Hironobu Eguchi

A Secreted Type of β1,6-N-Acetylglucosaminyltransferase V (GnT-V) Induces Tumor Angiogenesis without Mediation of Glycosylation A NOVEL FUNCTION OF GnT-V DISTINCT FROM THE ORIGINAL GLYCOSYLTRANSFERASE ACTIVITY

Angiogenesis is the first regulatory step of tumor progression. Herein, we report on some findings that show that β1,6-N-acetylglucosaminyltransferase V (GnT-V) functions as an inducer of angiogenesis that has a novel and completely different function from the original function of glycosyltransferase. A secreted type of GnT-V protein itself promoted angiogenesis in vitro and in vivo at physiological concentrations. The highly basic domain of GnT-V induced the release of fibroblast growth factor-2 from heparan sulfate proteoglycan on the cell surface and/or extracellular matrix, leading to angiogenesis. These findings provide some novel information on the relationship between GnT-V and tumor metastasis. The inhibition of GnT-V secretion or its expression represents a novel potential strategy for the inhibition of tumor angiogenesis.

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-Lea 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 (Galβ1–3GlcNAcβ1–3Galβ1–4Glc) was first converted to 3′-sulfolacto-N-fucopentaose II (sulfo-3Galβ1–3(Fucα1–4)GlcNAcβ1–3Galβ1–4Glc)-PA by sequential reactions with GP3ST and FucT-III. The 3′-sulfolacto-N-fucopentaose III (sulfo-3Galβ1–4(Fucα1–3)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 α1,3-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-Lex epitopes, respectively, suggesting that GP3ST is responsible for their biosynthesis in vivo. The expression of the 3′-sialyl-Lex epitope on Chinese hamster ovary cells was attenuated by the introduction of GP3STgene, indicating that GP3ST and α2,3-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.

The action of N-acetylglucosaminyltransferase-V is prevented by the bisecting GlcNAc residue at the catalytic step

Using a purified protein and bisected acceptor oligosaccharides, we demonstrate that N‐acetylglucosaminyltransferase (GnT)‐V transfers a N‐acetylglucosamine residue via a β1,6‐linkage to the bisected oligosaccharides. We also kinetically characterized the substrate specificity of GnT‐V with respect to the bisected oligosaccharide. Although the K m values for the bisected acceptors were comparable to that for a non‐bisected acceptor, the V max values for the bisected acceptors were much lower than that for the non‐bisected acceptor. These findings suggest that the acceptor specificity of GnT‐V is determined by the catalytic process rather than by its binding to the substrate. It was also found that the presence of the 2‐N‐acetyl group in the bisecting monosaccharide moiety plays a critical role in determining the catalytic efficiency of the enzyme.