Molecular cloning and characterization of the Caenorhabditis elegans alpha1,3-fucosyltransferase family.
The genome of Caenorhabditis elegans encodes five genes with homology to known alpha1,3 fucosyltransferases (alpha1,3FTs), but their expression and functions are poorly understood. Here we report the molecular cloning and characterization of these C. elegans alpha1,3FTs (CEFT-1 through -5). The open-reading frame for each enzyme predicts a type II transmembrane protein and multiple potential N-glycosylation sites. We prepared recombinant epitope-tagged forms of each CEFT and found that they had unusual acceptor specificity, cation requirements, and temperature sensitivity. CEFT-1 acted on the N-glycan pentasaccharide core acceptor to generate Manalpha1-3(Manalpha1-6)Manbeta1-4GlcNAcbeta1-4(Fucalpha1-3)GlcNAcbeta1-Asn. In contrast, CEFT-2 did not act on the pentasaccharide acceptor, but instead utilized a LacdiNAc acceptor to generate GalNAcbeta1-4(Fucalpha1-3)GlcNAcbeta1-3Galbeta1-4Glc, which is a novel activity. CEFT-3 utilized a LacNAc acceptor to generate Galbeta1-4(Fucalpha1-3)GlcNAcbeta1-3Galbeta1-4Glc without requiring cations. CEFT-4 was similar to CEFT-3, but its activity was enhanced by some divalent cations. Recombinant CEFT-5 was well expressed, but did not act on available acceptors. Each CEFT was optimally active at room temperature and rapidly lost activity at 37 degrees C. Promoter analysis showed that CEFT-1 is expressed in C. elegans eggs and adults, but its expression was restricted to a few neuronal cells at the head and tail. We prepared deletion mutants for each enzyme for phenotypic analysis. While loss of CEFT-1 correlated with loss of pentasaccharide core activity and core alpha1,3-fucosylated glycans in worms, loss of other enzymes did not correlate with any phenotypic changes. These results suggest that each of the alpha1,3FTs in C. elegans has unique specificity and expression patterns.
Nguyen
Kiem
K
Department of Biochemistry and Molecular Biology, Oklahoma Center for Medical Glycobiology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA.
van Die
Irma
I
Grundahl
Kiely M
KM
Kawar
Ziad S
ZS
Cummings
Richard D
RD
D000595:Amino Acid Sequence; D000818:Animals; D017173:Caenorhabditis elegans / Q000201:enzymology / Q000235:genetics*; D002240:Carbohydrate Sequence; D003001:Cloning, Molecular; D017124:Conserved Sequence; D000939:Epitopes; D005647:Fucosyltransferases / Q000145:classification / Q000235:genetics* / Q000378:metabolism*; D017238:Genes, Helminth; D006031:Glycosylation; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013696:Temperature
D000939:Epitopes; D011994:Recombinant Proteins; D005647:Fucosyltransferases