Sequential synthesis of chondroitin oligosaccharides by immobilized chondroitin polymerase mutants

Sugiura, Norío; Shimokata, Satoshi; Minamisawa, Toshikazu; Hirabayashi, Jun; Kimata, Koji; Watanabe, Hideto

doi:10.1007/s10719-008-9105-0

Cited by 28 publications

(19 citation statements)

References 32 publications

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“…Chondroitin polymerase from Escherichia coli strain K4 (K4CP) and point mutants of the enzyme were prepared as described previously [23]. Recombinant chondroitin sulfate sulfotransferases C4ST-1, C6ST-1, GalNac4S-6ST, and UA2ST were prepared as described previously [22].…”

Section: Methodsmentioning

confidence: 99%

Molecular dissection of placental malaria protein VAR2CSA interaction with a chemo-enzymatically synthesized chondroitin sulfate library

et al. 2016

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Placental malaria, a serious infection caused by the parasite Plasmodium falciparum, is characterized by the selective accumulation of infected erythrocytes (IEs) in the placentas of the pregnant women. Placental adherence is mediated by the malarial VAR2CSA protein, which interacts with chondroitin sulfate (CS) proteoglycans present in the placental tissue. CS is a linear acidic polysaccharide composed of repeating disaccharide units of D-glucuronic acid and N-acetyl-D-galactosamine that are modified by sulfate groups at different positions. Previous reports have shown that placental-adhering IEs were associated with an unusually low sulfated form of chondroitin sulfate A (CSA) and that a partially sulfated dodecasaccharide is the minimal motif for the interaction. However, the fine molecular structure of this CS chain remains unclear. In this study, we have characterized the CS chain that interacts with a recombinant minimal CS-binding region of VAR2CSA (rVAR2) using a CS library of various defined lengths and sulfate compositions. The CS library was chemo-enzymatically synthesized with bacterial chondroitin polymerase and recombinant CS sulfotransferases. We found that C-4 sulfation of the N-acetyl-D-galactosamine residue is critical for supporting rVAR2 binding, whereas no other sulfate modifications showed effects. Interaction of rVAR2 with CS is highly correlated with the degree of C-4 sulfation and CS chain length. We confirmed that the minimum structure binding to rVAR2 is a tri-sulfated CSA dodecasaccharide, and found that a highly sulfated CSA eicosasaccharide is a more potent inhibitor of rVAR2 binding than the dodecasaccharides. These results suggest that CSA derivatives may potentially serve as targets in therapeutic strategies against placental malaria.

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Section: Methodsmentioning

confidence: 99%

Molecular dissection of placental malaria protein VAR2CSA interaction with a chemo-enzymatically synthesized chondroitin sulfate library

et al. 2016

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“…CS (1 mol) solution in 1.8 M HCl containing 100 mol of HMDA (100 l) was heated at 65°C for 2 h, followed by the addition of sodium cyanoborohydrate (150 mol), and further heating at 65°C for 16 h. The HMDA-conjugated saccharides were purified by gel filtration chromatography on a Superdex 30 HR16/600 column (1.6 ϫ 60 cm) using 0.2 M ammonium acetate as an eluent and obtained with freeze-drying repeated three times. The structure of purified CH6-HMDA was confirmed using a MALDI-TOF MS spectrometer (Autoflex; Bruker, Bremen, Germany) (18). The disaccharide compositions of CS-HMDA were measured using fluorometric post-column HPLC after digestion with chondroitinase ABC or AC II, as described below.…”

Section: Expression and Purification Of Recombinant Chondroitinmentioning

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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“…Betaglycan from linkage region TOF First synthesis of the tetraosyl hexapeptide Chondroitin oligosaccharides R-TOF (DHB), TOF/TOF, MS/MS Sequential synthesis by immobilized chondroitin polymerase mutants (Sugiura, et al, 2008a) Chondroitin sulfate E octasaccharide TOF, ESI Synthesis from an acetamide-type disaccharide unit (Tamura, et al, 2008) Heparin fragments…”

Section: Glycosaminoglycans and Related Compoundsmentioning

confidence: 99%

Analysis of carbohydrates and glycoconjugates by matrix‐assisted laser desorption/ionization mass spectrometry: An update for 2007–2008

Harvey

2011

Mass Spectrometry Reviews

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This review is the fifth update of the original review, published in 1999, on the application of MALDI mass spectrometry to the analysis of carbohydrates and glycoconjugates and brings coverage of the literature to the end of 2008. The first section of the review covers fundamental studies, fragmentation of carbohydrate ions, use of derivatives and new software developments for analysis of carbohydrate spectra. Among newer areas of method development are glycan arrays, MALDI imaging and the use of ion mobility spectrometry. The second section of the review discusses applications of MALDI MS to the analysis of different types of carbohydrate. Specific compound classes that are covered include carbohydrate polymers from plants, N- and O-linked glycans from glycoproteins, biopharmaceuticals, glycated proteins, glycolipids, glycosides and various other natural products. There is a short section on the use of MALDI mass spectrometry for the study of enzymes involved in glycan processing and a section on the use of MALDI MS to monitor products of the chemical synthesis of carbohydrates with emphasis on carbohydrate-protein complexes and glycodendrimers. Corresponding analyses by electrospray ionization now appear to outnumber those performed by MALDI and the amount of literature makes a comprehensive review on this technique impractical. However, most of the work relating to sample preparation and glycan synthesis is equally relevant to electrospray and, consequently, those proposing analyses by electrospray should also find material in this review of interest.

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Sequential synthesis of chondroitin oligosaccharides by immobilized chondroitin polymerase mutants

Cited by 28 publications

References 32 publications

Molecular dissection of placental malaria protein VAR2CSA interaction with a chemo-enzymatically synthesized chondroitin sulfate library

Molecular dissection of placental malaria protein VAR2CSA interaction with a chemo-enzymatically synthesized chondroitin sulfate library

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

Analysis of carbohydrates and glycoconjugates by matrix‐assisted laser desorption/ionization mass spectrometry: An update for 2007–2008

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