STRUCTURAL STUDIES ON THE CHONDROITINASE ABC-RESISTANT SULFATED TETRASACCHARIDES ISOLATED FROM VARIOUS CHONDROITIN SULFATE ISOMERS

Various commercially available chondroitin sulfates, including an A is omer from whale cartilage, C and D isomers from shark cartilage, and a n E isomer from squid cartilage, were exhaustively digested with a com mercial highly purified Proteus vulgaris chondroitinase ABC. Gel chrom atography of all digests yielded a disaccharide and an oligosaccharide fraction which was resistant to the enzyme digestion and which accoun ts for 20-31 mol% of the produced total oligosaccharides. Variably sul fated tetrasaccharides were isolated from the oligosaccharide fraction of each chondroitin sulfate isomer by HPLC, then characterized chemic ally and enzymatically. One disulfated and three trisulfated component s were also characterized by 500-MHz one- and two-dimensional H-1 NMR spectroscopy. The structures of one tetrasulfated, four trisulfated, a nd five disulfated tetrasaccharides with the common core structure, al pha-L-Delta(4,5)HexpA-(1 --> 3)-beta-D-GalpNAc-(1 --> 4)-beta-D-GlcpA- (1 --> 3)-D-GalpNAc, were determined. Air isolated tetrasaccharides we re resistant to the highly purified enzyme, but susceptible to the con ventional, commercial chondroitinase ABC. The former was also inactive towards alpha-L-Delta(4,5)HexpA-(1 --> 3)-beta-D-GalpNAc-(1 --> 4)-be ta-D-GlcpA-(1 --> 3)-D-GalpNAc isolated from chondroitin, beta-D-GlcpA -(1 --> 3)-beta-D-GlcpNAc-(1 --> 4)-beta-D-GlcpA-(1 --> 3)-D-GlcpNAc f rom hyaluronan, and alpha-L-Delta(4,5)HexgA-(1 --> 3)-beta-D-GalpNAc4S O(3)(-)(1 --> 4)-alpha-L-IdopA-(1 --> 3)-D-GalpNAc4SO(3)(-) from derma tan sulfate. These results indicate that, unlike the conventional enzy me, highly purified chondroitinase ABC cannot degrade tetrasaccharides irrespective of their sulfation profiles. The enzymatic action is siz e-dependent.