Date of Award

2020

Document Type

Thesis

Degree Name

Master of Science

College

College of Arts and Sciences

Program

Biology, MS

First Advisor

Peter Lyons

Second Advisor

Denise L. Smith

Third Advisor

Benjamin Navia

Abstract

Carboxypeptidase O (CPO) is a membrane-bound peptidase that cleaves acidic and polar C-terminal amino acids of peptides; however, its biological function remains unknown. CPO is strongly expressed in the small intestine, where it has been proposed to participate in digestion events at the brush border by extracellular cleavage of dietary peptides. At the subcellular level, CPO is anchored to the inner leaflet of the endoplasmic reticulum (ER) membrane, where it has been shown to associate with lipid droplets (LDs). The ER membrane also represents the site of iv chylomicron formation, a process that shares many similarities with that of LDs biogenesis. We sought to investigate a potential role of CPO in LD and chylomicron formation. Using a stablyexpressing human colon carcinoma (Caco-2) cell model, we evaluated CPO association with LDs and apoB, a chylomicron biomarker, by immunofluorescence microscopy. The association of CPO with LDs was dependent on the time elapsed post-feeding (ANOVA, p < 0.05), and was lowest at 6 h (17.9%), reaching a maximum at 12 h (29.8%) and later slightly decreasing at 24 h (26.3%).

Our results indicate that CPO associated with apoB, further showing a similar association pattern as that of LDs at the three time points. However, we express a limited level of confidence in these results since the immunocytochemistry images presumably suggest lower association levels than those reported in the quantification data. Overall, our findings strengthen the hypothesis that CPO plays an intracellular role in the formation of lipid droplets in enterocytes.

To better understand the potential biological role of CPO, we performed an in-depth analysis of data from several gene expression databases and platforms. RNA-Seq and microarray expression data showed high expression in the ileum epithelium and mucosa. Modulation of CPO expression, which was mainly characterized by downregulation in the ileum, was found in ulcerative colitis and Crohn’s disease, with the latter showing stronger modulation. RNA-Seq data also showed prominent expression in Peyer’s patches and basophils, which suggested a promising role of CPO in immunity.

Subject Area

Carboxypeptidases; Chylomicrons; Lipoproteins; Lipid droplets

DOI

https://dx.doi.org/10.32597/theses/182/

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