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From Vitamin C to Metabolite Repair: The Role of Novel Sugar Nucleotide Phosphorylases

  • Author(s): Adler, Lital Noah
  • Advisor(s): Clarke, Steven G
  • et al.
Abstract

The pcm-1 gene in the soil nematode Caenorhabditis elegans that encodes the L- isoaspartyl methyltransferase has roles in protein repair as well as in the worm's insulin-like signaling pathway. Interestingly, the pcm-1 gene displays overlapping exon sequences with a previously undescribed gene (mcp-1, C10F3.4) that is transcribed from the opposite strand. This rare topology in the C. elegans genome (less than 0.05% of all genes) led us to ask whether the expression of the pcm-1 and mcp-1 genes may be co-regulated or whether their products may have a related function. PSI-BLAST analyses revealed that MCP-1 has homologs in plants, vertebrates and invertebrates and that all the homologs share a conserved HIT (histidine triad) motif. Mutations in the Arabidopsis thaliana homolog, vtc2, lead to vitamin C deficiency in these plants.

In this thesis, I have characterized the enzymatic activity of VTC2 from A. thaliana as a GDP-L-galactose phosphorylase, the last missing step in the Smirnoff-Wheeler pathway for vitamin C biosynthesis in plants. Additional work has been done to show that the green alga Chlamydomonas reinhardtii shares homologs with all of the genes in the Smirnoff-Wheeler pathway as well as the specific activity of GDP-L-galactose phosphorylase.

While vitamin C is an apparent important antioxidant and enzymatic cofactor in C. elegans, no reports had been made as to the ability of this nematode to produce it. In my work I found out that C. elegans extracts contain low levels of vitamin C. Nevertheless, as levels of vitamin C in the mcp-1 deletion strain are not different from the values in the control animals and as C. elegans lack key homologs in the biosynthesis pathway, I concluded that mcp-1 was not involved in vitamin C synthesis in C. elegans.

Enzymatic analysis revealed that C. elegans ’ mcp-1 and its mammalian homologs encode GDP-D-glucose phosphorylases. mcp-1 mutant worms that lacked this activity accumulated GDP-D-glucose. This unique metabolite, which shares high similarity with GDP-D-mannose, does not have a reported role in invertebrates or mammals. With my collaborators, I suggested that GDP-D-glucose could compete with GDP-D-mannose for binding to glycosyltransferases in the first steps of protein N-glycosylation in the cell, thus potentially replacing mannose residues with glucose residues in a step that could lead to N-glycosylation defect. Thus, MCP-1 may serve as a metabolite repair enzyme that eliminates adventitiously formed GDP-D-glucose from the cytosol to ensure correct protein N-glycosylation. I have tested this hypothesis by observing morphological changes in mcp-1 mutant worms and by directly comparing the N-glycan profiles of mcp-1 and control animals.

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