N-Linked Oligosaccharides on Chondroitin 6-Sulfotransferase-1 Are Required for Production of the Active Enzyme, Golgi Localization, and Sulfotransferase Activity toward Keratan Sulfate

Yusa, Akiko; Kitajima, Ken; Habuchi, Osami

doi:10.1074/jbc.m600140200

Cited by 23 publications

(19 citation statements)

References 40 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…However, the CS6OST showed significant differences between the two culture conditions and the length of time in culture, with a fourfold increase in expression under MM culture conditions. The CS6OST enzyme transfers the sulfate to the C-6 position of the Nacetylgalactosamine, with mutations in this gene resulting in chondrodysplasia with major involvement of the spine (Yusa et al, 2006; Fig. 5C).…”

Section: Gene Expression Of Sulfotransferases Associated With Osteogementioning

confidence: 96%

The heparan sulfate proteoglycan (HSPG) glypican‐3 mediates commitment of MC3T3‐E1 cells toward osteogenesis

Haupt¹,

Murali²,

Mun³

et al. 2009

Journal Cellular Physiology

View full text Add to dashboard Cite

Heparan sulfate (HS) sugar chains attached to core proteoglycans (PGs) termed HSPGs mediate an extensive range of cell-extracellular matrix (ECM) and growth factor interactions based upon their sulfation patterns. When compared with non-osteogenic (maintenance media) culture conditions, under established osteogenic culture conditions, MC3T3-E1 cells characteristically increase their osteogenic gene expression profile and switch their dominant fibroblast growth factor receptor (FGFR) from FGFR1 (0.5-fold decrease) to FGFR3 (1.5-fold increase). The change in FGFR expression profile of the osteogenic-committed cultures was reflected by their inability to sustain an FGF-2 stimulus, but respond to BMP-2 at day 14 of culture. The osteogenic cultures decreased their chondroitin and dermatan sulfate PGs (biglycan, decorin, and versican), but increased levels of the HS core protein gene expression, in particular glypican-3. Commitment and progress through osteogenesis is accompanied by changes in FGFR expression, decreased GAG initiation but increased N- and O-sulfation and reduced remodeling of the ECM (decreased heparanase expression) resulting in the production of homogenous (21 kDa) HS chain. With the HSPG glypican-3 expression strongly upregulated in these processes, siRNA was used to knockdown this gene to examine the effect on osteogenic commitment. Reduced glypican-3 abrogated the expression of Runx2, and thus differentiation. The reintroduction of this HSPG into Runx2-null cells allowed osteogenesis to proceed. These results demonstrate the dependence of osteogenesis on specific HS chains, in particular those associated with glypican-3.

show abstract

Section: Gene Expression Of Sulfotransferases Associated With Osteogementioning

confidence: 96%

The heparan sulfate proteoglycan (HSPG) glypican‐3 mediates commitment of MC3T3‐E1 cells toward osteogenesis

Haupt¹,

Murali²,

Mun³

et al. 2009

Journal Cellular Physiology

View full text Add to dashboard Cite

show abstract

“…It is imperative to remove the viral chondroitinase contamination from the enzyme preparations [17] as the chondroitinase degrades chondroitin oligosaccharide backbones,the substrates used by both CS4OST and CS6OST.T he expression of CS6OST in E. coli was unsuccessful possibly owing to the fact that glycosylation is required for the sulfotransferase activity. [18] Thep roducts were analyzed in terms of their purity and structure by high-resolution anion-exchange HPLC,e lectrospray ionization mass spectrometry (ESI-MS), and NMR spectroscopy.T he majority of products gave rise to as ingle symmetric peak in the HPLC trace,c onfirming their purity (Table S1 and Figures S1-S60). One exception is compound 4, which displayed ap urity of only 59 %b ecause the trifluoroacetyl group on residue Di sl abile,a nd am ixture of compound 4 and detrifluoroacetylated compound 6 was obtained, as demonstrated by ESI-MS analysis ( Figure S13).…”

Section: Zuschriftenmentioning

confidence: 99%

“…The chondroitinase degrades chondroitin oligosaccharide backbones, the substrates used by both CS4OST and CS6OST. The expression of CS6OST in E. coli was unsuccessful possibly due to the fact that glycosylation is required for the sulfotransferase activity [18] .…”

mentioning

confidence: 99%

Enzymatic Synthesis of Homogeneous Chondroitin Sulfate Oligosaccharides

Liu

2017

Angew Chem Int Ed

View full text Add to dashboard Cite

Chondroitin sulfate (CS) is a sulfated polysaccharide that plays essential physiological roles. Here, we report to utilize an enzyme-based method to synthesize a library of CS oligosaccharides consisting of 15 different CS oligosaccharides. The library covers 4-O-sulfated and 6-O-sulfated oligosaccharides with the size ranging from trisaccharide to nonasaccharide. We also demonstrate the synthesis of unnatural 6-O-sulfated CS pentasaccharides containing either a 6-O-sulfo 2-azido galactosamine or a 6-O-sulfo galactosamine residue. The availability of structurally defined CS oligosaccharides offers a novel approach to investigate the biological functions of CS.

show abstract

“…Several enzymes involved in the biosynthesis of dermatan sulfate have been shown to be dependent on N-glycosylation for proper function (27,28). We therefore investigated the importance of N-glycosylation for enzymatic activity of DS-epimerase 1.…”

Section: N-glycosylation Is Required For Ds-epimerase 1 Activity-mentioning

confidence: 99%

Identification of the Active Site of DS-epimerase 1 and Requirement of N-Glycosylation for Enzyme Function

Pacheco

Maccarana

Malmström

et al. 2009

Journal of Biological Chemistry

View full text Add to dashboard Cite

Dermatan sulfate is a highly sulfated polysaccharide and has a variety of biological functions in development and disease. Iduronic acid domains in dermatan sulfate, which are formed by the action of two DS-epimerases, have a key role in mediating these functions. We have identified the catalytic site and three putative catalytic residues in DS-epimerase 1, His-205, Tyr-261, and His-450, by tertiary structure modeling and amino acid conservation to heparinase II. These residues were systematically mutated to alanine or more conserved residues, which resulted in complete loss of epimerase activity. Based on these data and the close relationship between lyase and epimerase reactions, we propose a model where His-450 functions as a general base abstracting the C5 proton from glucuronic acid. Subsequent cleavage of the glycosidic linkage by Tyr-261 generates a 4,5-unsaturated hexuronic intermediate, which is protonated at the C5 carbon by His-205 from the side of the sugar plane opposite to the side of previous proton abstraction. Concomitant recreation of the glycosidic linkage ends the reaction, generating iduronic acid. In addition, we show that proper N-glycosylation of DS-epimerase 1 is required for enzyme activity. This study represents the first description of the structural basis for epimerization by a glycosaminoglycan epimerase.Chondroitin/dermatan sulfate proteoglycans are ubiquitous components of extracellular matrices and have been implicated in several biological processes, such as cell proliferation, cell signaling, wound healing, blood coagulation and neurite outgrowth (1, 2). Chondroitin/dermatan sulfate glycosaminoglycan chains consist of a repeated disaccharide unit of GlcA and GalNAc, where in DS, 3 in addition to ␤-D-GlcA, its epimer ␣-Liduronic acid (IdoA) can be found to various extents. The chain can also be modified by sulfation of position 2 of GlcA/IdoA and of positions 4 and 6 of GalNAc by sulfotransferases that are specific for each position (3). The degree of epimerization and sulfation and the position of the sulfate groups vary during development and among tissues and are regulated by growth factors (4).The presence of IdoA is critical in mediating several biological functions of dermatan sulfate, such as the binding to hepatocyte growth factor and heparin cofactor II (1). IdoA is also required for self-interaction of DS chains, and the self-interactions of decorin side chains are believed to be important in collagen fibrillogenesis (5, 6). This can be explained by the fact that IdoA increases the glycosaminoglycan chains conformational flexibility, which facilitates interactions (7).Epimerization of GlcA into IdoA in dermatan sulfate is catalyzed by the DS-epimerase, which we recently purified and cloned (8). Based on sequence similarity with the DS-epimerase, a second epimerase has also been identified, and its activity has been established. 4 Here, the two enzymes are referred to as DS-epimerase 1 (encoded by the DSE gene) and DS-epimerase 2 (encoded by the DSEL gene), respectively...

show abstract

N-Linked Oligosaccharides on Chondroitin 6-Sulfotransferase-1 Are Required for Production of the Active Enzyme, Golgi Localization, and Sulfotransferase Activity toward Keratan Sulfate

Cited by 23 publications

References 40 publications

The heparan sulfate proteoglycan (HSPG) glypican‐3 mediates commitment of MC3T3‐E1 cells toward osteogenesis

The heparan sulfate proteoglycan (HSPG) glypican‐3 mediates commitment of MC3T3‐E1 cells toward osteogenesis

Enzymatic Synthesis of Homogeneous Chondroitin Sulfate Oligosaccharides

Identification of the Active Site of DS-epimerase 1 and Requirement of N-Glycosylation for Enzyme Function

Contact Info

Product

Resources

About