Urinary FSH glycoform evaluation by automated Western blotting
Citation
Katta, Sahithi. 2019. Urinary FSH glycoform evaluation by automated Western blotting -- In Proceedings: 15th Annual Symposium on Graduate Research and Scholarly Projects. Wichita, KS: Wichita State University
Abstract
FSH is a critical hormone for fertility in women. Its structure and function have been studied for
years. In females, FSH enables ovarian follicle development, thereby producing mature oocytes at
ovulation. Recently, studies emanating from Dr. Bousfield’s laboratory have shown that human FSH
exists as two major glycoforms, fully-glycosylated FSH24, which possesses two α-subunit and two
β-subunit asparagine-linked oligosaccharides, and hypo-glycosylated FSH21 or FSH18 glycoforms,
which both possess one FSHβ oligosaccharide and two α-subunit oligosaccharides. Pituitary FSH21
abundance exhibits reduced relative abundance with increasing age in women. FSH glycoform
concentrations have been reported to vary in serum during the human menstrual cycle. As FSH21
exhibits greater FSH biologic activity than FSH24, age- and cycle-related changes in glycoform
abundance may regulate fertility. The goal of this project is to evaluate FSH glycoform ratios in
female urine samples. Human FSH and other urinary proteins will be precipitated with ethanol, FSH
will be captured by immuno-affinity chromatography using the anti-FSHβ monoclonal antibody,
15-1.E3.E5, followed by gel filtration. Automated Western blotting of 50 ng FSH samples will be
used to measure the relative abundance of both FSH glycoforms. First, 200 µg of purified
recombinant hFSH were added to ethanol-precipitated urinary proteins and immunocaptured with the
15-1.E3.E5 antibody column. The bound fraction was subjected to Superdex 75 gel filtration and
fractions collected by hand. FSH recoveries were measured by an FSH ELISA. In the second series of
experiments, 200 ng samples of recombinant hFSH were added to 35-mL samples of precipitated urinary
proteins, FSH affinity purified, and FSH quantified by ELISA. Half of the 200-µg added to urinary
proteins was bound by the antibody column. Superdex 75 chromatography verified the purity of the
FSH and indicated that >80% was heterodimer. When 200 ng FSH samples were added to urinary proteins
the column captured most of the FSH, as indicated by the low amounts of FSH immunoactivity in the
breakthrough fraction. Most FSH was recovered in the pH 2.7 fraction. Monoclonal antibody 15-1.
E3.E5 can capture the majority of 200 ng FSH samples in precipitated urinary protein samples. As
automated Western blotting can detect 50 ng FSH samples, it is feasible to measure FSH glycoform
abundance in
urinary protein samples.
Description
Presented to the 15th Annual Symposium on Graduate Research and Scholarly Projects (GRASP) held at the Rhatigan Student Center, Wichita State University, April 26, 2019.
Research completed in the Department of Biological Sciences, Fairmount College of Liberal Arts and Sciences