitin N-acetylgalactosaminyltransferase-1 (ChGn-1) was previously shown to reduce the number of chondroitin sulfate (CS) chains, leading to skeletal dysplasias in mice, suggesting that ChGn-1 regulates the number of CS chains for normal cartilage development. Recently, we demonstrated that 2-phosphoxylose phosphatase (XYLP) regulates the number of CS chains by dephosphorylating the Xyl residue in the glycosaminoglycan-protein linkage region of proteoglycans. However, the relationship between ChGn-1 and XYLP in controlling the number of CS chains is not clear. In this study, we for the first time detected a phosphorylated tetrasaccharide linkage structure, GlcUA?1-3Gal?1-3Gal?1-4Xyl(2-O-phosphate), in ChGn-1(-/-) growth plate cartilage but not in ChGn-2(-/-) or wild-type growth plate cartilage. In contrast, the truncated linkage tetrasaccharide GlcUA?1-3Gal?1-3Gal?1-4Xyl was detected in wild-type, ChGn-1(-/-), and ChGn-2(-/-) growth plate cartilage. Consistent with the findings, ChGn-1 preferentially transferred N-acetylgalactosamine to the phosphorylated tetrasaccharide linkage in vitro. Moreover, ChGn-1 and XYLP interacted with each other, and ChGn-1-mediated addition of N-acetylgalactosamine was accompanied by rapid XYLP-dependent dephosphorylation during formation of the CS linkage region. Taken together, we conclude that the phosphorylated tetrasaccharide linkage is the preferred substrate for ChGn-1 and that ChGn-1 and XYLP cooperatively regulate the number of CS chains in growth plate cartilage.

SUBMITTER: Izumikawa T

PROVIDER: S-EPMC4342460 | biostudies-literature | 2015 Feb

REPOSITORIES: biostudies-literature

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GlcUAβ1-3Galβ1-3Galβ1-4Xyl(2-O-phosphate) is the preferred substrate for chondroitin N-acetylgalactosaminyltransferase-1.

Izumikawa Tomomi T Sato Ban B Mikami Tadahisa T Tamura Jun-ichi J Igarashi Michihiro M Kitagawa Hiroshi H

The Journal of biological chemistry 20150107 9

A deficiency in chondroitin N-acetylgalactosaminyltransferase-1 (ChGn-1) was previously shown to reduce the number of chondroitin sulfate (CS) chains, leading to skeletal dysplasias in mice, suggesting that ChGn-1 regulates the number of CS chains for normal cartilage development. Recently, we demonstrated that 2-phosphoxylose phosphatase (XYLP) regulates the number of CS chains by dephosphorylating the Xyl residue in the glycosaminoglycan-protein linkage region of proteoglycans. However, the re ...[more]

PMID: 25568321

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Project description:Chondroitin sulfate (CS) is a sulfated glycosaminoglycan (GAG) chain. In cartilage, CS plays important roles as the main component of the extracellular matrix (ECM), existing as side chains of the major cartilage proteoglycan, aggrecan. Six glycosyltransferases are known to coordinately synthesize the backbone structure of CS; however, their in vivo synthetic mechanism remains unknown. Previous studies have suggested that two glycosyltransferases, Csgalnact1 (t1) and Csgalnact2 (t2), are critical for initiation of CS synthesis in vitro. Indeed, t1 single knockout mice (t1 KO) exhibit slight dwarfism and a reduction in CS content in cartilage compared with wild-type (WT) mice. To reveal the synergetic roles of t1 and t2 in CS synthesis in vivo, we generated systemic single and double knockout (DKO) mice and cartilage-specific t1 and t2 double knockout (Col2-DKO) mice. DKO mice exhibited postnatal lethality, whereas t2 KO mice showed normal size and skeletal development. Col2-DKO mice survived to adulthood and showed severe dwarfism compared with t1 KO mice. Histological analysis of epiphyseal cartilage from Col2-DKO mice revealed disrupted endochondral ossification, characterized by drastic GAG reduction in the ECM. Moreover, DKO cartilage had reduced chondrocyte proliferation and an increased number of apoptotic chondrocytes compared with WT cartilage. Conversely, primary chondrocyte cultures from Col2-DKO knee cartilage had the same proliferation rate as WT chondrocytes and low GAG expression levels, indicating that the chondrocytes themselves had an intact proliferative ability. Quantitative RT-PCR analysis of E18.5 cartilage showed that the expression levels of Col2a1 and Ptch1 transcripts tended to decrease in DKO compared with those in WT mice. The CS content in DKO cartilage was decreased compared with that in t1 KO cartilage but was not completely absent. These results suggest that aberrant ECM caused by CS reduction disrupted endochondral ossification. Overall, we propose that both t1 and t2 are necessary for CS synthesis and normal chondrocyte differentiation but are not sufficient for all CS synthesis in cartilage.

Project description:O-Glycan biosynthesis is initiated by the transfer of N-acetylgalactosamine (GalNAc) from a nucleotide sugar donor (UDP-GalNAc) to Ser/Thr residues of an acceptor substrate. The detailed transfer mechanism, catalyzed by the UDP-GalNAc polypeptide:N-acetyl-alpha-galactosaminyltransferases (ppGalNAcTs), remains unclear despite structural information available for several isoforms in complex with substrates at various stages along the catalytic pathway. We used all-atom molecular dynamics simulations with explicit solvent and counterions to study the conformational dynamics of ppGalNAcT-2 in several enzymatic states along the catalytic pathway. ppGalNAcT-2 is simulated both in the presence and in the absence of substrates and reaction products to examine the role of conformational changes in ligand binding. In multiple 40-ns-long simulations of more than 600 ns total run time, we studied systems ranging from 45,000 to 95,000 atoms. Our simulations accurately identified dynamically active regions of the protein, as previously revealed by the X-ray structures, and permitted a detailed, atomistic description of the conformational changes of loops near the active site and the characterization of the ensemble of structures adopted by the transferase complex on the transition pathway between the ligand-bound and ligand-free states. In particular, the conformational transition of a functional loop adjacent to the active site from closed (active) to open (inactive) is correlated with the rotameric state of the conserved residue W331. Analysis of water dynamics in the active site revealed that internal water molecules have an important role in enhancing the enzyme flexibility. We also found evidence that charged side chains in the active site rearrange during site opening to facilitate ligand binding. Our results are consistent with the single-displacement transfer mechanism previously proposed for ppGalNAcTs based on X-ray structures and mutagenesis data and provide new evidence for possible functional roles of certain amino acids conserved across several isoforms.

Project description:It is suggested that most cases of low back pain are related to degeneration of intervertebral discs. Disc degeneration is a chronic and progressive disease and the search for effective medical treatments continues. Neurotropin is widely used in Japan and China to treat low back pain and neck-shoulder-arm syndrome. The present study aimed to investigate the effect of Neurotropin on glycosaminoglycan synthesis in nucleus pulposus cells. Cultured human nucleus pulposus cells were treated with Neurotropin every second day for two weeks. Production of glycosaminoglycan was assessed using a dimethyl-methylene blue assay and PicoGreen was used to measure DNA content. Microarray analysis, real-time PCR, and western blotting were performed to assess the biological processes related to Neurotropin-stimulated glycosaminoglycan synthesis. The results showed that the level of glycosaminoglycan normalized to DNA content was significantly upregulated by the addition of Neurotropin. Gene expression profiling showed over two-fold upregulation of 697 genes in response to Neurotropin treatment. Among these genes, ontological analysis suggested significant implication of phosphatidylinositol 3-kinase signaling, and analysis focused on this pathway demonstrated marked upregulation of angiopoietin 1 and insulin-like growth factor 1. Activation of phosphorylation of the signal transducer protein AKT was detected by western blotting. Of the genes related to sulfated glycosaminoglycan synthesis, the greatest increase in mRNA levels was observed for chondroitin sulfate N-acetylgalactosaminyltransferase 1, an enzyme initiating synthesis of chondroitin sulfate side chains attached to a core protein of aggrecan, which is a predominant disc matrix component. These findings suggest that Neurotropin may activate the phosphatidylinositol 3-kinase-AKT pathway and stimulate glycosaminoglycan synthesis through upregulation of expression of mRNA for chondroitin sulfate N-acetylgalactosaminyltransferase 1. Because there was no cytotoxic cellular growth inhibition, Neurotropin treatment might offer an accessible therapeutic strategy for intervertebral disc degeneration.

Dataset Information

GlcUA?1-3Gal?1-3Gal?1-4Xyl(2-O-phosphate) is the preferred substrate for chondroitin N-acetylgalactosaminyltransferase-1.

Publications

GlcUAβ1-3Galβ1-3Galβ1-4Xyl(2-O-phosphate) is the preferred substrate for chondroitin N-acetylgalactosaminyltransferase-1.

Similar Datasets

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