A remodeling system of the 3′-sulfo-Lewis a and 3′-sulfo-Lewis x epitopes

It has been reported that the chemically synthesized 3'-sulfo-Le(a) and 3'-sulfo-Le(x) 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 beta Ga1-3-O-sulfotransferase GP3ST, hitherto known alpha1,3/1,4-fucosyltransferases (FucT-III, IV, V, VI, VII, and IX) and arylsulfatase A. The pyridylaminated (PA) lacto-N-tetraose (Gal beta1-3GlcNAc beta1-3Gal beta1-4Glc) was first converted to 3'-sulfolacto-N-fucopentaose II (sulfo-3Gal beta1-3(Fuc alpha1-4)GlcNAc beta1-3Gal beta1-4Glc)-PA by sequential reactions with GP3ST and FucT-III. The 3'-sulfolacto-N-fucopentaose III (sulfo-3Gal beta1-4(Fuc alpha1-3)GlcNAc beta1-3Gal beta1-4Glc)-PA was then synthesized from lacto-N-neotetraose (Gal beta1-4GlcNAc beta1-3Gal beta1-4Glc)-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 alpha1,3-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 alpha2,3-sialyltransferase compete for the common Gal beta1-4GlcNAc-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-Le(x) (type 2 chain) but not that on the 3'-sulfo-Le(a) (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.