Site-specific characterization of N-glycans
For intact N-glycopeptide analysis, chymotryptic digests of both hFSHs were subjected under UPLC equipped with a HILIC column designed to retain hydrophilic peptides, such as glycosylated peptides. MS2 spectra containing glycan fragment ions representing markers of specific glycopeptides were applied for database searching and glycan elucidation (Figure 4a). Highly heterogeneous glycosylation patterns were observed for each N-glycosylation site of both hFSHs. Supporting Information Table 3 summarizes the glycan structures found at each glycosylation site. Mono-, bi-, tri-, and tetra-antennary sialylated glycans were present in both subunits. These glycans were predominantly of the complex type, although some hybrid forms could also be observed. A few glycan chains at αN78 and βN24 of rhFSH and at αN52 of uhFSH showed high fucosylation level. Several structural differences were apparent in the glycan of rhFSH and uhFSH. In rhFSH, sulfated glycans were present at αN52, as well as in both sites in the β-subunit, whereas no sulfated glycan was observed in uhFSH. Moreover, the glycans containing
NeuGc were identified at αN78 and in both sites of the β-subunit, whereas this structure was not observed in uhFSH. Penta-antennary oligosaccharides are possibly present at αN52 and βN24 of uhFSH. Figure 4b shows the glycans attached at αN52 of each hFSH. The αN52 of rhFSH possessed sulfated complex glycans with and without sialylation,whereas highly fucosylated glycans and hybrid type glycans were observed at the same site in uhFSH.
A previous study has reported that the binding between hFSH and G protein-coupled receptors that results in hormonal response is greatly affected by the glycans at particular sites. For instance, the site-directed mutagenesis that occurred at αN52 of hFSH to selectively eliminate the glycan at this position resulted either in increased or in an unaltered receptor binding activity with a reduced receptor activation and signal transduction.32 Therefore, characterization of rhFSH and uhFSH glycopeptides can provide valuable information on their functions.
Discovery of O-glycosylation
Compared with N-glycosylation, O-glycosylation attached to S or T does not have a consensus motif for potential sites, does not have a common core structure, and are more heterogeneous. To identify O-glycosylation, the hFSH samples were treated with PNGase F to remove the N-glycans and then digested with trypsin. The tryptic peptides were analyzed under DDA with product ion triggered ETD (HCD-pd-ETD). The HCD data are rich in glycan fragments, along with a few peptide cleavage b and y ions, whereas the
complementary ETD preferentially induces cleavages along the peptide backbone providing c and z ions. The most common mucin-type O-linked oligosaccharides were used as a glycan database for data processing.
Table 1 shows the identified O-glycopeptides in both hFSHs. Two O-glycosylation sites were assigned in both subunits of rhFSH, whereas five O-glycosylation sites were identified in both subunits of uhFSH. Two glycoforms, namely, HexNAc2Hex1 and HexNAc, were identified in βT6 of rhFSH by both fragmentation modes. Figure 5 shows the b/y and c/z ions of O-glycopeptide NSCELTNITIAIEKEEC#R with a HexNAc2Hex1
residue. Although an N-glycosylation site is also located at the peptide, the presence of b6 and c6 ions confirmed that the HexNAc2Hex1 was attached to βT6. The presence of target glyco-oxonium ion, HexNAc, further confirmed the presence of O-glycans in rhFSH.Only one glycoform, HexNAc2Hex1, was identified in βT6 of uhFSH. The O-glycosylation site, αS43, was also identified in both hFSHs, while αS34, αS39 and βT34 were only assigned in uhFSH. O-glycosylation has not yet been reported in hFSH. Compared with N-glycans, O-glycans are more heterogeneous and exhibit no common core structures;O-glycosylation characterization is thus a great challenge and a systematic approach in analyzing glycosylation is called for. We employed DDA with HCD-pd-ETD on Orbitrap Tribrid MS to obtain reliable information on O-glycosylation. The βT6 of rhFSH and βT6 and βT34 of uhFSH were located close to the N-glycosylation sites. Thus, O-glycosylation, which is accompanied by N-glycosylation, possibly plays a key role in determining the functional properties of hFSH.
In vivo bioassay of the biological activity of hFSH
The oligosaccharides in hFSH determine the half-life of plasma and consequently the in vivo bioactivity of the secreted hormone.6 The in vivo bioactivity of hFSH was further studied herein through the ovarian weight augmentation test. Table 2 shows the protein contents, in vivo bioactivity, and specific activity of rhFSH and uhFSH. The measured in vivo bioactivity of rhFSH corresponded closely with the labeled amount. The specific activity of rhFSH was nearly similar to that of uhFSH. The investigation on the biological effects of hFSH glycosylation has also demonstrated that variations in FSH glycosylation have specific effects on follicular growth, antral formation, and estradiol secretion.7
Therefore, the biological effect of rhFSH and uhFSH must be further investigated.
Conclusions
Various strategies were used to map and compare the oligosaccharides of rhFSH and uhFSH. The following were investigated in this study: (a) analysis of intact protein and subunit masses; (b) profiling of N-glycan structures; (c) characterization of site-specific Nand O-glycosylation; (d) determination of N- and O-glycosylation sites; and (e) analysis of charge variations, sialic acid contents, and specific activities. High level of sialylation, antennary, and macro-heterogeneity were observed in uhFSH. Moreover, NeuGc and sulfated glycans were detected in rhFSH. We also elucidated for the first time the
O-glycosylation in both hFSHs. Comparative study on the biological effect of rhFSH and hFSH is further required to reveal the critical structural features of glycans.
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