THE SULFATED CARBOHYDRATE-PROTEIN LINKAGE REGION ISOLATED FROM CHONDROITIN 4-SULFATE CHAINS OF INTER-ALPHA-TRYPSIN INHIBITOR IN HUMAN PLASMA

Inter-alpha-trypsin inhibitor (ITI) in human plasma has a unique struc tural architecture composed of three polypeptide chains (H-1, H-2 and L chains), which are linked to each other through a chondroitin 4-sulp hate chain. The structure of the carbohydrate-protein linkage region o f the chondroitin 4-sulphate chain attached to the L chain was investi gated. The peptide-chondroitin sulphate fraction was isolated by anion -exchange chromatography after exhaustive digestion with lysyl endopep tidase and then V8 protease. The chondroitin 4-sulphate chain was rele ased from the peptides by beta-elimination using (NaBH4)-H-3 and then digested with chondroitinase ABC. These treatments resulted in a singl e H-3-labelled hexasaccharide alditol fraction derived from the linkag e region which had been associated with the L chain. Chemical and enzy matic analyses as well as fast-atom bombardment-mass spectrometry (FAB -MS) analysis revealed that the H-3-labelled hexasaccharide alditol ha d the following structure: Delta HexA-alpha 1-3GalNAc(4-sulphate) beta 1-4GlcA beta 1-3Gal(4-sulphate)beta 1-3Gal beta 1-4Xyl-ol (where Delt a HexA is 4-deoxy-alpha-L-threo-hex-4-enepyranosyluronic acid and Xyl- ol is xylitol). The structure contained the novel 4-sulphated Gal resi due, which was previously demonstrated in a linkage hexasaccharide iso lated from chondroitin 4-sulphate of rat chondrosarcoma (Sugahara et a l., J, Biol. Chem., 263, 10168-10174, 1988) and of whale cartilage (Su gahara et al., Eur. J. Biochem., 202, 805-811, 1991). The above disulp hated hexasaccharide alditol was the only component detected in the li nkage region fraction of the chondroitin 4-sulphate chain of ITI, whic h implies some biological significance of this novel structure.