Cloning of Gb3 Synthase, the Key Enzyme in Globo-series Glycosphingolipid Synthesis, Predicts a Family of α1,4-Glycosyltransferases Conserved in Plants, Insects, and Mammals

Jeremy J Keusch,Stephen M Manzella,Kwame A Nyame,Richard D Cummings,Jacques U Baenziger

doi:10.1074/jbc.m002630200

Jeremy J Keusch, Stephen M Manzella + Show 3 more

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https://doi.org/10.1074/jbc.m002630200

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Abstract

We have cloned Gb(3) synthase, the key alpha1, 4-galactosyltransferase in globo-series glycosphingolipid (GSL) synthesis, via a phenotypic screen, which previously yielded iGb(3) synthase, the alpha1,3-galactosyltransferase required in isoglobo-series GSL (Keusch, J. J., Manzella, S. M., Nyame, K. A., Cummings, R. D., and Baenziger, J. U. (2000) J. Biol. Chem. 33). Both transferases act on lactosylceramide, Galbeta1,4Glcbeta1Cer (LacCer), to produce Gb(3) (Galalpha1,4LacCer) or iGb(3) (Galalpha1, 3LacCer), respectively. GalNAc can be added sequentially to either Gb(3) or iGb(3) yielding globoside and Forssman from Gb(3), and isogloboside and isoForssman from iGb(3). Gb(3) synthase is not homologous to iGb(3) synthase but shows 43% identity to a human alpha1,4GlcNAc transferase that transfers a UDP-sugar in an alpha1, 4-linkage to a beta-linked Gal found in mucin. Extensive homology (35% identity) is also present between Gb(3) synthase and genes in Drosophila melanogaster and Arabidopsis thaliana, supporting conserved expression of an alpha1,4-glycosyltransferase, possibly Gb(3) synthase, throughout evolution. The isolated Gb(3) synthase cDNA encodes a type II transmembrane glycosyltransferase of 360 amino acids. The highest tissue expression of Gb(3) synthase RNA is found in the kidney, mesenteric lymph node, spleen, and brain. Gb(3) glycolipid, also called P(k) antigen or CD77, is a known receptor for verotoxins. CHO cells that do not express Gb(3) and are resistant to verotoxin become susceptible to the toxin following transfection with Gb(3) synthase cDNA.

Highlights

We have cloned Gb3 synthase, the key ␣1,4-galactosyltransferase in globo-series glycosphingolipid (GSL) synthesis, via a phenotypic screen, which previously yielded iGb3 synthase, the ␣1,3-galactosyltransferase required in isoglobo-series GSL
Isolation of a Rat Gb3 Synthase cDNA via Expression Cloning—Using a phenotypic screen based on recognition of terminal GalNAc by the Monoclonal antibodies (mAb) SMLDN1.1, we identified two independent and nonhomologous ␣-galactosyltransferases that act on LacCer
We have cloned Gb3 synthase, an ␣1,4-galactosyltransferase that acts on LacCer to form Gb3 GSL, known as CD77 or blood group Pk antigen [2, 4]

Summary

Introduction

We have cloned Gb3 synthase, the key ␣1,4-galactosyltransferase in globo-series glycosphingolipid (GSL) synthesis, via a phenotypic screen, which previously yielded iGb3 synthase, the ␣1,3-galactosyltransferase required in isoglobo-series GSL CHO cells that do not express Gb3 and are resistant to verotoxin become susceptible to the toxin following transfection with Gb3 synthase cDNA.

Results

Conclusion