Enzymatic Reconstruction of Dermatan Sulfate

Takagaki, Keiichi; Munakata, Hidekazu; Kakizaki, Ikuko; Majima, Mitsuo; Endo, Masahiko

doi:10.1006/bbrc.2000.2452

Cited by 32 publications

(14 citation statements)

References 20 publications

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“…Custom Synthesis of Chimeric Oligosaccharides-Chimeric oligosaccharides were synthesized using the transglycosylation reaction of testicular hyaluronidase as described previously (22,23). A typical transglycosylation reaction was carried out as follows.…”

Section: Methodsmentioning

confidence: 99%

“…The resulting product was purified by HPLC, and its structure was confirmed by a method described previously (9,10). Using systematic combinations of the donor and acceptor molecules, we prepared many chimeric type GAG oligosaccharides consisting of different combinations of disaccharide units (22,23).…”

Section: Methodsmentioning

confidence: 99%

“…To study the effect of structural differences at the nonreducing terminal on binding to type V collagen, new model oligosaccharides were custom synthesized by using the transglycosylation reaction of testicular hyaluronidase, as described previously (22,23). Ch, Ch4S, Ch6S, or desulfated DS as donors containing various disaccharide units, and a PA-hexasaccharide (PA-oligosaccharide number 6), GlcA-GalNAc(4S,6S)-GlcA-GalNAc(4S,6S)-GlcA-GalNAc(6S)-PA as acceptor, were incubated with hyaluronidase under optimal conditions (0.15 M Tris-HCl buffer, pH 7.0, in the absence of NaCl at 37°C for 60 min).…”

Section: -J)mentioning

confidence: 99%

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Domain Structure of Chondroitin Sulfate E Octasaccharides Binding to Type V Collagen

Takagaki

Munakata

Kakizaki

et al. 2002

Journal of Biological Chemistry

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We demonstrated previously that chondroitin sulfate E (ChS-E) binds to type V collagen (Munakata, H., Takagaki, K., Majima, M., and Endo, M. (1999) Glycobiology 9, 1023-1027). In this study, we investigated the structure and binding of ChS-E oligosaccharides. Eleven oligosaccharides were isolated from ChS-E by gel filtration chromatography and anion-exchange high performance liquid chromatography after hydrolysis with testicular hyaluronidase. Separately, seven oligosaccharides were custom synthesized using the transglycosylation reaction of testicular hyaluronidase. Structural analysis was performed by enzymatic digestions in conjunction with high performance liquid chromatography and mass spectrometry. This library of 18 oligosaccharides was used as a source of model molecules to clarify the structural requirements for binding to type V collagen. Binding was analyzed by a biosensor based on surface plasmon resonance. The results indicated that to bind to type V collagen the oligosaccharides must have the following carbohydrate structures: 1) octasaccharide or larger in size; 2) a continuous sequence of three GlcA␤1-3GalNAc(4S,6S) units; 3) a GlcA␤1-3GalNAc(4S,6S) unit, GlcA␤1-3GalNAc(4S) unit or GlcA␤1-3GalNAc(6S) unit at the reducing terminal; 4) a GlcA␤1-3GalNAc(4S,6S) unit at the nonreducing terminal. It is likely that these characteristic oligosaccharide sequences play key roles in cell adhesion and extracellular matrix assembly.Proteoglycan exists in tissues of many mammalian species and is widely distributed in the cell surface and extracellular matrix. It is known that its carbohydrate chain, chondroitin sulfate (ChS), 1 has various important biological activities in areas such as cell migration, recognition, and morphogenesis (1-5). Recently, greater attention has been directed toward the functions of ChS in the brain, optic nerves, and chondrocytes (6 -8). ChS has many types of structural domains that are known to participate in specific physiological functions. However, the relationship between the biological function and structure of ChS domains is not yet fully understood. Recently, we used a surface plasmon resonance (SPR) biosensor to investigate the interaction of glycosaminoglycans (GAGs) with collagens and glycoproteins from the extracellular matrix (9). It was found that chondroitin sulfate E (ChS-E) has specific affinity for type V collagen, and that a novel characteristic sequence included in ChS-E is probably involved in binding to type V collagen. ChS-E from squid cartilage consists mainly of the disaccharide units GlcA␤1-3GalNAc(4S,6S), GlcA␤1-3GalNAc(4S), GlcA␤1-3GalNAc(6S), and GlcA␤1-3GalNAc, where 4S and 6S represent 4-O-and 6-O-sulfate, respectively (10, 11). Thus, since ChS-E has a variety of structures, it is likely that some specific feature of its structure is necessary for binding to type V collagen. However, the structural features within ChS-E that bind to type V collagen remain unclear.In the present work, we have prepared various oligosaccharides by testicular hyaluronidase...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: -J)mentioning

confidence: 99%

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Domain Structure of Chondroitin Sulfate E Octasaccharides Binding to Type V Collagen

Takagaki

Munakata

Kakizaki

et al. 2002

Journal of Biological Chemistry

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“…Bovine testicular hyaluronidase (BTH) is useful for biochemical and histochemical analysis of glycosaminoglycans, and recently has also become an extremely important tool in glycotechnology [3][4][5][6][7][8] whereby its transglycosylation reaction can be used together with hydrolysis to reconstruct various glycosaminoglycan chains such as HA and Ch, in a similar way to gene technology. This has opened a new avenue of glycotechnology.…”

Section: Introductionmentioning

confidence: 99%

Novel products in hyaluronan digested by bovine testicular hyaluronidase

et al. 2008

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When the products of hyaluronan (HA) digested by bovine testicular hyaluronidase (BTH) were analyzed by high-performance liquid chromatography (HPLC), minor peaks were detected just before the main even-numbered oligosaccharide peaks. The amount of each minor peak was dependent on the reaction conditions for transglycosylation, rather than hydrolysis, by the BTH. Mainly based on HPLC and MS analysis, each minor peak was found to correspond to its oligosaccharide with one N-acetyl group removed from the reducing terminal N-acetylglucosamine. Enzymatic studies showed that the N-deacetylation activity was closely related to reaction temperature, pH, and the concentration of NaCl contained in the buffer, and glycosaminoglycan types and chain lengths of substrates. These findings strongly suggest that the N-deacetylation reaction in minor peaks was due to a novel enzyme contaminant in the BTH, N-deacetylase, that carries out N-deacetylation at the reducing terminal N-acetylglucosamine of oligosaccharides and is dependent on HA hydrolysis by BTH.

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“…To present, the study of DS structure has been approached from two points of view. Some attempts have been made by investigating the synthetic model of DS by reconstruction of the chain using transglycosylation reaction of specific glycosidases [7], whereas others try to cleave the DS chain using specific enzymes and analyze the products using various analytical techniques, including electrophoresis, fluorophore-assisted carbohydrate electrophoresis, HPLC, NMR, and mass spectroscopy [3,[8][9][10][11][12][13][14][15].…”

Section: Introductionmentioning

confidence: 99%

Identification of oligomeric domains within dermatan sulfate chains using differential enzymic treatments, derivatization with 2-aminoacridone and capillary electrophoresis

Mitropoulou¹,

Lamari²,

Syrokou³

et al. 2001

Electrophoresis

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Galactosaminoglycans, i.e. dermatan sulfate (DS) and chondroitin sulfate, are linear heteropolysaccharides consisting of repeating disaccharide units of L-iduronic acid (L-IdoA) or D-glucuronic acid (D-GlcA) residues linked to N-acetyl-galactosamine. High-performance capillary electrophoresis (HPCE or CE) has been successfully used for determining the disaccharide composition of glycosaminoglycans. However, only limited information is available on how to identify oligomeric domains rich in D-GlcA or L-IdoA. The aim of this study was therefore to develop a rapid and accurate CE procedure by which such oligosaccharides can be determined together with the variously sulfated disaccharides. Isolated dermatan sulfates of human origin were separately digested with chondroitinases ABC, AC and B and the enzymic products were derivatized with 2-aminoacridone. CE analysis of these products was performed using a phosphate buffer, pH 3.0, and reversed polarity at 30 kV. The derivatization enabled their detection with laser-induced fluorescence (LIF) and UV at 260 nm at much higher sensitivity than the detection of nonderivatized delta-saccharides at 232 nm and therefore components undetectable at 232 nm were nicely detected after derivatization. Except for delta-disaccharides, altogether five distinct oligosaccharides with differences in charge density were identified. Depending on the lyase that produced these oligomers, information on the presence of L-IdoA- or D-GlcA-containing domains within the DS chain and the sulfation pattern of these oligomeric domains was obtained. This CE method could also be useful in studying the functional oligomeric domains in galactosaminoglycan chains.

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Enzymatic Reconstruction of Dermatan Sulfate

Cited by 32 publications

References 20 publications

Domain Structure of Chondroitin Sulfate E Octasaccharides Binding to Type V Collagen

Domain Structure of Chondroitin Sulfate E Octasaccharides Binding to Type V Collagen

Novel products in hyaluronan digested by bovine testicular hyaluronidase

Identification of oligomeric domains within dermatan sulfate chains using differential enzymic treatments, derivatization with 2-aminoacridone and capillary electrophoresis

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