Ion-spray mass spectrometric analysis of glycosaminoglycan oligosaccharides

Takagaki, Keiichi; Kojima, Kaoru; Majima, Mitsuo; Nakamura, Toshiya; Kato, Ikuo; Endo, Masahiko

doi:10.1007/bf00731162

Cited by 73 publications

(54 citation statements)

References 28 publications

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“…Preparation of the Oligosaccharides-Oligosaccharides containing the GlcA␤1-3GalNAc(4S,6S) disaccharide unit were prepared according to the procedure described in a previous report (18). ChS-E was hydrolyzed by testicular hyaluronidase.…”

Section: Methodsmentioning

confidence: 99%

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%

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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“…4-Methylumbelliferyl GAG (GAG-MU), which is an artificial substrate for endo-␤-xylosidase, was prepared from cultured medium of human skin fibroblasts by the method previously reported (26). After digestion of GAG-MU with hyaluronidase from Streptomyces hyalurolyticus (Seikagaku Kogyo Co., Tokyo, Japan), Gal␤1-4Xyl␤1-MU and Gal␤1-3Gal␤1-4Xyl␤1-MU were purified by Bio-Gel P-4 (Bio-Rad) column chromatography as described previously (27,28). To obtain ⌬GlcA␤1-3GalNAc␤1-3GlcA␤1-3Gal␤1-3Gal␤1-4Xyl␤1-MU and ⌬GlcA␤1-3Gal␤1-3Gal␤1-4Xyl␤1-MU, GAG-MU was digested with chondroitinase ABC and chondroitinase ACII (Seikagaku Kogyo Co.) as described by Saito et al (29) and then purified by Sephadex G-50 (Amersham Biosciences) column chromatography.…”

Section: Methodsmentioning

confidence: 99%

“…Ion Spray Mass Spectrometry-Mass spectra were obtained on an API-100 triple-quadruple mass spectrometer (PE SCIEX, Ontario, Canada) equipped with an atmospheric pressure ionization source, as described previously (27,37). The samples were dissolved in 50% methanol and injected at 2 l/min with a micro-HPLC syringe pump.…”

Section: Methodsmentioning

confidence: 99%

Cleavage of the Xylosyl Serine Linkage between a Core Peptide and a Glycosaminoglycan Chain by Cellulases

Takagaki¹,

Iwafune²,

Kakizaki³

et al. 2002

Journal of Biological Chemistry

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We previously found that endo-␤-xylosidase from Patinopecten is an endo-type glycosidase that cleaves the xylosyl serine linkage between a glycosaminoglycan chain and its core protein (Takagaki, K., Kon, A., Kawasaki, H., Nakamura, T., Tamura, S., and Endo, M. (1990) J. Biol. Chem. 265, 854 -860). Screening for endo-␤-xylosidase activity in several cellulases detected this activity in the enzymes from Aspergillus niger, Penicillium funiculosum, Trichoderma reesei, Trichoderma viride, and Irpex lacteus. The cellulase derived from A. niger was purified, and its molecular weight was determined to be 26,000 by SDS-PAGE. Examination of the specificity of the cellulase revealed that 1) the enzyme acts on the linkage region (xylosyl serine) between a core peptide and a glycosaminoglycan chain; 2) enzymatic activity is greater with shorter glycosaminoglycan chains; 3) the enzyme readily hydrolyzes the linkage in glycosaminoglycan peptides, but intact proteoglycan is cleaved only slowly; and 4) the activity is unaffected by the glycosaminoglycan component (chondroitin sulfate, dermatan sulfate, and heparan sulfate). Judging from these enzymatic characteristics, this cellulase is different from the endo-␤-xylosidase of Patinopecten. We believe that this cellulase will become a useful tool in the further development of glycotechnology, because, like the endo-␤-xylosidase of Patinopecten, it enables the release of intact glycosaminoglycans from glycosaminoglycan peptides.

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“…The abundances of the charge states observed under each condition are dictated by factors including the molecular conformation of the ion in solution 14 , instrumental ion source conditions [15], pH [16], solvent polarity [17], and solvent volatility [18,19]. In these experiments the pH and instrument operating conditions were held constant.…”

Section: Esi Conditions For Production Of Highly Charged Ions From Sumentioning

confidence: 99%

“…The abundances of highly charged ions were found to increase as the volatility of the solvent decreased, with optimal results (maximum charge states) observed when samples were analyzed in 5% isopropanol, 0.1% ammonium hydroxide. A possible explanation is that low-volatility droplets have greater surface tension and are therefore able to bear a greater amount of charge than are those with higher volatility [18].…”

Section: Esi Conditions For Production Of Highly Charged Ions From Sumentioning

confidence: 99%

Competing fragmentation processes in tandem mass spectra of heparin-like glycosaminoglycans

Naggar

Costello

Zaia

2004

J. Am. Soc. Mass Spectrom.

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Heparin-like glycosaminoglycans (HLGAGs) are highly sulfated, linear carbohydrates attached to proteoglycan core proteins and expressed on cell surfaces and in basement membranes. These carbohydrates bind several families of growth factors and growth factor receptors and act as coreceptors for these molecules. Tandem mass spectrometry has the potential to increase our understanding of the biological significance of HLGAG expression by providing a facile means for sequencing these molecules without the need for time-consuming total purification. The challenge for tandem mass spectrometric analysis of HLGAGs is to produce abundant ions derived via glycosidic bond cleavages while minimizing the abundances of ions produced from elimination of the fragile sulfate groups. This work describes the competing fragmentation pathways that result from dissociation of high negative charge state ions generated from HLGAGs. Glycosidic bond cleavage ion formation competes with losses of equivalents of H 2 SO 4 , resulting in complex ion patterns. For the most highly sulfated structure examined, an octasulfated tetramer, an unusual loss of charge from the precursor ion was observed, accompanied by low abundance ions originating from subsequent backbone cleavages. These results demonstrate that fragmentation processes competing with glycosidic bond cleavages are more favored for highly sulfated HLGAG ions. In conclusion, reduction of charge-charge repulsions, such as is achieved by pairing the HLGAG ions with metal cations, is necessary in order to minimize the abundances of ions derived via fragmentation processes that compete with glycosidic bond cleavages. Sequence determination, including localization of sulfate groups on HLGAGs, is of significant interest, as their biological activities are believed to be expressed through patterns of sulfation and uronic acid epimerization [4,5].HLGAGs have traditionally proven difficult to analyze using mass spectrometry. Efforts to analyze these molecules using fast atom bombardment MS resulted in the observation of losses of SO 3 and NaSO 3 from the precursor ion, in addition to glycosidic bond cleavage ions [6 -10]. The poor sensitivity, relative to that achieved for peptides of comparable size, and lack of an intact precursor ion meant that tandem MS was not a viable analysis option when FAB ionization was employed. Although HLGAGs produce weak signals by matrix-assisted laser desorption/ionization (MALDI), much more abundant ions are detected from complexes between these molecules and a basic peptide [11][12][13]. In conjunction with enzymatic and/or chemical degradation, this MALDI sample preparation technique has been effectively used to sequence highly purified HLGAGs [14 -17]. Because the ions are detected in the positive mode, however, the charge is carried by the basic peptide and tandem MS of the HLGAG is not possible.Because HLGAGs and other sulfated carbohydrates produce abundant negative ions using electrospray ionization (ESI) without requiring basic peptide complexation [18...

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Ion-spray mass spectrometric analysis of glycosaminoglycan oligosaccharides

Cited by 73 publications

References 28 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

Cleavage of the Xylosyl Serine Linkage between a Core Peptide and a Glycosaminoglycan Chain by Cellulases

Competing fragmentation processes in tandem mass spectra of heparin-like glycosaminoglycans

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