Abstract
The large array of different glycolipids described in mammalian tissues is a reflection, in part, of diverse glycosyltransferase expression. Herein, we describe the cloning of a UDP-galactose: beta-d-galactosyl-1,4-glucosylceramide alpha-1, 3-galactosyltransferase (iGb(3) synthase) from a rat placental cDNA expression library. iGb(3) synthase acts on lactosylceramide, LacCer (Galbeta1,4Glcbeta1Cer) to form iGb(3) (Galalpha1,3Galbeta1, 4Glcbeta1Cer) initiating the synthesis of the isoglobo-series of glycosphingolipids. The isolated cDNA encoded a predicted protein of 339 amino acids, which shows extensive homology (40-50% identity) to members of the ABO gene family that includes: murine alpha1, 3-galactosyltransferase, Forssman (Gb(5)) synthase, and the ABO glycosyltransferases. In contrast to the murine alpha1, 3-galactosyltransferase, iGb(3) synthase preferentially modifies glycolipids over glycoprotein substrates. Reverse transcriptase-polymerase chain reaction revealed a widespread tissue distribution of iGb(3) synthase RNA expression, with high levels observed in spleen, thymus, and skeletal muscle. As an indirect consequence of the expression cloning strategy used, we have been able to identify several potential glycolipid biosynthetic pathways where iGb(3) functions, including the globo- and isoglobo-series of glycolipids.
Highlights
There are many diverse glycans found in nature that may be synthesized on different aglycone substrates, including proteins and lipids
Following the synthesis of Gb3 (Gal␣1,4Gal1,4Glc1Cer) or iGb3 (Gal␣1,3Gal1,4Glc1Cer), sequential addition of GalNAc residues by Gb4 synthase and Gb5 synthase leads to the production of both Gb4 and Gb5 or iGb4 and iGb5, respectively
Chinese hamster ovary (CHO) cells transfected with iGb3 synthase cDNA synthesize Gal␣1,3Gal on GSL, primarily LacCer (Gal1,4Glc1Cer), producing iGb3 that is subsequently converted to iGb4 and iGb5
Summary
Glycosyltransferases, iGb3 Synthase, That Directs the Synthesis of Isoglobo-glycosphingolipids*. We describe the cloning of a UDP-galactose: -D-galactosyl-1,4-glucosylceramide ␣-1,3-galactosyltransferase (iGb3 synthase) from a rat placental cDNA expression library. IGb3 synthase acts on lactosylceramide, LacCer (Gal1,4Glc1Cer) to form iGb3 (Gal␣1,3Gal1,4Glc1Cer) initiating the synthesis of the isoglobo-series of glycosphingolipids. As a first step to this approach we have cloned Gb3 synthase and iGb3 synthase, two transferases that act on LacCer and initiate the synthesis of the globo- and isoglobo-series of GSL. Using a mAb SMLDN1.1 that detects GalNAc on these downstream products, we have cloned Gb3 and iGb3 synthases from a rat placental cDNA expression library. In this paper we described the cloning of iGb3 synthase, an ␣1,3-galactosyltransferase, that shares high homology with other glycosyltransferases in the histo-blood group ABO gene family but differs in its substrate specificity
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