Comparison of hFSH Glycosylation by Electrospray Ionization Mass Spectrometry.

Abstract

FSH is a highly heterogenous glycoprotein hormone, which possesses 4 N-glycosylation sites, each decorated with a family of glycans. Glycosylation heterogeneity is usually evaluated by fractionation methods that separate on the basis of negative charge. From the pattern of isoform isoelectric points, differences in glycosylation have been inferred. For example, because different isoform patterns were encountered when pituitary extract, serum samples, and urine samples were fractionated by chromatofocusing then evaluated by FSH RIA, glycosylation of FSH from all three sources was assumed to differ. Recent advances in mass spectrometry now permit direct comparison of FSH glycans. In the present study, we compared FSH glycosylation in human FSH preparations derived from pituitary glands, postmenopausal urine, and recombinant hFSH expressed in the rat GH3 cell line. The glycan populations released from reduced, carboxymethylated hFSH preparations obtained from the pituitary and from postmenopausal urine were virtually identical. The major glycans were identical, the major differences occurred in relatively minor glycan populations, including a 35% decrease in sulfated glycans, 27% decrease in glycans with a bisecting GlcNAc residue, and a 55% decrease in fucosylated glycans. The great similarity in pituitary and urinary hFSH glycan populations is a highly significant finding as it the absence of major changes in glycan popultations between pituitary and urinary FSH means that serum FSH glycans must also be the same. Therefore, examination of urinary hFSH glycosylation is directly relevant to serum hFSH glycosylation. We also compared glycosylation of tetra-glycosylated hFSH and two di-glycosylated hFSH preparations. The di-glycosylated hFSH derived from FSH preparations possessed a glycan population that was highly similar to that from tetra-glycosylated hFSH. Glycans from both glycoform preparations were very similar to pituitary hFSH, except the sulfated glycan populations were greater in the glycoform preparations. Glycosylation of di-glycosylated hFSH isolated from hLH preparations was strikingly different from all other hFSH preparations. The glycans were largely high mannose and in combinantion with their abundance, it could be argued that these represent an intracellular form. Glycosylation of recombinant hFSH revealed the absence of tetra-antennary glycans, which comprised 15% of pituitary hFSH glycans. Moreover, recombinant hFSH triantennary glycans possessed a third branch linked beta1-6 to the alpha1-6 mannose residue, which is due to the action of GlcNAc transferase V. In pituitary this branch is linked beta1-4 to the alpha1-3 mannose residue, as a consequence of GlcNAc transferase IV. This suggested the absence of GlcNAc transferase IV in rat GH3 cells. However, RT PCR found evidence for expression of two isoforms of this enzyme, while failing to detect GlcNAc transferase V. In order to make a more native-like recombinant hFSH, GH3 cells will have to be engineered to express the more human pituitary-like glycosyltransferase population. Supported by NIH grant P01 AG029531. (poster)