Novel Sulfated Octa- and Decasaccharides from Squid Cartilage Chondroitin Sulfate E: Sequencing and Application for Determination of the Epitope Structure of the Monoclonal Antibody MO-225
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- 作者：Sarama Sathyaseelan Deepa ; Kittiwan Kalayanamitra ; Yumi Ito ; Prachya Kongtawelert ; Shigeyuki Fukui ; Shuhei Yamada ; Tadahisa Mikami ; Kazuyuki Sugahara
- 刊名：Biochemistry
- 出版年：2007
- 出版时间：March 6, 2007
- 年：2007
- 卷：46
- 期：9
- 页码：2453 - 2465
- 全文大小：142K
- 年卷期：v.46,no.9(March 6, 2007)
- ISSN：1520-4995
文摘
A mixture of octa- and decasaccharides obtained by the digestion with the hyaluronidase ofchondroitin sulfate E derived from squid cartilage was subfractionated into 20 and 23 different components,respectively, by anion-exchange HPLC. MALDI-TOF/MS was used to assign the sugar and sulfatecomposition of the putative octa- and decasaccharides, and a disaccharide composition analysis revealedthe building blocks to be A- [GlcUA
1-3GalNAc(4S)], C- [GlcUA1-3GalNAc(6S)], and E- [GlcUA1-3GalNAc(4S,6S)] units, where 4S and 6S represent 4-O- and 6-O-sulfate, respectively. The sequences ofthese octa- and decasaccharides were determined at low picomole amounts by a combination of enzymaticdigestions with chondroitinases in conjunction with anion-exchange HPLC. Sequencing revealed that eachfraction is a mixture of a major component together with one to three minor components, reflecting theheterogeneity of the parent polysaccharide. Among the 11 different octasaccharide sequences reportedhere, 8 are novel, while all of the 6 decasaccharide sequences are novel, and this is the first report of thesequencing of CS oligosaccharides longer than octasaccharides. The reactivity of the monoclonal antibodyMO-225 with octa- and decasaccharides tested with an oligosaccharide microarray revealed that a CS-Edecasaccharide is the minimal requirement for antibody recognition. Among the 6 decasaccharides, onlyE-E-E-E-C was recognized by MO-225, suggesting the requirement of a C-unit at the reducing end andalso the importance of chain length, which in turn may indicate the importance of the conformation acquiredby this specific sequence for antibody recognition.
1-3GalNAc(4S)], C- [GlcUA1-3GalNAc(6S)], and E- [GlcUA1-3GalNAc(4S,6S)] units, where 4S and 6S represent 4-O- and 6-O-sulfate, respectively. The sequences ofthese octa- and decasaccharides were determined at low picomole amounts by a combination of enzymaticdigestions with chondroitinases in conjunction with anion-exchange HPLC. Sequencing revealed that eachfraction is a mixture of a major component together with one to three minor components, reflecting theheterogeneity of the parent polysaccharide. Among the 11 different octasaccharide sequences reportedhere, 8 are novel, while all of the 6 decasaccharide sequences are novel, and this is the first report of thesequencing of CS oligosaccharides longer than octasaccharides. The reactivity of the monoclonal antibodyMO-225 with octa- and decasaccharides tested with an oligosaccharide microarray revealed that a CS-Edecasaccharide is the minimal requirement for antibody recognition. Among the 6 decasaccharides, onlyE-E-E-E-C was recognized by MO-225, suggesting the requirement of a C-unit at the reducing end andalso the importance of chain length, which in turn may indicate the importance of the conformation acquiredby this specific sequence for antibody recognition.