Demonstration of the immature glycosaminoglycan tetrasaccharide sequence GlcAβ1-3Galβ1-3Galβ1-4Xyl on recombinant soluble human α-thrombomodulin: An oligosaccharide structure on a 'part-time' proteoglycan

Abstract

Thrombomodulin (TM), a cell surface glycoprotein, is a critical mediator of endothelial anticoagulant defenses occurring both as a chondroitin sulfate proteoglycan (β-TM) and a protein (α-TM) unsubstituted by chondroitin sulfate (CS), hence its description as a 'part-time' proteoglycan (PG) (Fransson, L. A. (1987) Trends Biochem. Sci. 12, 406-411). Sugar analysis was performed on α-TM to investigate a possible biosynthetic mechanism for part- time PGs. Recombinant human α-TM, which was expressed in CHO-K1 cells, separated by anion-exchange chromatography from β-TM, and purified by immunoaffinity chromatography (Nawa, K., Sakano, K., Fujiwara, H., Sato, Y., Sugiyama, N., Teruuchi, T., Iwamoto, M., and Marumoto, Y. (1990) Biochem. Biophys. Res. Commun. 171, 729-737), was used for analysis. Preliminary sugar composition analysis after acid hydrolysis showed Xyl in addition to Gal, GalNAc, GlcNAc, Man, Fuc, and Glc. O-Glycosidically-linked oligosaccharides were liberated by mild alkaline treatment and purified. The isolated oligosaccharide fraction was derivatized with a fluorophore 2-aminobenzamide (2AB), resulting in two fluorescent components, a 2AB-oligosaccharide and a putative 2AB-Glc. Based on structural analysis by a combination of sequential exoglycosidase digestion and 500-MHz 1H NMR spectroscopy of the 2AB- oligosaccharide, the structure of the oligosaccharide was elucidated as GlcAβ1-3Galβ1-3Galβ1-4Xyl, which turned out to represent a glycosaminoglycan (GAG)-protein linkage region tetrasaccharide common to various PGs and was considered to be a biosynthetic intermediate of an immature GAG chain. The results may indicate that at least one class of the so-called part-time PGs bear the linkage tetrasaccharide at the GAG attachment sites and that the critical determining step or the rate-limiting step for PG biosynthesis is the transfer of the fifth sugar residue, the first hexosamine, rather than xylose.