Demonstration of the immature glycosaminoglycan tetrasaccharide sequence GlcA beta 1-3Gal beta 1-3Gal beta 1-4Xyl on recombinant soluble human alpha-thrombomodulin - An oligosaccharide structure on a "part-time" proteoglycan

Thrombomodulin (TM), a cell surface glycoprotein, is a critical mediator of endothelial anticoagulant defenses occurring both as a chondroitin sulfate proteoglycan (beta-TRI) and a protein (alpha-TRI) unsubstituted by chondro itin 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 alpha-TM to investigate a possible biosynthetic mechanism for part-time PGs. Recombinant human alpha-TM, which was expressed in CHO- K1 cells, separated by anion-exchange chromatography from beta-TM, and puri fied by immunoaffinity chromatography (Nawa, K,, Sakano, K., Fujiwara, H., Sate, Y., Sugiyama, N,, Teruuchi, T,, Iwamoto, M., and Marumoto, Y. (1990) Biochem. Biophys. Res. Commun. 171, 729-737), was used for analysis. Prelim inary sugar composition analysis after acid hydrolysis showed Xyl in additi on to Gal, GalNAc, GlcNAc, Man, Fuc, and Glc. O-Glycosidically-linked oligo saccharides were liberated by mild alkaline treatment and purified. The iso lated oligosaccharide fraction was derivatized with a fluorophore 2-aminobe nzamide (2AB), resulting in two fluorescent components, a 2AB-oligosacchari de and a putative 2AB-Glc. Based on structural analysis by a combination of sequential exoglycosidase digestion and 500-MHz H-1 NMR spectroscopy of th e 2AB-oligosaccharide, the structure of the oligosaccharide was elucidated as GlcA beta 1-3Gal beta 1-3Gal beta 1-4Xyl, 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 imma ture 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 hex osamine, rather than xylose.