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

Abstract

A mixture of octa- and decasaccharides obtained by the digestion with the hyaluronidase of chondroitin 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 sulfate composition of the putative octa- and decasaccharides, and a disaccharide composition analysis revealed the building blocks to be A- [GlcUAbeta1-3GalNAc(4S)], C- [GlcUAbeta1-3GalNAc(6S)], and E- [GlcUAbeta1-3GalNAc(4S,6S)] units, where 4S and 6S represent 4-O- and 6-O-sulfate, respectively. The sequences of these octa- and decasaccharides were determined at low picomole amounts by a combination of enzymatic digestions with chondroitinases in conjunction with anion-exchange HPLC. Sequencing revealed that each fraction is a mixture of a major component together with one to three minor components, reflecting the heterogeneity of the parent polysaccharide. Among the 11 different octasaccharide sequences reported here, 8 are novel, while all of the 6 decasaccharide sequences are novel, and this is the first report of the sequencing of CS oligosaccharides longer than octasaccharides. The reactivity of the monoclonal antibody MO-225 with octa- and decasaccharides tested with an oligosaccharide microarray revealed that a CS-E decasaccharide is the minimal requirement for antibody recognition. Among the 6 decasaccharides, only E-E-E-E-C was recognized by MO-225, suggesting the requirement of a C-unit at the reducing end and also the importance of chain length, which in turn may indicate the importance of the conformation acquired by this specific sequence for antibody recognition.

Functions of Chondroitin Sulfate/Dermatan Sulfate Chains in Brain Development

“…The original study by Yamagata et al (64) showed the reactivity of MO-225 with the tetrasaccharides ⌬A-D and ⌬E-D. Recently, Ito et al (65) have shown that MO-225 could strongly react with octasaccharide and larger oligosaccharide fractions derived from CS-D of shark cartilage, and Deepa et al (41) showed that MO-225 reacted with a decasaccharide sequence rich in E units, prepared from squid cartilage CS-E.…”

“…41. b Plus (ϩ) and minus (Ϫ) indicate positive and negative inhibitory activity, respectively, against the binding of GD3G7 to immobilized CS-E in ELISA as described in the legend to Fig.…”

Effect of chondroitin sulfate-E on the osteoclastic differentiation of RAW264 cells

“…These results suggest that at least four repeating units of CS-E have the capacity to regulate osteoblast and osteoclast differentiation through distinct mechanisms. In a previous study, it was reported that various oligosaccharides of CS-E enhanced neurite outgrowth 28,29) . Determining the correlation between CS-E oligosaccharide structure and the inhibitory activity on osteoclast formation requires further investigation.…”

Involvement of Human Natural Killer-1 (HNK-1) Sulfotransferase in the Biosynthesis of the GlcUA(3-O-sulfate)-Gal-Gal-Xyl Tetrasaccharide Found in α-Thrombomodulin from Human Urine

“…CS-K from king crab cartilage was prepared as reported previously (21). The octa-, hexa-, and tetrasaccharides derived from Chn were prepared by treatment with sheep testicular hyaluronidase as described (22).The structurally defined tet-…”