Ju Tongzhong, Zheng Qinlong, Cummings Richard D
Department of Biochemistry and Molecular Biology, Oklahoma Center for Medical Glycobiology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA.
Glycobiology. 2006 Oct;16(10):947-58. doi: 10.1093/glycob/cwl008. Epub 2006 Jun 8.
The common O-glycan core structure in animal glycoproteins is the core 1 disaccharide Galbeta1-3GalNAcalpha1-Ser/Thr, which is generated by the addition of Gal to GalNAcalpha1-Ser/Thr by core 1 UDP-alpha-galactose (UDP-Gal):GalNAcalpha1-Ser/Thr beta1,3-galactosyltransferase (core 1 beta3-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.
动物糖蛋白中常见的O-聚糖核心结构是核心1二糖Galβ1-3GalNAcα1-Ser/Thr，它是由核心1 UDP-α-半乳糖（UDP-Gal）：GalNAcα1-Ser/Thr β1,3-半乳糖基转移酶（核心1 β3-Gal-T或T-合酶，EC2.4.1.122）将Gal添加到GalNAcα1-Ser/Thr上生成的。尽管O-聚糖在脊椎动物中发挥着重要作用，但从诸如自由生活的线虫秀丽隐杆线虫等模式生物中仍有许多有待了解的地方，这些模式生物在探索O-聚糖结构/功能方面具有许多优势。在此，我们报告了秀丽隐杆线虫T-合酶（Ce-T-合酶）的克隆及酶学特性。通过使用人类T-合酶蛋白序列进行BlastP搜索，在GenBank中鉴定出一个假定的秀丽隐杆线虫T-合酶基因C38H2.2。以秀丽隐杆线虫cDNA文库为模板，通过聚合酶链反应生成的Ce-T-合酶全长cDNA包含1170 bp，包括终止密码子TAA。该cDNA编码一个389个氨基酸的蛋白质，具有典型的II型膜拓扑结构，与人类T-合酶有42.7%的显著同源性。Ce-T-合酶在腔内结构域有七个半胱氨酸残基，包括所有直系同源物中的六个保守半胱氨酸残基。Ce-T-合酶有四个潜在的N-糖基化序列，而哺乳动物直系同源物缺乏N-糖基化序列。在全基因组搜索中仅鉴定出一个Ce-T-合酶基因，它包含八个外显子。使用绿色荧光蛋白（GFP）构建体对Ce-T-合酶进行启动子分析表明，该基因在所有发育阶段均有表达，且似乎在所有细胞中都有表达。出乎意料的是，从多种哺乳动物细胞系分泌的重组可溶性Ce-T-合酶中仅恢复了极低的活性，而在Hi-5昆虫细胞中表达的可溶性Ce-T-合酶中恢复了强大的酶活性。脊椎动物T-合酶需要分子伴侣Cosmc，但我们的结果表明Ce-T-合酶不需要Cosmc，可能需要无脊椎动物特异性因子来形成最佳活性酶。这些结果表明，Ce-T-合酶在生成核心1 O-聚糖方面是人类T-合酶的功能直系同源物，并为在该模式生物中探索O-聚糖功能开辟了新途径。
