Expression Cloning of Mouse cDNA of CMP-NeuAc:Lactosylceramide α2,3-Sialyltransferase, an Enzyme That Initiates the Synthesis of Gangliosides

Abstract

Expression cloning of a cDNA for the ␣2,3-sialyltransferase (GM3 synthase) (EC 2.4.99.-) gene was performed using a GM3-lacking mouse fibroblast line L cell and anti-GM3 monoclonal antibody. Plasmids from a cDNA library generated with poly(A) ؉ RNA of a mouse fibrosarcoma line CMS5j and pdl3027 (polyoma T antigen) were co-transfected into L cells. The isolated cDNA clone pM3T-7 predicted a type II membrane protein with 13 amino acids of cytoplasmic domain, 17 amino acids of transmembrane region, and a large catalytic domain with 329 amino acids. Introduction of the cDNA clone into L cells resulted in the neo-synthesis of GM3 and high activity of ␣2,3-sialyltransferase. Among glycosphingolipids, only lactosylceramide showed significant activity as an acceptor, indicating that this gene product is a sialyltransferase specific for the synthesis of GM3. An amino acid sequence deduced from the cloned cDNA showed the typical sialyl motif with common features among ␣2,3-sialyltransferases. Among various mouse tissues, brain, liver, and testis showed relatively high expression of a 2.3-kilobase mRNA, whereas all tissues, more or less, expressed this gene.

Identification of Physiologically Relevant Substrates for Cloned Gal: 3-O-Sulfotransferases (Gal3STs)

“…On the other hand, a similar enzyme (STZ) cloned by Kitagawa and Paulson (32) utilized Gal␤1,3GlcNAc␤1,3Gal␤1,4Glc-Cer 5-fold more efficiently than nLC4Cer. Another cloned ␣2,3-sialyltransferase was found to be specific for the synthesis of GM3, NeuAc␣2,3Gal␤1,4Glc-Cer (33). A cloned human ST-3GalVI exhibited high acceptor specificity toward terminal Gal␤1,4GlcNAc sequence in glycoproteins and glycolipids (34).…”

Ganglioside GM3 Participates in the Pathological Conditions of Insulin Resistance

“…However, there was no obvious effect on serine phosphorylation of IRS-1 so far examined (data not shown), suggesting the differences of endogenous versus artificial exogenous GM3 on the effects of serine phosphorylation of IRS-1. To solve this issue, we are trying to transfect the synthase gene for GM3 (23,60) into adipocytes and other cell lines to identify the direct target(s) of endogenous GM3 responsible for insulin resistance.…”

Characterization of Mouse Sialyltransferase Genes: Their Evolution and Diversity

“…3). [33][34][35][36][37] ST3Gal V knockout mice showed heightened sensitivity to insulin due to enhanced insulin receptor phosphorylation in the skeletal muscle. 38) These mice were protected from high-fat diet induced insulin resistance, indicating that GM3 ganglioside is a negative regulator of insulin signaling.…”