Glycobiology Advance Access originally published online on June 8, 2006
Glycobiology 2006 16(10):947-958; doi:10.1093/glycob/cwl008
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Identification of core 1 O-glycan T-synthase from Caenorhabditis elegans
Department of Biochemistry and Molecular Biology, Oklahoma Center for Medical Glycobiology, University of Oklahoma Health Sciences Center, 975 N.E. 10th Street, BRC Rm. 417, Oklahoma City, OK 73104
1 To whom correspondence should be addressed; e-mail: richard-cummings{at}ouhsc.edu
Received on February 27, 2006; revised on May 25, 2006; accepted on May 26, 2006
The common O-glycan core structure in animal glycoproteins is the core 1 disaccharide Galß1-3GalNAc
1-Ser/Thr, which is generated by the addition of Gal to GalNAc1-Ser/Thr by core 1 UDP--galactose (UDP-Gal):GalNAc1-Ser/Thr ß1,3-galactosyltransferase (core 1 ß3-Gal-T or T-synthase, EC2.4.1.122). Although O-glycans play important roles in vertebrates, much remains to be learned from model organisms such as the free-living nematode Caenorhabditis elegans, which offer many advantages in exploring O-glycan structure/function. Here, we report the cloning and enzymatic characterization of T-synthase from C. elegans (Ce-T-synthase). A putative C. elegans gene for T-synthase, C38H2.2, was identified in GenBank by a BlastP search using the human T-synthase protein sequence. The full-length cDNA for Ce-T-synthase, which was generated by polymerase chain reaction using a C. elegans cDNA library as the template, contains 1170 bp including the stop TAA. The cDNA encodes a protein of 389 amino acids with typical type II membrane topology and a remarkable 42.7% identity to the human T-synthase. Ce-T-synthase has seven Cys residues in the lumenal domain including six conserved Cys residues in all orthologs. The Ce-T-synthase has four potential N-glycosylation sequons, whereas the mammalian orthologs lack N-glycosylation sequons. Only one gene for Ce-T-synthase was identified in the genome-wide search, and it contains eight exons. Promoter analysis of the Ce-T-synthase using green fluorescent protein (GFP) constructs shows that the gene is expressed at all developmental stages and appears to be in all cells. Unexpectedly, only minimal activity was recovered in the recombinant, soluble Ce-T-synthase secreted from a wide variety of mammalian cell lines, whereas robust enzyme activity was recovered in the soluble Ce-T-synthase expressed in Hi-5 insect cells. Vertebrate T-synthase requires the molecular chaperone Cosmc, but our results show that Ce-T-synthase does not require Cosmc and might require invertebrate-specific factors for the formation of the optimally active enzyme. These results show that the Ce-T-synthase is a functional ortholog to the human T-synthase in generating core 1 O-glycans and open new avenues to explore O-glycan function in this model organism.
Key words: Caenorhabditis elegans / cloning / core 1 O-glycan / galactosyltransferase / T-synthase
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