Macro- and Micro-heterogeneity in Pituitary and Urinary Follicle-Stimulating Hormone Glycosylation

Abstract

FSH glycosylation macroheterogeneity in pituitary and urinary hFSH samples was evaluated by Western blotting. Microheterogeneity in two highly purified urinary and pituitary hFSH preparations was evaluated by nano-electrospray mass spectrometry of peptide-N-glycanase-released oligosaccharides. An age-related loss of hypo-glycosylated hFSH in individual female pituitaries was indicated by progressively reduced abundance of hFSH21 relative to hFSH24. Urinary hFSH was evaluated as a potentially non-invasive indicator of glycoform abundance, as the only way for pituitary FSH to reach the urine is through the blood. Both highly purified and crude postmenopausal urinary hFSH preparations possessed the same amount of hFSH21 as postmenopausal pituitary gland FSH. Considerable microheterogeneity was encountered in both pituitary and urinary hFSH glycan populations, as 84 pituitary hFSH glycan ions were observed as compared with 68 urinary hFSH glycans. The biggest quantitative differences between the two populations were reduced abundance of bisecting GlcNAc-containing and fucosylated glycans, along with sulfated glycans in the urinary hFSH glycan population. The relative abundance of sialic acid and glycan antenna did not rationalize the retarded electrophoretic mobilities of the urinary hFSHβ21- and α-subunit bands relative to the corresponding pituitary hFSH bands, as the most abundant glycans in the former possessed only 2 more branches and the same sialic content as in the latter. Site-specific glycosylation information will probably be necessary.

Figures

Figure 1. Determination of FSH glycoform content of pituitary hFSH21 by Western blot and dual tandem Superdex 75 gel filtration

A. Isolation of hFSH immunopurified from a 55 year old male pituitary by Superdex 75 gel filtration using a single, 10 × 300 mm column. Inset. Representative Western blot using anti-hFSHβ antibody, RFSH20, performed in triplicate. B. Representative chromatogram measuring glycoform abundance using two Superdex 75 columns combined in series and monitoring the effluent at 210 nm. C. Representative chromatogram measuring FSH glycoform abundance on the same columns, monitoring absorbance at 280 nm. The percentages in each panel indicate the mean of three determinations of the partially glycosylated hFSH21 variant relative abundance.

Figure 2. Relative abundance of hFSH21 in pituitaries of 12 women as a function of age

A. Collage of representative Western blots from each individual. Lanes 1-12 were included in the correlation analysis. Lanes a-c were excluded due to steroid treatment, which appeared to increase the abundance of the hFSHβ21 band in 2 of 3 cases. B. Plot of average (±Std. Dev.) relative abundance of the hFSHβ21 band determined by Western blots for three or four 1-μg samples of each hFSH preparation. A highly significant negative correlation was associated with increasing age.

Figure 3

Pituitary and urinary hFSH Western blots. Pooled human pituitary hFSH, commercially prepared postmenopausal gonadotropin, Pergonal, and individual first void urine characterized by Western blotting. A. Urinary hFSH lots analyzed with anti-hFSHβ antibody RFSH20. B. Urinary hFSH lots analyzed with anti-hFSHα antibody HT13. Lane 1, 1 μg hFSH (AFP4161B); lane 2, Pergonal lot A; lane 3, Pergonal lot B; lane 4, Pergonal lot C. C. RFSH20 Western blot of individual urinary hFSH preparations shown in panel E, below. D. HT13 Western blots of urinary hFSH, as above. Column fractions as indicated below. E. Superdex 75 chromatograms showing purification of anti-hFSHβ 46.3H6.B7-bound hFSH from first void urine samples. The bars show portions of the chromatograms analyzed by Western blotting in panels C and D, above.

Figure 4

SDS-PAGE and Western blot of pituitary and urinary hFSH preparations. Samples consisting of 1 or 10 μg hFSH were reduced in the presence of 5% β-mercaptoethanol and 1% SDS, and subjected to electrophoresis on 15% polyacrylamide gels. A. Proteins visualized by Coomassie blue staining. Based on these results, the pituitary hFSH preparation AFP7298A was selected for glycan analysis. Lane 1, Bio-Rad Precision Plus high MW pre-stained MW markers; lane 2, 10 μg pituitary hFSH (AFP7220); lane 3, 10 μg pituitary hFSH (AFP7298A); lane 4, 10 μg pituitary hFSH (AFP5720); lane 5, 10 μg urinary hFSH. B. FSHβ subunits visualized by Western blotting using monoclonal antibody RFSH20, diluted 1:10,000. C. FSHα subunit Western blot using monoclonal antibody HT13. Lane 1, 1 μg pituitary hFSH (AFP7298A); lane 2, urinary hFSH. While all images were acquired with a BioRad VersaDoc 4000, the positions in the instrument and lenses employed for transillumination and chemiluminescence imaging were different, thereby producing different sized images.

Figure 5

ESI-mass spectrometry analysis of intact pituitary and urinary hFSH glycans. A. Negative ion ESI spectrum of pituitary hFSH glycans. B. Negative ion ESI spectrum of urinary hFSH oligosaccharides. Neutral and acidic glycan masses along with compositions are listed in Tables 1 and 2.

Figure 6

Glycan structures representing the neutral cores of 52 glycans as well as 12 sialylated glycans. The composition (H = hexose, N = amino sugar, S = sialic acid, F = fucose) and calculated mass of the neutral core glycan are given. The letters P and U correspond pituitary and urinary hFSH, respectively. Those in parentheses were not found in the original data tables. The structures follow the system proposed by the Oxford Glycobiology Institute [63]. Monosaccharide symbols are: closed square, GlcNAc; open circle, Mannose; open diamond, Galactose; closed diamond, GalNAc; dotted diamond, fucose; closed star, sialic acid. Linkages are indicated by solid lines for β and dashed lines for α.

Figure 7

Comparison of pituitary and urinary hFSH glycan populations. A. Relative abundance of glycans based on glycan core structure indicated by gray scale. Each bar represents the relative abundance of the sum of glycan variants possessing the same underlying glycan structure for a given hormone (Table 1) both as shaded (0 = white, 100% of max. = black) and numerical values. B. Core glycan structures for the 6 most abundant glycan families in panel A. The m/z value and rank order of relative abundance are given below each. C. Glycan variant abundance within each glycan family (wide bars). Narrow bars indicate relative abundance of each variant using gray scale 0-100% and shown in the same order listed in Table 1. The glycan variant m/z and relative abundance within each group are indicated beside each bar and are intended for digital format, which permits zooming in to read individual values. The wide bars represent each glycan family listed in panel A, and are identified by the m/z values on the right. These serve to separate each group of individual glycan variants. D. Glycan structures for the 6 most abundant glycan variants in each FSH preparation. When the same variant is most abundant in both pituitary and urinary hFSH, the structures are centered. When they differ, pituitary glycans are shown on the left and urinary glycans on the right. The numbers indicate the order of abundance within each FSH preparation. Families 3-5 and variants 3-5 were very similar in abundance.