Analysis of oligosaccharyltransferase isoform function in human follicle stimulating hormone via siRNA knockdown
Chambers, Carrie Anne
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Follicle-stimulating hormone (FSH) is a glycoprotein hormone with two subunits, alpha and beta, and is required for gamete development. FSH is now known to exist as a ratio of two glycoforms that varies during the menstrual cycle and in premenopausal periods in women. A newly identified glycoform, referred to as diglycosylated FSH, is characterized by the presence of two glycans attached to the alpha subunit while none are attached to the beta subunit. Preliminary data suggest that estrogen is responsible for reducing the glycosylation of FSHbeta in reproductive-age women, thus yielding higher concentrations of diglycosylated FSH than is found in older women. This selective reduction of glycosylation of the FSHbeta subunit is suspected to be due to activity of different oligosaccharyltransferase (OST) subunit isoforms. To investigate how the OST subunit isoforms STT3A and STT3B subunit isoforms affect FSH glycosylation, we used a stably transfected GH3 cell line that produces recombinant hFSH, and selectively knocked down each isoform via siRNA transfection, and examined the glycosylation patterns of the hFSH produced. We used a liposomal transfection methodology for siRNA transfection, and chromatographic methods for FSH purification. Following transfection, RNA from transfected cells was isolated and evaluated using reverse transcription and quantitative real-time PCR (RT-qPCR) to measure expression levels of the two isoforms and glycosylation patterns of FSH subunits were examined via Western blot. Ultimately, use of siRNA to selectively knock-down STT3 isoforms did not produce observable variance in FSH subunit glycosylation patterns when compared to controls. Although the RT-qPCR results suggest that isoform knock-down was achieved on some level, the subunit glycoforms captured on the Western blots did not show significant variation in glycoform abundance.
Thesis (M.S.)--Wichita State University, College of Liberal Arts and Sciences, Dept. of Biological Sciences