Molecular size and charge as dimensions to identify and characterize circulating glycoforms of human FSH, LH and TSH

Abstract

Background: FSH, LH, and TSH are glycoprotein hormones secreted from the pituitary as fully and low-asparagine-glycosylated hormones. These glycoforms of the hormones exist as a large number of isoforms varying in their glycan contents of terminal anionic monosaccharides (AMS), i.e. sialic acid (SA) and sulfonated N-acetylgalactosamine (SU). Due to the immense heterogeneity and the low concentrations in serum it has been a challenge to develop reliable analytical methods to measure and characterize the circulating glycoforms of these hormones.

Methods: The hormones were separated with respect to AMS content per molecule by calibrated 0.1% agarose suspension electrophoreses. Glycoforms in separated fractions were then analyzed with respect to size by 180 calibrated Sephadex G-100 gel filtrations. The hormones were measured with time-resolved sandwich fluoroimmunoassays. All separations and assays were performed in veronal buffer at pH 8.7. Sera and fractions were also analyzed after removal of terminal SA.

Results: In addition to the fully glycosylated FSH, LH, and TSH, also tri-glycosylated FSH and di-glycosylated LH and TSH forms could be identified in serum samples. The low- and fully glycosylated hormones differed both with respect to size and to median number of AMS per molecule. Algorithms, based on the distributions by electrophoreses, were developed for each hormone to estimate percent low-glycosylated forms in serum. The median numbers of SA and SU per glycoform molecule were estimated using results obtained after desialylation.

Conclusion: The methods can be used for identification and characterization of glycoforms of circulating FSH, LH, and TSH in physiological and clinical studies.

Keywords: FSH; LH; TSH; glycoforms; glycosylation; sialic acid; sulfonation.

Figure 1.

Schematic drawings of structures of the heterodimeric glycoforms of human FSH, LH, and TSH consisting of one α and one β peptide chain. The number of amino acids of the peptide chains is given in parentheses, and the peptide positions of the glycans are indicated.

Figure 2.

Calibration of Sephadex G-100 gel filtration. The patterns of proteins and hormones in relation to fraction eluted are shown in the left panel. The position where the first protein peak, measured at 280 nm, passed 30% of its top level was given an arbitrary value of 100, and the corresponding position on the slope of the albumin peak was given an arbitrary value of 57. The patterns expressed as size in arbitrary units are shown in the right panel.

Figure 3.

Size of glycoforms of FSH, LH, and TSH in serum, as estimated by gel filtration, in relation to number of anionic monosaccharides (AMS) per molecule, as estimated by electrophoresis. Number of gel filtrations within parentheses. Mean values of glycoform size, in arbitrary units, ranging from 37.9 to 57.4, are shown.

Figure 4.

Estimation of percent TSHdi by gel filtration in a serum sample from a 27-year-old woman with hypothyroidism. Distribution of TSH in pg per 25 µL eluate in relation to AMS per molecule by electrophoresis in the lower panel. Results of estimations of size of TSH glycoforms in fractions after electrophoresis in the upper panel, including measurements where TSHdi and TSHtri overlap.

Figure 5.

Electrophoresis of TSH in a serum sample from a 34-year-old euthyroid woman. The TSH in femtogram per 25 µL eluate is plotted in peaks in relation to number of AMS per molecule, and the distribution in percent of total amount of TSH eluted is indicated. The distributions of TSHdi and TSHtri from size estimations by gel filtration, and the percent TSHdi estimated by electrophoresis using an algorithm, are shown.

Figure 6.

The percent low-glycosylated glycoforms, as estimated by electrophoresis using the three algorithms (see text), was plotted against the corresponding percentage, as estimated by gel filtration. The slopes and the coefficients of correlation were close to 1.