A Unique Nonreducing Terminal Modification of Chondroitin Sulfate by N-Acetylgalactosamine 4-Sulfate 6-O-Sulfotransferase

Ohtake, Shiori; Kimata, Koji; Habuchi, Osami

doi:10.1074/jbc.m306132200

Cited by 45 publications

(38 citation statements)

References 37 publications

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“…Although the enzyme was reported to preferentially catalyze GalNAc(4S) residues at the nonreducing end of CSA (6,30), it also catalyzed inside 4S units. Although UA2ST sulfated a GlcUA residue with only 4% of the 6S units in sCSC-HMDA, it transferred ϳ90% of the 6S units in sCSAC-HMDA, consistent with a previous report (31) showing that UA2ST preferentially sulfates the GlcUA residue of GalNAc(4S)-GlcUAGalNAc(6S).…”

Section: Discussionmentioning

confidence: 99%

Construction of a Chondroitin Sulfate Library with Defined Structures and Analysis of Molecular Interactions

Sugiura

Shioiri

Chiba

et al. 2012

Journal of Biological Chemistry

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Background: Chondroitin sulfate (CS) is a linear polysaccharide, composed of repeating disaccharide units and modified with sulfate groups at various positions.Results: A CS library was constructed with defined structures using CS polymerase and sulfotransferases. Conclusion: The CS library provided details of interactions with CS-binding molecules. Significance: Chemo-enzymatic synthesis provides a useful tool for studying the biological functions of CS.Chondroitin sulfate (CS) is a linear acidic polysaccharide, composed of repeating disaccharide units of glucuronic acid and N-acetyl-D-galactosamine and modified with sulfate residues at different positions, which plays various roles in development and disease. Here, we chemo-enzymatically synthesized various CS species with defined lengths and defined sulfate compositions, from chondroitin hexasaccharide conjugated with hexamethylenediamine at the reducing ends, using bacterial chondroitin polymerase and recombinant CS sulfotransferases, including chondroitin-4-sulfotransferase 1 (C4ST-1), chondroitin-6-sulfotransferase 1 (C6ST-1), N-acetylgalactosamine 4-sulfate 6-sulfotransferase (GalNAc4S-6ST), and uronosyl 2-sulfotransferase (UA2ST). Sequential modifications of CS with a series of CS sulfotransferases revealed their distinct features, including their substrate specificities. Reactions with chondroitin polymerase generated non-sulfated chondroitin, and those with C4ST-1 and C6ST-1 generated uniformly sulfated CS containing >95% 4S and 6S units, respectively. GalNAc4S-6ST and UA2ST generated highly sulfated CS possessing ϳ90% corresponding disulfated disaccharide units. Sequential reactions with UA2ST and GalNAc4S-6ST generated further highly sulfated CS containing a mixed structure of disulfated units. Surprisingly, sequential reactions with GalNAc4S-6ST and UA2ST generated a novel CS molecule containing ϳ29% trisulfated disaccharide units. Enzyme-linked immunosorbent assay and surface plasmon resonance analysis using the CS library and natural CS products modified with biotin at the reducing ends, revealed details of the interactions of CS species with anti-CS antibodies, and with CS-binding molecules such as midkine and pleiotrophin. Chemo-enzymatic synthesis enables the generation of CS chains of the desired lengths, compositions, and distinct structures, and the resulting library will be a useful tool for studies of CS functions. Chondroitin sulfate (CS)2 is a glycosaminoglycan that is a linear acidic polysaccharide composed of repeating disaccharide units (-4 D-glucuronic acid (GlcUA) ␤ 1-3 N-acetyl-dgalactosamine (GalNAc)␤1-) n and modified with sulfate groups at various positions on the sugar residues (1). The average molecular weight (M r ) of CS is 10,000ϳ100,000 consisting of 40ϳ400 saccharide residues as a mature molecule (Table 1).The major disaccharide structures (see Fig. 1A) of CS are as follows: a non-sulfated unit (0S, GlcUA-GalNAc), a monosulfated unit at the C-4 position of the GalNAc residue (4S, GlcUA-GalNAc(4S)), a monosulfated unit at ...

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Section: Discussionmentioning

confidence: 99%

Construction of a Chondroitin Sulfate Library with Defined Structures and Analysis of Molecular Interactions

Sugiura

Shioiri

Chiba

et al. 2012

Journal of Biological Chemistry

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“…The Chst15 gene has highest sequence identity to human and rat type II transmembrane N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase (GalNAc4S6ST) found in the Golgi system required for biogenesis of glycoproteins (Ohtake et al 2001). It catalyzes the transfer of sulfate from 3′-phosphoadenosine 5′-phosphosulfate to position 6 of the non-reducing GalNAc(4SO(4)) residues contained in proteins such as chondroitin sulfate A and dermatan sulfate (Ohtake et al 2001(Ohtake et al , 2003. The extent of contribution of each of these genes to the head bobber phenotype will require further investigation.…”

Section: Discussionmentioning

confidence: 99%

Head Bobber: An Insertional Mutation Causes Inner Ear Defects, Hyperactive Circling, and Deafness

Somma

Alger

McGuire

et al. 2012

JARO

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The head bobber transgenic mouse line, produced by pronuclear integration, exhibits repetitive head tilting, circling behavior, and severe hearing loss. Transmitted as an autosomal recessive trait, the homozygote has vestibular and cochlea inner ear defects. The space between the semicircular canals is enclosed within the otic capsule creating a vacuous chamber with remnants of the semicircular canals, associated cristae, and vestibular organs. A poorly developed stria vascularis and endolymphatic duct is likely the cause for Reissner's membrane to collapse post-natally onto the organ of Corti in the cochlea. Molecular analyses identified a single integration of~3 tandemly repeated copies of the transgene, a short duplicated segment of chromosome X and a 648 kb deletion of chromosome 7(F3). The three known genes (Gpr26, Cpxm2, and Chst15) in the deleted region are conserved in mammals and expressed in the wild-type inner ear during vestibular and cochlea development but are absent in homozygous mutant ears. We propose that genes critical for inner ear patterning and differentiation are lost at the head bobber locus and are candidate genes for human deafness and vestibular disorders.

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“…The →4)-β-D-GlcA (1→3)-β-D-GalNAc4S6S (1→ (S E ) sequence is involved in critical receptor binding functions 73 and is also believed to terminate CS-A chains. 74 The GAG chain modifying enzymes in HS biosynthesis are a much more complex story because of the multiple isoforms of the 6-OST and 3-OST enzymes involved late in the biosynthetic pathway. 71,75 These can afford the enormous structural diversity responsible for the interaction of HS with many different proteins.…”

Section: Discussionmentioning

confidence: 99%

Glycomics of Proteoglycan Biosynthesis in Murine Embryonic Stem Cell Differentiation

et al. 2007

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Glycosaminoglycans (GAGs) play a critical role in binding and activation of growth factors involved in cell signaling critical for developmental biology. The biosynthetic pathways for GAGs have been elucidated over the past decade and now analytical methodology makes it possible to determine GAG composition in as few as 10 million cells. A glycomics approach was used to examine GAG content, composition, and the level of transcripts encoding for GAG biosynthetic enzymes as murine embryonic stem cells (mESCs) differentiate to embryoid bodies (EBs) and to extraembryonic endodermal cells (ExE) to better understand the role of GAGs in stem cell differentiation. Hyaluronan synthesis was enhanced by 13-and 24-fold, most likely due to increased expression of hyaluronan synthase-2. Chondroitin sulfate (CS)/dermatan sulfate (DS) synthesis was enhanced by 4-and 6-fold, and heparan sulfate (HS) synthesis was enhanced by 5-and 8-fold following the transition from mESC to EB and ExE. Transcripts associated with the synthesis of the early precursors were largely unaltered, suggesting other factors account for enhanced GAG synthesis. The composition of both CS/DS and HS also changed upon differentiation. Interestingly, CS type E and highly sulfated HS both increase as mESCs differentiate to EBs and ExE. Differentiation was also accompanied by enhanced 2-sulfation in both CS/DS and HS families. Transcript levels for core proteins generally showed increases or remained constant upon mESC differentiation. Finally, transcripts encoding selected enzymes and isoforms, including GlcNAc-4,6-O-sulfotransferase, C5-epimerases, and 3-O-sulfotransferases involved in late GAG biosynthesis, were also enriched. These biosynthetic enzymes are particularly important in introducing GAG fine structure, essential for intercellular communication, cell adhesion, and outside-in signaling. Knowing the changes in GAG fine structure should improve our understanding the biological properties of differentiated stem cells.

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A Unique Nonreducing Terminal Modification of Chondroitin Sulfate by N-Acetylgalactosamine 4-Sulfate 6-O-Sulfotransferase

Cited by 45 publications

References 37 publications

Construction of a Chondroitin Sulfate Library with Defined Structures and Analysis of Molecular Interactions

Construction of a Chondroitin Sulfate Library with Defined Structures and Analysis of Molecular Interactions

Head Bobber: An Insertional Mutation Causes Inner Ear Defects, Hyperactive Circling, and Deafness

Glycomics of Proteoglycan Biosynthesis in Murine Embryonic Stem Cell Differentiation

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