The Uniform Galactose 4‐Sulfate Structure in the Carbohydrate‐Protein Linkage Region of Human Urinary Trypsin Inhibitor

Abstract

The carbohydrate-protein linkage region of a chondroitin 4-sulfate chain attached to urinary trypsin inhibitor (UTI) was isolated from human urine and characterized structurally. The chondroitin 4–sulfate chain was released from UTI by β-elimination using alkaline NaBH₄ then digested with chondroitinase ABC. These treatments resulted in only a single hexasaccharide alditol derived from the carbohydrate-protein linkage region. Chemical and enzymic analyses and 600–MHz ¹H-NMR spectroscopy revealed that the hexasaccharide alditol had the following structure: ΔHexAα1-3GalNAc(4-sulfate)β1-4GlcAβ1-3Gal(4-sulfate)β1-3Galβ1-4Xyl-ol, where ΔHexA, GlcA and Xyl-ol represent 4-deoxy-α-L-threo -hex-4–enepyranosyluronic acid, D-glucuronic acid and D-xylitol, respectively. This structure contained the novel 4-sulfated Gal residue, which was first demonstrated in one of the three linkage hexasaccharide-serines isolated from chondroitin 4-sulfate of rat chondrosarcoma [Sugahara, K., Yamashina, I., de Waard, P., Van Halbeek, H. & Vliegenthart, J. F. G. (1988) J. Biol Chem. 263, 10168–10174]. This disulfated structure was recently identified as the sole structural component in the linkage hexasaccharide alditol fraction isolated from inter-α-trypsin inhibitor (ITI) in human plasma [Yamada, S., Oyama, M., Kinugasa, H., Nakagawa, T., Kawasaki, T., Nagasawa, S., Khoo, K.-H., Morris, H. R., Dell, A. & Sugahara, K. (1995) Glycobiology 5, 335–341]. The structural uniformity in the linkage hexasaccharide structure of ITI and UTI is in marked contrast to the heterogeneity demonstrated in the linkage hexasaccharides isolated from cartilaginous chondroitin sulfate whose linkage regions are sometimes but not always phosphorylated on the Xyl residue or sulfated on the Gal residue(s). The uniform structure containing the novel 4-sulfated Gal residue in the linkage region of UTI and ITI may imply its significance in the biosynthetic mechanism of chondroitin sulfate.