A Remodeling System of the 3′-Sulfo-Lewis a and 3′-Sulfo-Lewis x Epitopes

Abstract

It has been reported that the chemically synthesized 3′-sulfo-Le a and 3′-sulfo-Lex epitopes have a high potential as a ligand for selectins. To elucidate the physiological functions of 3′-sulfated Lewis epitopes, a remodeling system was developed using a combination of a βGal-3-O-sulfotransferase GP3ST, hitherto known α1,3/1,4-fucosyltransferases (FucT-III, IV, V, VI, VII, and IX) and arylsulfatase A. The pyridylaminated (PA) lacto-N-tetraose (Galβ 1-3GlcNAcβ1-3Galβ1-4Glc) was first converted to 3′ -sulfolacto-N-fucopentaose II (sulfo-3Galβ1-3(Fucα1-4)GlcNAcβ 1-3Galβ1-4Glc)-PA by sequential reactions with GP3ST and FucT-III. The 3′-sulfolacto-N-fucopentaose III (sulfo-3Galβ1-4(Fucα 1-3)GlcNAcβ1-3Galβ1-4Glc)-PA was then synthesized from lacto-N-neotetraose (Galβ1-4GlcNAcβ1-3Galβ1-4Glc)-PA by GP3ST and FucT-III, -IV, -V, -VI, -VII, or -IX in a similar manner. The substrate specificity for the 3′-sulfated acceptor of the α 1,3-fucosyltransferases was considerably different from that for the non-substituted and 3′-sialylated varieties. When the GP3ST gene was introduced into A549 and Chinese hamster ovary cells expressing FucT-III, they began to express 3′-sulfo-Lea and 3′-sulfo-Le x epitopes, respectively, suggesting that GP3ST is responsible for their biosynthesis in vivo. The expression of the 3′-sialyl-Le x epitope on Chinese hamster ovary cells was attenuated by the introduction of GP3ST gene, indicating that GP3ST and α 2,3-sialyltransferase compete for the common Galβ1-4GlcNAc-R oligosaccharides. Last, arylsulfatase A, which is a lysosomal hydrolase that catalyzes the desulfation of 3-O-sulfogalactosyl residues in glycolipids, was found to hydrolyze the sulfate ester bond on the 3′-sulfo-Lex (type 2 chain) but not that on the 3′-sulfo-Lea (type 1 chain). The present remodeling system might be of potential use as a tool for the study of the physiological roles of 3′-sulfated Lewis epitopes, including interaction with selectins.