Hypo-glycosylated human follicle-stimulating hormone (hFSH(21/18)) is much more active in vitro than fully-glycosylated hFSH (hFSH(24))

Abstract

Hypo-glycosylated hFSH(21/18) (possesses FSHβ(21) and FSHβ(18)bands) was isolated from hLH preparations by immunoaffinity chromatography followed by gel filtration. Fully-glycosylated hFSH(24) was prepared by combining the fully-glycosylated FSHβ(24) variant with hCGα and isolating the heterodimer. The hFSH(21/18) glycoform preparation was significantly smaller than the hFSH(24) preparation and possessed 60% oligomannose glycans, which is unusual for hFSH. Hypo-glycosylated hFSH(21/18) was 9- to 26-fold more active than fully-glycosylated hFSH(24) in FSH radioligand assays. Significantly greater binding of (125)I-hFSH(21/18) tracer than hFSH(24) tracer was observed in all competitive binding assays. In addition, higher binding of hFSH(21/18) was noted in association and saturation binding assays, in which twice as much hFSH(21/18) was bound as hFSH(24). This suggests that more ligand binding sites are available to hFSH(21/18) in FSHR than to hFSH(24). Hypo-glycosylated hFSH(21/18) also bound rat FSHRs more rapidly, exhibiting almost no lag in binding, whereas hFSH(24) specific binding proceeded very slowly for almost the first hour of incubation.

Keywords: FSH isoforms; FSHR; Oligosaccharide.

Copyright © 2013 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.

Figures

Figure 1. Hypo-glycosylated hFSH isolation from hLH preparations

FSH bound to anti-hFSHβ immunoaffinity columns was applied to a 10 X 300 mm Superdex 75 column and the chromatogram developed as described under Methods. A. FSHβ and FSHα Western blots of reduced samples from the chromatogram in panel C. Anti-FSHβ: Lane 1, 1 μg hFSH (AFP7298A); lane 2, 1 μg fraction A; lane 3, 1 μg fraction B; lane 4, 1 μg fraction C. Anti-α: Lane 5, 1 μg hFSH (AFP7298); lane 2, 1 μg fraction A; lane 3, 1 μg fraction B; lane 4, 1 μg fraction C. B. FSHβ Western blot of non-reduced samples from the chromatogram in panel C. Lane 1, 1 μg hFSH (AFP7298A); lane 2, 1 μg fraction A; lane 3, 1 μg fraction B; lane 4, 1 μg fraction C. C. First round of hFSH isolation. D. Second round of hFSH isolation. E. Third round of hFSH isolation. The open bars show portions of each chromatogram containing aggregated hFSH (A) hFSH heterodimer (B), and subunits (C).

Figure 2. Hypo- and fully-glycosylated hFSH isolation

A. Immunopurified hFSH fractionated by Superdex 75 chromatography and fractions analyzed by Western blotting. Fractions indicated by bars and numbers. Insets 1 & 2. Western blot analysis of 1 μg samples of reduced Superdex 75 fractions with FSHβ- and FSHα-specific antibodies RFSH20 and HT13, respectively. Lane 1, hFSH AFP7298A; lane 2, fraction A; lane 3, fraction B; lane 4, fraction C; lane 5, fraction D. The arrows show the positions of the hFSHβ21,000 and 24,000 Mr bands, as indicated. B. The 24,000 Mr hFSHβ was combined with hCGα and incubated for 72 hr at 37°C. The mixture was reduced to <200 μl and applied to a Superdex 75 column as described under Methods. Insets 3 and 4, FSHβ and FSHα Western blot analysis of reduced Superdex 75 fractions, respectively. Lane 1, hFSH24; lane 2, pituitary hFSH (AFP7298A).

Figure 3. FSH receptor-binding assays of fully- and hypo-glycosylated hFSH preparations

A. 125I-fully-glycosylated hFSH24 tracer and rat testis homogenate. B. 125I-hypo-glycosylated hFSH21/18 tracer with rat testis homogenate. C. 125I-hypo-glycosylated hFSH21/18 tracer with CHO cells expressing hFSHR. Receptor-binding assays carried out as described under Methods. Quantitative results are shown in Table 1.

Figure 4. Saturation binding of hypo- and fully-glycosylated hFSH preparations to rat testis FSH receptors

The same rat testis homogenate was used to measure specific binding of both glycoforms. Closed circles, saturation binding of 125I-hypo-glycosylated hFSH. Closed squares, saturation binding of 125I-fully-glycosylated hFSH. Specific binding (Bo – Bn) was determined using 2000-fold excess cold eFSH. Representative data from two experiments.

Figure 5. Association kinetics for hypo- and fully-glycosylated hFSH binding to rat testis FSH receptors

FSH glycoform tracers, in the presence and absence of 2000-fold cold eFSH, were incubated with rat testis FSH receptors at 37°C for the indicated times, then placed in an ice water bath. When all the tubes were incubated, they were centrifuged to separate bound from free hormone, the supernatants aspirated and 125I-hFSH glycoform bound to the pellets counted in a gamma counter. Specific binding is plotted against incubation time. Representative data from three experiments.

Figure 6. Comparison of recombinant insect hFSH subunit electrophoretic mobilities with those of pituitary FSH glycoforms

A. FSHβ-Western blot comparing mobilities of hFSH glycoforms with those of recombinant insect cell hFSHβ subunit preparations. The primary antibody was RFSH20. B. FSHα-Western blot using HT13 primary antibody. The same preparations were compared in both blots. Lane 1, pituitary hFSH (AFP4161B); lane 2, hypo-glycosylated hFSH; lane 3, fully-glycosylated hFSH; lane 4, wt recombinant insect hFSH; lane 5, αT54A mutant recombinant insect hFSH; lane 6, αT80A mutant recombinant insect hFSH; lane 7, βT26A mutant recombinant insect hFSH; lane 8, recombinant bacterial hFSHβ. Pre-stained BioRad MW marker positions are indicated by lines.