Galactosyltransferases are enzymes which transfer galactose from UDP-Gal to various acceptors with either retention of the anomeric configuration to form alpha1,2-, alpha1,3-, alpha1,4-, and alpha1, 6-linkages, or inversion of the anomeric configuration to form beta1, 3-, beta1,4-, and beta1-ceramide linkages. During the last few years, several (c)DNA sequences coding for galactosyltransferases became available. We have retrieved these sequences and conducted sequence similarity studies. On the basis of both the nature of the reaction catalyzed and the protein sequence identity, these enzymes can be classified into twelve groups. Using a sensitive graphics method for protein comparison, conserved structural features were found in some of the galactosyltransferase groups, and other classes of glycosyltransferases, resulting in the definition of five families. The lengths and locations of the conserved regions as well as the invariant residues are described for each family. In addition, the DxD motif that may be important for substrate recognition and/or catalysis is demonstrated to occur in all families but one.

The transplantation of organs from other species into humans is considered to be a potential solution to the shortage of human donor organs. Organ transplantation from pig to human, however, results in hyperacute rejection, initiated by the binding of human natural antidonor antibody and complement. The major target antigen of this natural antibody is the terminal disaccharide Galalphal,3Gal, which is synthesized by Galbeta1,4GlcNAc alpha1,3-galactosyltransferase. Here we review our current knowledge of this key enzyme. A better understanding of structure, enzyme properties, and expression pattern of alpha1,3-galactosyltransferase has opened up several novel therapeutic approaches to prevent hyperacute vascular rejection. Cloning, and expression in vitro of the corresponding cDNA, has allowed to develop strategies to induce immune tolerance, and deplete or neutralize the natural xenoreactive antibody. Elucidation of the genomic structure has led to the production of transgenic animals that are lacking alpha1,3-galactosyltransferase activity. A detailed knowledge of the enzyme properties has formed the basis of approaches to modify donor organ glycosylation by intracellular competition. Study of the expression pattern of alpha1,3-galactosyltransferase has helped to understand the mechanism of hyperacute rejection in discordant xenotransplantation, and that of complement-mediated, natural immunity against interspecies transmission of retroviruses.

In recent years, several glycosyltransferase genes and cDNAs have been cloned and characterized. Although the glycosyltransferases seem to share the same general architecture, there is only little sequence similarity between the various enzymes. Moreover, a comparison of the organization of the genes shows that there is no common pattern of intron-exon structure. In addition, there seems to be little or no correlation between glycosyltransferase exons and protein domains. Taken together, these observations suggest that many of the glycosyltransferase genes evolved independently. So far, only two glycosyltransferase gene families have been described. These families may have evolved by exon-shuffling, or by gene duplication and subsequent divergence. For specific glycosyltransferases, mechanisms such as alternative splicing and alternative promoter usage play a role in the production of multiple protein isoforms from a single gene. These isoenzymes may differ in their enzymatic properties or cellular localization.

The choice for a heterologous expression system to produce glycoprotein therapeutics highly depends on its potential to perform mammalian-like posttranslational modifications such as glycosylation. To gain more insight into the glycosylation potential of the baculovirus mediated insect cell expression system, we have studied the expression of glycosyltransferases involved in complex-type N-glycosylation. Lepidopteran insect cell lines derived from Trichoplusia ni, Spodoptera frugiperda, and Mamestra brassicae were found to express a beta 1-->4- N-acetylgalactosaminyltransferase (beta 4-GalNAcT) that catalyzes the transfer of GalNAc from UDP-GalNAc to oligosaccharides and glycoproteins carrying a terminal beta-linked GlcNAc residue. These results suggest that Lepidopteran insect cells are capable of synthesizing complex-type carbohydrate chains containing GalNAc beta 1-->4GlcNAc (LacdiNAc) units. Baculovirus infection of the cells, however, resulted in a decrease in the activity of beta 4-GalNAcT from 80 to <1 pmol.min-1 mg-1 protein within 48 h post infection. Furthermore, considerable beta-N-acetylgalactosaminidase and beta-N-acetylglucosaminidase activity was observed in insect cells, whether or not infected with baculovirus, as well as in the culture medium. These enzyme activities could be responsible for degradation of complex-type oligosaccharide chains containing LacdiNAc units. Our findings provide an enzymatic basis for the observation that most recombinant glycoproteins produced by baculovirus infected insect cells carry oligomannosidic-type N-linked glycans, in spite of the fact that uninfected insect cells have the potential for the synthesis of mammalian-like complex-type glycans.

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