Structural analysis of unsaturated hexasaccharides isolated from shark cartilage chondroitin sulfate D that are substrates for the exolytic action of chondroitin ABC lyase.
The enzymatic action of highly purified chondroitin ABC lyase from Proteus vulgaris is dependent on the size of the substrate, and the enzyme does not cleave tetrasaccharides, irrespective of their sulfation profiles [Sugahara, K., Shigeno, K., Masuda, M., Fujii, N., Kurosaka, A. & Takeda, K. (1994) Carbohydr. Res. 255, 145-163]. To characterize the enzyme action in more detail, we isolated nine sulfated hexasaccharides from commercial shark cartilage chondroitin sulfate D, after partial digestion with highly purified chondroitin ABC lyase, by means of gel chromatography and HPLC on an amine-bound silica column. Structural analysis by 500-MHz H-NMR spectroscopy, and enzymatic digestion in conjunction with HPLC, demonstrated that these hexasaccharides, with the common core saccharide structure delta 4 HexA (alpha 1-3)GalNAc(beta 1-4)GlcA(beta 1-3)GalNAc(beta 1-4) GlcA(beta 1-3)GalNAc(where delta 4 HexA and GlcA represent 4-deoxy-alpha-L-threo-hex-4-enepyranosyluronic acid and glucuronic acid, respectively) bear three or four sulfate groups in different combinations. In the hexasaccharides, the D, disaccharide unit GlcA2-SO3 (beta 1-3) GalNAc4SO(3-) which is characteristic of chondroitin sulfate D, was arranged on the reducing side of the A disaccharide unit GlcA(beta 1-3)GalNAc4SO(3)-, and thus formed an A-D tetrasaccharide sequence GlcA(beta 1-3)GalNAc4SO(3)-(beta 1-4)GlcA2SO(3)-(beta 1-3) GalNAc6SO(3)-. Analysis of the degradation products of these hexasaccharides with highly purified chondroitin ABC lyase indicated that the enzyme preferentially acted on the unsaturated hexasaccharides in an exolytic fashion and removed an unsaturated disaccharide unit from the non-reducing termini, irrespective of the sulfation profiles of the hexasaccharides.
Sugahara
K
K
Department of Biochemistry, Kobe Pharmaceutical University, Japan.
Nadanaka
S
S
Takeda
K
K
Kojima
T
T
D000818:Animals; D001483:Base Sequence; D002356:Cartilage / Q000737:chemistry*; D002808:Chondroitin Lyases / Q000378:metabolism*; D002809:Chondroitin Sulfates / Q000737:chemistry* / Q000378:metabolism; D004187:Disaccharides / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D009005:Monosaccharides / Q000737:chemistry; D009844:Oligosaccharides / Q000737:chemistry* / Q000378:metabolism; D012754:Sharks; D013379:Substrate Specificity
D004187:Disaccharides; D009005:Monosaccharides; D009844:Oligosaccharides; C065068:chondroitin sulfate D; D002809:Chondroitin Sulfates; D002808:Chondroitin Lyases