Abstract
We have previously reported the molecular cloning of beta1, 3-galactosyltransferase-V (beta3GalT-V), which catalyzes the transfer of Gal to GlcNAc-based acceptors with a preference for the core3 O-linked glycan GlcNAc(beta1,3)GalNAc structure. Further characterization indicated that the recombinant beta3GalT-V enzyme expressed in Sf9 insect cells also utilized the glycolipid Lc3Cer as an efficient acceptor. Surprisingly, we also found that beta3GalT-V catalyzes the transfer of Gal to the terminal GalNAc unit of the globoside Gb4, thereby synthesizing the glycolipid Gb5, also known as the stage-specific embryonic antigen-3 (SSEA-3). The SSEA-3 synthase activity of beta3GalT-V was confirmed in vivo by stable expression of the human beta3GalT-V gene in F9 mouse teratocarcinoma cells, as detected with the monoclonal antibody MC-631 by flow cytometry analysis and immunostaining of extracted glycolipids. The biological relation between SSEA-3 formation and beta3GalT-V was further documented by showing that F9 cells treated with the differentiation-inducing agent retinoic acid induced the expression of both the SSEA-3 epitope and the endogenous mouse beta3GalT-V gene. This study represents the first example of a glycosyltransferase, which utilizes two kinds of sugar acceptor substrates without requiring any additional modifier molecule.
Highlights
From the Institute of Physiology, University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland and the §Eunice Kennedy Shriver Center, Waltham, Massachusetts 02452
During our investigation of the donor and acceptor substrate specificity of the 3GalT-V enzyme, we have found that this enzyme exhibited a preference for GlcNAc-based acceptors [10]
The human teratocarcinoma cell line NCCIT showed a high level of stage-specific embryonic antigen-3 (SSEA-3) expression as reported earlier [16]
Summary
We found that 3GalT-V catalyzes the transfer of Gal to the terminal GalNAc unit of the globoside Gb4, thereby synthesizing the glycolipid Gb5, known as the stage-specific embryonic antigen-3 (SSEA-3). The lgtA glycosyltransferase from Neisseria meningitidis represents such an exception, as it can transfer two donors, GlcNAc and GalNAc, to Gal-based acceptors [1] Another notable exception is represented by the 1,4-galactosyltransferase-I enzyme, which is able to change its acceptor substrate specificity from GlcNAc to Glc by interacting with ␣-lactalbumin [2, 3]. Previous work from this group and other laboratories has revealed a family of structurally related 1,3-glycosyltransferase enzymes (4 –10). We have established the SSEA-3 synthase activity of 3GalT-V both in vitro and in vivo in F9 mouse teratocarcinoma cells as a model system
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