Characterization of recombinant hFSH glycosylation

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Issue Date
2012-04-03
Authors
Rose, Emily
White, William K.
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Abstract

Human FSH stimulates ovarian follicle development in females and regulates testicular Sertoli cell function in males. Our laboratory has identified two major hFSH forms differing in glycosylation and binding affinity to FSH receptors. The larger hFSH glycoform possesses all 4 N-glycans (tetra-glycosylated hFSH). The smaller glycoform consists of non-glycosylated FSHβ subunit combined with glycosylated α subunit (di-glycosylated hFSH). As FSH glycosylation is essential for FSH receptor activation, this difference in glycosylation could affect activation of intracellular signal pathways. Currently, we express recombinant human FSH in stably transfected GH3 rat anterior pituitary somatotrope cells. The goal of the present study is to compare recombinant hFSH glycosylation with human pituitary-derived FSH glycosylation. FSH preparations immunopurified from conditioned medium and separated into high and low MW fractions by gel filtration were evaluated. Goals included verifying the presence of both glycoforms and defining the nature of the glycan populations decorating each glycoform. Reverse-phase, high performance liquid chromatography carried out on high and low MW recombinant hFSH fractions revealed both factions possessed mostly the tetra-glycosylated glycoform. Total amino acid and carbohydrate analysis indicated the glycan populations were largely bi-antennary, while human pituitary FSH possesses predominantly tri- and tetra-antennary N-glycans. The smaller recombinant hFSH glycans may explain why the two hFSHβ bands in these preparations corresponding to pituitary hFSH 21 and 24 kDa bands migrate faster than those derived from pituitary hFSH.

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Second place winner of oral presentations in the Natural Science/Engineering section at the 12th Annual Undergraduate Research and Creative Activity Forum (URCAF) held at the Rhatigan Student Center , Wichita State University, April 3, 2012
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