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 p-elimination using alkaline NaBH, then digested with chondroitinase ABC. These treatments resulted in only a single hexasaccharide alditol derived from the carbohydrateprotein linkage region. Chemical and enzymic analyses and 600-MHz 'H-NMR spectroscopy revealed that the hexasaccharide alditol had the following structure: AHexAal-3GalNAc(4-sulfate)~1-4GlcA~l-3Gal(4-sulfate)~1-3Gal~l-4Xyl-o1, where AHexA, GlcA and Xyl-ol represent 4-deoxy-a-~-threo-hex-4-enepyranosyluronic acid, D-ghCUroniC 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. Bid. Chem. 263, 10168-101741. This disulfated structure was recently identified as the sole structural component in the linkage hexasaccharide alditol fraction isolated from inter-rx-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 -3411. The structural uniformity in the linkage hexasaccharide structure of IT1 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 IT1 may imply its significance in the biosynthetic mechanism of chondroitin sulfate.Keywords: chondroitin sulfate; 'H-NMR; inter-a-trypsin inhibitor; sulfated oligosaccharides; urinary trypsin inhibitor.Urinary trypsin inhibitor (UTI) is a serine-protease inhibitor present in human urine and is a proteoglycan bearing an undersulfated chondroitin 4-sulfate chain (Balduyck et al., 1986: Toyoda et al., 1993. The purified UTI has been used therapeutically in patients with rheumatoid arthritis. Intraarticular injection of UTI improves the symptoms and clinical signs of rheumatoid arthritis markedly, which is ascribed to the inhibition of a plasminogen activator of the urokinase type (Kikuchi et al., 1987). The inhibitory activity of UTI i s attributable to the protein moiety (Gebhard et al., 1989), which consists of 143 amino acids (Wachter and Hochstrasser, 1981) identical in amino acid sequence to that of the L chain, bikunin, of inter-a-trypsin inhibitor (ITI) (Morii and Travis, 1985: Reisinger et al., 1985). IT1 is considered to be a metabolic precursor of UTI (Hochstrasser Corre.~pondeizce fo K. Sugahara, Department of Biochemistry, Kobe Pharmaceutical University, H...

Sulfation of the Bikunin Chondroitin Sulfate Chain Determines Heavy Chain·Hyaluronan Complex Formation

“…Previous reports of the bikunin CS linkage region structure are based on analysis of urine and plasma that eluted from an anion exchange column with a salt gradient up to 0.5 M NaCl (1,2,43). These fractions have been reported to contain the disulfated CS linkage region hexasaccharide structure ⌬HexA␣1-3GalNAc(4S)␤1-4GlcA␤1-3Gal(4S)␤1-3Gal␤1-4Xyl-ol (24,25,44,45). The 2B6 antibody is known to react with the linkage region hexasaccharide ⌬HexA␣1-3GalNAc(4S)␤1-4GlcA␤1-3Gal␤1-3Gal␤1-4Xyl-ol (46).…”

“…The CS chain linkage region of bikunin in urine and plasma has been shown to contain a disulfated hexasaccharide (24,25) on its non-reducing end, whereas other groups have suggested that the non-reducing end is monosulfated on the GalNAc residue (9,28). HC1 and HC2 are located relatively close to each other on the bikunin CS chain in I␣I with HC2 located toward the reducing end of the CS chain and HC1 located further toward the non-reducing end (26).…”

“…The CS chains of urinary bikunin and plasma I␣I have been found to contain 15 Ϯ 3 disaccharides of which ϳ5 disaccharides are 4-sulfated on GalNAc and clustered near the reducing end (23)(24)(25)(26)(27). The CS chain linkage region of bikunin in urine and plasma has been shown to contain a disulfated hexasaccharide (24,25) on its non-reducing end, whereas other groups have suggested that the non-reducing end is monosulfated on the GalNAc residue (9,28).…”

Positive Mode LC-MS/MS Analysis of Chondroitin Sulfate Modified Glycopeptides Derived from Light and Heavy Chains of The Human Inter-α-Trypsin Inhibitor Complex*

“…The linkage region of this GAG chain has been the target for several studies concluding a uniform galactose-4-sulfate tetrasaccharide structure (GlcA␤3Gal␤3(4-O-SO 3 )-Gal␤4Xyl␤-O-) in both human plasma and urinary samples (22,23). The I␣I complex is a unique macromolecular arrangement of structurally related proteins, i.e.…”

Variations in the Chondroitin Sulfate-Protein Linkage Region of Aggrecans from Bovine Nasal and Human Articular Cartilages

“…Shibata et al (12) also observed that the first disaccharide repeat was mostly unsulfated, and more so if the preceding xylose was phosphorylated. From the CS-substituted trypsin inhibitors of urine and plasma (15,16), a uniformly GalNAc 4-O-sulfated and Gal 4-O-sulfated linkage region was obtained. In shark cartilage aggrecan CS, which has mainly 6-O-sulfated GalNAc, the first Gal of the linkage region and sometimes both the first and second Gal can carry 6-O-sulfate (17,18).…”