David H. Joziasse

Bovine α1,3-galactosyltransferase catalytic domain structure and its relationship with ABO histo-blood group and glycosphingolipid glycosyltransferases

α1,3‐galactosyltransferase (α3GalT, EC 2.4.1.151) is a Golgi‐resident, type II transmembrane protein that transfers galactose from UDP‐α‐galactose to the terminal N‐acetyllactosamine unit of glycoconjugate glycans, producing the Galα1,3Galβ1,4GlcNAc oligosaccharide structure present in most mammalian glycoproteins. Unlike most other mammals, humans and Old World primates do not possess α3GalT activity, which is relevant for the hyperacute rejection observed in pig‐to‐human xenotransplantation. The crystal structure of the catalytic domain of substrate‐free bovine α3GalT, solved and refined to 2.3 Å resolution, has a globular shape with an α/β fold containing a narrow cleft on one face, and shares a UDP‐binding domain (UBD) with the recently solved inverting glycosyltransferases. The substrate‐bound complex, solved and refined to 2.5 Å, allows the description of residues interacting directly with UDP‐galactose. These structural data suggest that the strictly conserved residue E317 is likely to be the catalytic nucleophile involved in galactose transfer with retention of anomeric configuration as accomplished by this enzyme. Moreover, the α3GalT structure helps to identify amino acid residues that determine the specificities of the highly homologous ABO histo‐blood group and glycosphingolipid glycosyltransferases.

Enzymes associated with glycosylation

Abstract Recent advances in the enzymology and molecular biology of glycosyltransferases — enzymes that catalyze the individual sugar attachment steps in the process of protein and lipid glycosylation — have begun to yield insights into how the expression of these enzymes is regulated. These results also provide us with an integrated view of the enzymatic and molecular relationships of several glycosyltransferases and identify a number of novel glycosylation pathways.

Xenotransplantation: the importance of the Galα1,3Gal epitope in hyperacute vascular rejection

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.

Lymphocyte triggering via L-selectin leads to enhanced galectin-3-mediated binding to dendritic cells

For proper immune surveillance, naive lymphocytes are recruited from the blood into secondary lymphoid organs. L‐selectin expressed on lymphocytes plays an important role in the initial attachment of these cells to high endothelial venules (HEV) in lymph nodes. Previously, we found that triggering via L‐selectin resulted in activation of lymphocytes, followed by an alteration in their adhesion capacity. This suggested that L‐selectin triggering might play a role in cell‐cell interactions after lymph node entry. Here, we identify a novel adhesion mechanism involving L‐selectin‐triggered lymphocytes and dendritic cells, and we show that enhanced binding to dendritic cells is mediated by galectin‐3 and not by integrins. Furthermore, it was shown that L‐selectin‐triggered T lymphocytes exhibited enhanced proliferation in an allogeneic mixed lymphocyte reaction. It is concluded that, in addition to a role for L‐selectin in tethering and rolling on endothelium, triggering of the molecule on the lymphocyte surface leads to changes that are pertinent for the function of the cell after passing the HEV. We argue that the described adhesion mechanism plays a role in optimizing the initial interaction between dendritic cells and lymphocytes.

One-pot enzymatic synthesis of the Gal alpha 1-->3Gal beta 1-->4GlcNAc sequence with in situ UDP-Gal regeneration.

The trisaccharide Galα1→3Galβ1→4GlcNAcβ1→O-(CH2)8COOCH3 was enzymatically synthesized, within situ UDP-Gal regeneration. By combination in one pot of only four enzymes, namely, sucrose synthase, UDP-Glc 4′-epimerase, UDP-Gal:GlcNAc β4-galactosyltransferase and UDP-Gal:Galβ1→4GlcNAc α3-galactosyltransferase, Galα1→3Galβ1→4GlcNAcβ1→O-(CH2)8COOCH3 was formed in a 2.2 µmol ml−1 yield starting from the acceptor GlcNAcβ1→O-(CH2)8COOCH3. This is an efficient and convenient method for the synthesis of the Galα1→3Galβ1→4GlcNAc epitope which plays an important role in various biological and immunological processes.

Specificity in the enzymic transfer of sialic acid to the oligosaccharide branches of BI- and triantennary glycopeptides of α1-acid glycoprotein

Abstract Partial in vitro sialylation of biantennary and triantennary glycopeptides of α1-acid glycoprotein using colostrum β-galactosideα(2→6) sialyltransferase followed by high resolution 1H-NMR spectroscopic analysis of the isolated products enabled the assignment of the Galβ(1→4)GlcNAcβ(1→2)Man α(1→3) Man branch as the most preferred substrate site for sialic acid attachment. The Galβ(1→4)GlcNAcβ(1→2)Man α(1→6) Man branch appeared to be much less preferred and the Galβ(1→4)GlcNAcβ(1→4)Manα(1→3)Man sequence of triantennary structures was of intermediate preference for the sialyltransferase. The specificity of the β-galactoside α(2→6) sialyltransferase is thus shown to extend to structural features beyond the terminal N -acetyllactosamine units on the oligosaccharide chains of serum glycoproteins.

The reducing end of αGal oligosaccharides contributes to their efficiency in blocking natural antibodies of human and baboon sera

Synthetic galactosyl oligosaccharides were tested for their ability to inhibit the cytotoxic reaction of human and baboon natural antibodies on PK15 cells in culture. Methyl-α-Gal gave weak inhibition, Galα1-3Gal substantially inhibited the reaction (400 μM), and Galα1-3Galβ2-4GlcNAc was ten times more efficient (30 μM). The modification from α to β anomeric configuration of the nonreducing end resulted in a complete loss of activity, while substitutions at the reducing end induced only a partial loss of activity. These observations suggest that natural anti-αGal antibodies recognize the epitope from its nonreducing end, but that substitutions at the reducing terminus can modify the antibody-binding capacity. Modified tri- and tetrasaccharides are better inhibitors than the disaccharide but not as good as Galα1-3Galβ1-4GlcNAc. The reducing terminus therefore contributes some energy to the reaction, indicating that certain oligosaccharides will be of more potential clinical use than others.

THE LIVING FACTORY : IN VIVO PRODUCTION OF N-ACETYLLACTOSAMINE CONTAINING CARBOHYDRATES IN E. COLI

Scientific and commercial interest in oligosaccharides is increasing, but their availability is limited as production relies on chemical or chemo-enzymatic synthesis. In search for a more economical, alternative procedure, we have investigated the possibility of producing specific oligosaccharides in E. coli that express the appropriate glycosyltransferases. The Azorhizobium chitin pentaose synthase NodC (a β(1,4)GlcNAc-transferase), and the Neisseria β(1,4)galactosyltransferase LgtB, were co-expressed in E. coli. The major oligosaccharide isolated from the recombinant strain, was subjected to LC-MS, FAB-MS and NMR analysis, and identified as βGal(1,4)[βGlcNAc(1,4)]4GlcNAc. High cell density culture yielded more than 1.0 gr of the hexasaccharide per liter of culture. The compound was found to be an acceptor in vitro for βGal(1,4)GlcNAc α(1,3)galactosyltransferase, which suggests that the expression of additional glycosyltransferases in E. coli will allow the production of more complex oligosaccharides.

REGULATION OF FUCOSYLTRANSFERASE-VII EXPRESSION IN PERIPHERAL LYMPH NODE HIGH ENDOTHELIAL VENULES

Binding of L‐selectin to the highly glycosylated peripheral lymph node addressins (PNAd) plays a central role in the normal recirculation of lymphocytes between the bloodstream and the lymph node. This interaction requires correct fucosylation of the PNAd, mediated by the recently identified fucosyltransferase‐VII (Fuc‐TVII). Here we show that during ontogeny Fuc‐TVII is absent at the day of birth, barely detectable on day 1, and clearly present from day 2 onwards. PNAd expression as detected by the MECA‐79 antibody precedes the expression of Fuc‐TVII. Furthermore, we demonstrate that in adult mice antigenic stimulation of peripheral lymph nodes leads to a temporary disappearance of Fuc‐TVII at days 2 and 3 after stimulation, followed by a complete reappearance by day 4, while expression of MECA‐79 is never completely absent during this period. Finally, occlusion of afferent lymphatics to peripheral lymph nodes resulted in a decreased expression of Fuc‐TVII in the high endothelial venules by day 5, and complete disappearance within 8 days. We conclude that the activity of Fuc‐TVII in cells of high endothelial venules is directly affected by afferent lymph and activation processes that occur in the lymph node after antigenic stimulation. The expression of Fuc‐TVII is therefore yet another level at which the function of high endothelial venules, and thus lymphocyte trafficking, can be regulated.

Assignment of two human α-1,3-galactosyltransferase gene sequences (GGTA1 and GGTA1P) to chromosomes 9q33–q34 and 12q14–q15

alpha-1,3-Galactosyltransferase is a terminal glycosyltransferase that is widely expressed in a variety of mammalian species, with the notable exception of man, apes, and Old World monkeys. Although transcripts for this enzyme are not detectable in humans, homologous sequences have been identified in human genomic DNA. These sequences correspond to a processed pseudogene that maps to chromosome 12 and the inactivated remnant of the once functional source gene that maps to chromosome 9. We have now established that the former sequence (GGTA1P) is localized to 12q14-q15 and the latter sequence (GGTA1) is localized to 9q33-q34 [corrected].