Molecular Cloning and Characterization of a Novel α1,2-Fucosyltransferase (CE2FT-1) from Caenorhabditis elegans

Abstract

Here we report the discovery of a unique fucosyltransferase (FT) in Caenorhabditis elegans. In studying the activities of FTs in extracts of adult C. elegans, we detected activity toward the unusual disaccharide acceptors Gal␤1-4Xyl-R and Gal␤1-6GlcNAc-R to generate products with the general structure Fuc␣1-2Gal␤1-R. We identified a gene encoding a unique ␣1,2FT (designated CE2FT-1), which contains an open reading frame encoding a predicted protein of 355 amino acids with the type 2 topology and domain structure typical of other glycosyltransferases. The predicted cDNA for CE2FT-1 has very low identity (5-10%) at the amino acid level to ␣1,2FT sequences in humans, rabbits, and mice. Recombinant CE2FT-1 expressed in human 293T cells has high ␣1,2FT activity toward the simple acceptor Gal␤-O-phenyl acceptor to generate Fuc␣1-2Gal␤-R, which in this respect resembles mammalian ␣1,2FTs. However, CE2FT-1 is otherwise completely different from known ␣1,2FTs in its acceptor specificity, since it is unable to fucosylate either Gal␤1-4Glc␤-R or free lactose and prefers the unusual acceptors Gal␤1-4Xyl␤-R and Gal␤1-6GlcNAc-R. Promoter analysis of the CE2FT-1 gene using green fluorescent protein reporter constructs demonstrates that CE2FT-1 is expressed in single cells of early stage embryos and exclusively in the 20 intestinal cells of L 1 -L 4 and adult worms. These and other results suggest that multiple fucosyltransferase genes in C. elegans may encode enzymes with unique activities, expression, and developmental roles.

Carbohydrates and glycosylation

“…These modifications result in an enormous molecular complexity that increases the capacity of GAGs to interact with proteins through varied arrangements of sulfated sugar residues (Turnbull et al, 2001). C. elegans produces heparan sulfate containing all modifications previously described in other organisms Yamada et al, 1999). In contrast, chondroitin appears not to be secondarily modified in C. elegans .…”

“…Although only a limited number of these genes have been functionally characterized, the degree of homology suggests that C. elegans possesses a repertoire of enzymes for the synthesis of a variety of complex carbohydrates. The occurrence of N-glycans [reviewed in Schachter (2004)], GalNAc-O-Ser/Thr glycans (Guerardel et al, 2001), glycosaminoglycans Yamada et al, 1999), glycolipids (Gerdt et al, 1999;Gerdt et al, 1997) and chitin (Veronico et al, 2001) has been verified in C. elegans in structural or biochemical studies. Although C. elegans N-and O-glycans have common features with vertebrate glycans in terms of their core glycan biosynthesis, their terminal structures show significant differences.…”

Molecular Basis of Anti-horseradish Peroxidase Staining in Caenorhabditis elegans

“…In C. elegans, expression of this epitope appears to be restricted to 10% of the neurons (12), and indeed fut-1 is apparently only expressed in specific neural cells (16). However, from the viewpoint of the wider importance of nematodes as parasites, more significant is that core ␣1,3-fucose is a known IgE epitope (39,57), and so the presence of this epitope on excretory-secretory antigens, for example (as shown previously by antibody binding for H. contortus (39)), may have a role in the interactions of parasitic nematodes with their hosts.…”

“…In particular, N-acetylgalactosaminyltransferases with roles in O-glycan (14) and, potentially, N-glycan and glycolipid biosynthesis (specifically, the ␤1,4-N-acetylgalactosaminyltransferase BRE-4) (15) have been expressed in recombinant form. An ␣1,2-fucosyltransferase with an unknown biological function (16), one N-acetylglucosaminyltransferase V homologue (17), one glucosyltransferase that potentially modifies O-glycans (18), and three genes encoding proteins with Nacetylglucosaminyltransferase I activity have also been described (19). The presence of the latter three is intriguing because plants, insects, and mammals would appear to only have one such gene.…”

Phylogenetic conservation of the cell-type-specific Lan3-2 glycoepitope in Caenorhabditis elegans

“…These neutral glycans are the most common glycan species found in whole worm extracts or extracts subjected to Concanavalin A affinity chromatography. While glycosyltransferases with cell-type-specific expression were identified using promoter analysis (Nguyen et al 2007;Zheng et al 2002), neutral glycans reflecting their restrictive expressions were not reported. Thus, in C. elegans, as well as in vertebrates (reviewed in Matani et al 2007), the function of negatively charged glycans such as glycosaminoglycans, has been extensively studied while, in contrast, the biological function of neutral glycans is not as well understood.…”