Date of Award

2021

Document Type

Thesis

Degree Name

Master of Science

College

College of Arts and Sciences

Program

Biology, MS

First Advisor

Peter J. Lyons

Second Advisor

Brian Y. Wong

Third Advisor

Robert Zdor

Abstract

Carboxypeptidase O (CPO) is a peptidase that is a member of the A/B subfamily of the metallocarboxypeptidases. This peptidase has been found in the brush border of the intestine and has specificity toward C-terminal acidic residues. CPO has been proposed to be involved in intestinal digestion and the formation of lipid droplets. Four copies of the CPO gene are present in the Xenopus tropicalis (tropical clawed frog) genome and are noted as CPO.1, CPO.2, CPO.3, and CPO.4. Not much is known about these genes regarding their properties and functions. In my research, I sought to investigate the properties and functions of these genes.

It was found that the predicted proteins of the four CPO genes from X. tropicalis have different properties than human CPO, a well-studied CPO enzyme. Phylogenetic analyses showed that CPO.3 is most likely the most ancient gene of the four CPO genes of X. tropicalis. Western blotting analyses showed that all of the CPO genes from X. tropicalis were translated into proteins with CPO.2 being the least expressed of the four genes. Carboxypeptidase assays iv were done on the protein samples obtained from HEK293T cells that were transfected with the CPO plasmids. No enzymatic activity was detected for any protein sample.

Human CPO has been documented to be found in the endoplasmic reticulum, however not much is known about the subcellular localization of the four CPO gene products from X. tropicalis. To determine the subcellular localization of each CPO protein, immunofluorescence microscopy was done on HEK293T cells that were transfected with both human CPO and X. tropicalis CPO plasmids. It was found that the X. tropicalis CPO proteins had similar distribution to proteins that were found in the endoplasmic reticulum and were distributed on average in puncta that resembled lipid droplets.

Subject Area

Carboxypeptidases; Xenopus

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