Structural determination of five novel tetrasaccharides containing 3-O-sulfated D-glucuronic acid and two rare oligosaccharides containing a beta-D-glucose branch isolated from squid cartilage chondroitin sulfate E.
Oversulfated chondroitin sulfate E (CS-E) derived from squid cartilage exhibits intriguing biological activities, which appear to reflect the biological activities of mammalian CS chains containing the so-called E disaccharide unit [GlcAbeta1-3GalNAc(4,6-O-disulfate)]. Previously, we isolated novel tetra- and hexasaccharides containing a rare GlcA(3-O-sulfate) at the nonreducing end after digestion of squid cartilage CS-E with testicular hyaluronidase. In this study, squid cartilage CS-E was extensively digested with chondroitinase AC-II, which yielded five highly sulfated novel tetrasaccharides and two odd-numbered oligosaccharides (tri- and pentasaccharides) containing D-Glc. Their structures were determined by fast atom bombardment mass spectrometry and (1)H NMR spectroscopy. The results revealed an internal GlcA(3-O-sulfate) residue for all the novel tetrasaccharide sequences, which rendered the oligosaccharides resistant to the enzyme. The results suggest that GlcA(3-O-sulfate) units are not clustered but rather interspersed in the CS-E polysaccahride chains, being preferentially located in the highly sulfated sequences. The predominant structure on the nearest nonreducing side of a GlcA(3-O-sulfate) residue was GalNAc(4-O-sulfate) (80%), whereas that on the reducing side was GalNAc(4,6-O-disulfate) (59%). The structural variety in the vicinity of the GlcA(3-O-sulfate) residue might represent the substrate specificity of the unidentified chondroitin GlcA 3-O-sulfotransferase. The results also revealed a trisaccharide and a pentasaccahride sequence, both of which contained a beta-d-Glc branch at the C6 position of the constituent GalNAc residue. Approximately 5 mol % of all disaccharide units were substituted by Glc in the CS-E preparation used.
Department of Biochemistry, Kobe Pharmaceutical University, Higashinada-ku, Kobe 658-8558, Japan.
Akiko
A
Kinoshita-Toyoda
Shuhei
S
Yamada
Stuart M
SM
Haslam
Kay-Hooi
KH
Khoo
Makiko
M
Sugiura
Howard R
HR
Morris
Anne
A
Dell
Kazuyuki
K
Sugahara
D000818:Animals; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002356:Cartilage / Q000737:chemistry*; D002808:Chondroitin Lyases / Q000737:chemistry / Q000378:metabolism; D002809:Chondroitin Sulfates / Q000737:chemistry*; D049832:Decapodiformes; D004187:Disaccharides / Q000737:chemistry*; D005947:Glucose / Q000737:chemistry* / Q000302:isolation & purification; D020723:Glucuronic Acid / Q000737:chemistry*; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D009844:Oligosaccharides / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D011522:Protons; D016339:Spectrometry, Mass, Fast Atom Bombardment; D013379:Substrate Specificity
D004187:Disaccharides; D009844:Oligosaccharides; D011522:Protons; C038142:chondroitin sulfate glycosaminoglycan; D020723:Glucuronic Acid; D002809:Chondroitin Sulfates; D002808:Chondroitin Lyases; D005947:Glucose