Graduate Program

Biological Sciences

Degree Name

Master of Science (MS)

Semester of Degree Completion

Summer 2020

Thesis Director

Gopal R. Periyannan

Thesis Committee Member

Britto P. Nathan

Thesis Committee Member

Gary A. Bulla

Abstract

Glutamate Carboxypeptidase II (GCPII) is a transmembrane, zinc metallopeptidase that is expressed in a wide range of organisms, including roundworms, mice, and humans. In humans, GCPII is primarily expressed in the prostate, kidneys, small intestine, and central nervous system. Within the small intestine, the expected function of GCPII is to aid in the absorption of dietary folate from the intestinal lumen. GCPII cleaves excess glutamates from folate to yield monoglutamated folate which is then readily transported into the enterocyte. Folate can then be used through the one carbon metabolic cycle for the synthesis of nucleotides, conversion of homocysteine to methionine, and serve as a precursor for methylation reactions. The human genome contains five paralogs of GCPII (hGCPII) that share a high degree of structural similarity to the three orthologs found in the roundworm, Caenorhabditis elegans (cGCPII). These orthologs are known as, gcp-2.1, gcp-2.2, and gcp-2.3. Three gcp-2 insertion mutant worm strains, the wild type (N2), RB1055 (gcp-2.1), TM6632 (gcp-2.2), and TM5414 (gcp-2.3), were used here to study the cGCPII paralogs’ role in reproduction, growth, and development.

There were two main goals in this study: (i) the functional characterization of the roles of the three cGCPII paralogs in folate metabolism-mediated reproduction and aging, and (ii) the determination of the expression patterns of the cGCPII paralogs in C. elegans to further explore its roles in C. elegans’ life stage development. Each of the C. elegans strains were fed a folate- limited E. coli OP50 bacterial diet to elucidate the differences in function of each cGCPII paralog. The results of this study demonstrate that each of the cGCPII paralogs plays a differential role in the biological processes of reproduction, development, and aging. The cGCPII paralogs gcp-2.1 and gcp-2.3 played important roles in folate metabolism mediated reproduction and aging. Additionally, an optimized, high throughput method of real-time gene expression analysis in C. elegans was produced and demonstrated increased expression of gcp-2.1 in wild type C. elegans compared to the other two paralogs. Ultimately, this study supports the use of C. elegans as a model for human GCPII and folate metabolism related mechanisms and functions. Overall, this study contributes to the understanding of the broader biological functions of GCPII in eukaryotic organisms.

Available for download on Saturday, July 16, 2022

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