UDP-N-acetylglucosamine: α-3-D-mannoside β-1, 2-N-acetylglucosaminyltransferase I (GnT I) is a key enzyme in the synthesis of Asn-linked complex and hybrid glycans. We have cloned cDNAs for three predicted C. elegans genes homologous to mammalian GnT I (designated gly-12, gly-13 and gly-14). All three cDNAs encode proteins with the domain structure typical of previously cloned Golgi-type glycosyltransferases. Expression in transgenic worms showed that they all encode active GnT I. GLY-13 is unique in that it utilizes only the physiological substrate Man5GlcNAc2-R as sugar acceptor. We have isolated mutants lacking these three genes by the method of UV irradiation in the presence of trimethylpsoralen (TMP). The gly-12 and gly-14 mutants as well as the gly-14; gly-12 double mutant displayed wild type phenotypes indicating that neither gly-12 nor gly-14 is necessary for worm development under standard laboratory conditions. This finding and other data indicate that GLY-13 is the major functional GnT I in C. elegans. The mutation lacking the gly-13 gene is partially lethal and the few survivors display severe morphological and behaviorial defects. We have shown that the observed phenotype co-segregates with the gly-13 deletion in genetic mapping experiments although a second mutation near the gly-13 gene cannot as yet be ruled out. Studies on mice with a null mutation in the GnT I gene have indicated that complex and hybrid N-glycans play critical roles in mammalian morphogenesis. Our data indicate that this may also be true for the nematode Caenorhabditis elegans. There are limitations to the use of null mutations of GnT I and other glycosyltransferases in determining the function of glycans in metazoan development. An alternative approach to the problem (functional post-translational proteomics) is required.
C. elegans is a well-defined and simple animal model for genetic dissection of the roles of O-linked glycoproteins in development. Polypeptide GalNAc transferases (ppGaNTases) are a family of enzymes in the Golgi apparatus that posttranslationally modify serine and threonine residues with an N-acetylgalactosamine sugar in proteins that are destined for the cell surface, extracellular matrix, or secretion. The C. elegans genome contains a total of nine genes that express thirteen glycosyltransferase mRNAs, all of which share significant sequence similarity and predicted structural topology with mammalian ppGaNTases. The size of this gene family suggests that, in animal cells, O-glycosylation of mucin-type protein sequences is regulated by tissue-specific distribution of different ppGaNTase isozymes. To understand how the repertoire of glycosyltransferases changes in a cell-specific pattern during embryonic and juvenile development, we have constructed transgenic animals that express lacZ or GFP reporters for each member of the ppGaNTase family in C. elegans. Preliminary transgene expression data on the spatiotemporal expression of these isoforms suggests that differential distribution of ppGaNTase isoforms regulates the specificity and potential of O-glycosylation in the cell during development.
In this review we use the example of the parasitic nematode, Trichinella spiralis to demonstrate the significance of glycans at the host-parasite interface. The history of research on the N-glycans of T. spiralis demonstrates the advantages of a multi-disciplinary approach, incorporating structural, synthetic, and immunologic methods to studies of parasitism. In common with many other nematodes, Trichinella secretes and displays on its cuticle a large number of proteins that display a rich repertoire of N-linked glycans. Many of these glycans are ubiquitous in nematodes, and, indeed, in many eukaryotes, but the most interesting are unique to Trichinella. The former group include high mannose and truncated structures, whilst the latter belong to the family of complex-type N-glycans. LacdiNAc is the preferred antenna building block in many helminths including Trichinella. The L1 stage of T. spiralis modifies lacdiNAc with phosphorylcholine moieties or by substitution with D-tyvelose (3, 6-dideoxy-D-arabino-hexose). Both modifications create highly immunogenic epitopes. Tyvelose is the target of protective immunity against T. spiralis and has been studied in greatest depth. Chemical synthesis of the terminal tetrasaccharide, containing an α- or β-linked tyvelose residue, was undertaken so that immunochemical experiments could be used to infer the stereochemistry of the terminal linkage in the naturally occurring glycan. The synthesis of these glycosides required the development of new methodology to introduce the unique capping 3, 6-dideoxy-β-D-arabino-hexopyranoside. Results of binding studies with synthetic glycans and protective antibodies have clarified the inhibitory actions of antibodies on T. spiralis.
Schistosomiasis is a parasitic disease inflicted by the digenetic blood flukes. Over the last decade, significant advance has been made in understanding the developmentally regulated glycosylation pattern of this parasitic trematode. Accumulating data indicate that the initial steps in N-glycosylation result in core structures similar to those well defined for the mammalian systems but the schistosomal glycans are distinguished by distinctive core modifications and variations in terminal fucosylated sequences. For the O-glycans, novel core type has been identified beside the conventional types 1 and 2. Several unusually long and complex O-glycan chains were found which are multifucosylated. In addition, the glycosphingolipids of Schistosoma mansoni appear to be based on a distinctive schisto-core and extended by unique structures. These advances toward a complete glycomic map of the parasitic schistosome are reviewed here.
Sialic acids, mainly N-acetylneuraminic acid, have been found during the development of a few insect species. In Drosophila melanogaster and the cicada Philaenus spumarius we detected polysialic acids, using lectins, antibodies, and various methods for the isolation and analysis of the monosaccharide subunits, in areas of neuronal development and in Malpighian tubules, respectively. However, sialic acids were not found in the adult animals of these species. Although the expression of these acidic sugars has unequivocally been demonstrated in vivo, their occurrence in insect cell cultures was, in most cases, not clearly demonstrated. The biosynthesis of sialic acids in cell cultures would be of great biotechnological significance, since the production of recombinant glycoproteins with mammalian type, complex, sialylated N-glycans using the baculovirus expression system would be of great benefit. From the studies dedicated to this problem it appears that the engineering of such glycans may only be possible by the expression of exogenous genes in insect cells. Several genes encoding sialic acid-metabolizing enzymes have therefore been transfected. The work on insect cells shows that sialic acids are not only restricted to the Deuterostomia branch of the animal kingdom but also occur in some Protostomia species, as in insects. This throws new light on the evolution of this acidic sugar.
In recent years, the fission yeast Schizosaccharomyces pombe has attracted interest as a promising unicellular eukaryote model for studies on the biosynthetic pathway and oligosaccharide structures of glycoproteins. The glycoproteins of S. pombe contain a large amount of galactose in addition to mannose, indicating that S. pombe is equipped with mechanisms for galactosylation of glycoprotein, like mammalian cells. To elucidate the physiological role of galactosylation, we isolated an S. pombe mutant (gms1) defective in protein galactosylation. We found that disruption of the gms1+ gene (Δgms1) in S. pombe led to a complete loss of cell surface galactosylation, due to a defect in the transport of UDP-galactose as substrate for galactosyltransferase from cytosol into the lumen of the Golgi apparatus. Therefore, the Δgms1 strain is very useful for the analysis of the phenotypes of S. pombe cells lacking galactose residues and for the elucidation of the galactosylation mechanism. Although galactose residues are not essential for growth of S. pombe cells, the galactosylation of protein is required for the maintenance of normal cell shape, sexual agglutination, tolerance toward various drugs, and non-sexual flocculation in S. pombe.
Galectins are a group of relatively small lectins, whose ability to bind to β-galactosides is evolutionarily conserved among extensive organisms. In our previous studies, two distinct types of galectins, i.e., 32-kDa galectin and 16-kDa galectin were identified from the nematode Caenorhabditis elegans, and their molecular properties were characterized. More recently, however, the presence of a number of galectin-like genes became evident after completion of the genome project of this organism. Some of these candidate genes were systematically designated lec-1-11 for further study, where the previously characterized 32-kDa and 16-kDa galectins were renamed LEC-1 and LEC-6, respectively. Four members showing a closer relationship to the 32-kDa galectin were defined as lec-2-5, and the others showing novel features as lec-7-11. Open reading frames encoded by these genes were established through cDNA cloning, and registered to the genome/proteome databases. To elucidate their physiological functions, detailed sugar-binding specificity was examined by reinforced frontal affinity chromatography, and identification of endogenous glycoprotein receptors was attempted by the recently developed “glyco-catch” method. By the former method, affinities to a series of fluorescently labeled oligosaccharides were quantitatively determined rapidly and reliably. By the latter method, we identified genes that encode glycoproteins specifically recognized by C. elegans galectins by combination of conventional lectin-affinity technique and in silico database search. Notably, these two methods are directly applicable to the “glycome project” originally proposed by the authors, which aims at making a “list of glycoproteins” along with the concept of genome science. By the glycocatch method, the following three points will be clarified: 1) Which genes are expressed as glycoproteins. 2) Which sites among potential glycosylation sites are actually glycosylated. 3) Which kind of glycans (N-linked or O-linked, high-mannose type or complex type) are attached to these sites. As a result of the application of the glycomic approach to both soluble and detergent extracts of C. elegans and by using concanavalin A and galectin LEC-6 as probes, a substantial number of glycoprotein genes have been assigned. Thus, the strategy is considered to be fully applicable to more complex genome organisms (organisms in which the entire genome has been sequenced), such as humans. In the end, the authors propose a novel strategy to specify glycan structures.
Proteins such as lectins, enzymes, and antibodies, have been employed in the investigation of protein-carbohydrate interaction. However, it is very difficult to design molecules that bind specifically to a target carbohydrate. In my study, selection of ganglioside-binding peptides from a combinatorial random peptide library was carried out. The selected peptides specifically bound to the ganglioside and were suggested to have interacted with carbohydrate through a consensus motif.