Viktor Y. Butnev

Structural features of mammalian gonadotropins

There are two species for which both pituitary and placental gonadotropins are readily available, humans and horses. The human gonadotropins are better characterized than equine gonadotropins. Nevertheless, the latter are very interesting because they provide exceptions to some of the general structure-function principles derived from studies on human and other mammalian gonadotropins. For example, separate genes encode the hLH beta and hCG beta subunits while a single gene encodes eLH beta and eCG beta. Thus, eCG and eLH differ only in their oligosaccharide moieties and eLH is the only LH that possesses the O-glycosylated C-terminal extension previously believed to be restricted to chorionic gonadotropins. Truncation experiments involving eLH beta and hCG beta have suggested the C-terminal extension has no effect on receptor binding. However, the largest of three eCG forms which differ only in the extent of O-glycosylation possessed reduced affinity for LH and FSH receptors. This result suggested that effects of O-glycosylation need to be considered when examining the glycosylation differences between eLH and eCG responsible for the 10-fold lower eCG receptor binding affinity compared with that of eLH. Contribution of alpha Asn56 N-linked oligosaccharides to the different biological activities of eLH and eCG has been evaluated following selective removal using peptide-N-glycanase digestion of native equine alpha-subunit preparations. Hormones-specific patterns of glycosylation were observed on alpha Asn56 of eLH, eFSH, and eCG. Removal of alpha Asn56 oligosaccharides increased the rate of subunit association, the extent of association, and receptor binding activity. Some unassociated alpha-subunit oligosaccharides were identified which may interfere with subunit association because they were more abundant in unassociated subunit oligosaccharide maps than in a total oligosaccharide map. This was most striking in the case of eCG alpha in which two minor peaks became the major oligosaccharide peaks detectable in the unassociated eCG alpha fraction following association with eLH beta and eFSH beta. The biological activities exhibited by hybrid hormones, eLH alpha reassociated with oLH beta and pLH beta, found to be greater than those of oLH and pLH provided an interesting exception to the general rule that the beta-subunit determines the potency of the heterodimer. LH receptor binding activities of eLH beta-chimeric ovine/equine alpha-subunits suggested that the equine alpha-subunit N-terminal domain may be responsible for this effect. Equine FSH has higher FSH receptor binding activity than human, ovine, and porcine FSH preparations. This probably results from two factors. First, the presence of the equine alpha-subunit promotes receptor binding as noted above. Second, the overall -2 charge of the eFSH beta determinant loop, which is less negative that the -3 observed in other species, results from the presence of an Asn residue at position 88 instead of Asp. This apparently facilitates binding to the FSH receptor.

Hypo-glycosylated human follicle-stimulating hormone (hFSH21/18) is much more active in vitro than fully-glycosylated hFSH (hFSH24)

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.

Identification of Twelve O-Glycosylation Sites in Equine Chorionic Gonadotropin β and Equine Luteinizing Hormone β by Solid-Phase Edman Degradation

Abstract The O-glycosylation sites for equine LHβ (eLHβ) and eCGβ were identified by solid-phase Edman degradation of four glycopeptides derived from the C-terminal region. Both subunits were O-glycosylated at the same 12 positions, rather than the 4–6 sites anticipated. These sites were partially glycosylated, with carbohydrate attachment ranging from 20% to 100% for eCGβ and from 10% to 100% for eLHβ. When the C-terminal peptide containing all but one of the O-linked oligosaccharides was removed by mild acid hydrolysis of either eLHβ or eCGβ, hybrid hormones could be obtained by reassociating eLHα,eFSHα, or eCGα with the truncated β subunit derivatives. These hybrid hormones were identical in LH receptor-binding activity when des(121-149)eLHβ or des(121-149)eCGβ were combined with the same α subunit preparation. Thus, O-glycosylation appears to be responsible for the β subunit contribution to the substantial difference in LH receptor-binding activity between eLH and eCG. Comparison of the equid LH/CGβ sequences with those available for the primate CGβ subunits indicated a greater conservation of glycosylation patterns in the former.

Production, purification, and characterization of recombinant hFSH glycoforms for functional studies

Previously, our laboratory demonstrated the existence of a β-subunit glycosylation-deficient human FSH glycoform, hFSH(21). A third variant, hFSH(18), has recently been detected in FSH glycoforms isolated from purified pituitary hLH preparations. Human FSH(21) abundance in individual female pituitaries progressively decreased with increasing age. Hypo-glycosylated glycoform preparations are significantly more active than fully-glycosylated hFSH preparations. The purpose of this study was to produce, purify and chemically characterize both glycoform variants expressed by a mammalian cell line. Recombinant hFSH was expressed in a stable GH3 cell line and isolated from serum-free cell culture medium by sequential, hydrophobic and immunoaffinity chromatography. FSH glycoform fractions were separated by Superdex 75 gel-filtration. Western blot analysis revealed the presence of both hFSH(18) and hFSH(21) glycoforms in the low molecular weight fraction, however, their electrophoretic mobilities differed from those associated with the corresponding pituitary hFSH variants. Edman degradation of FSH(21/18)-derived β-subunit before and after peptide-N-glycanase F digestion confirmed that it possessed a mixture of both mono-glycosylated FSHβ subunits, as both Asn(7) and Asn(24) were partially glycosylated. FSH receptor-binding assays confirmed our previous observations that hFSH(21/18) exhibits greater receptor-binding affinity and occupies more FSH binding sites when compared to fully-glycosylated hFSH(24). Thus, the age-related reduction in hypo-glycosylated hFSH significantly reduces circulating levels of FSH biological activity that may further compromise reproductive function. Taken together, the ability to express and isolate recombinant hFSH glycoforms opens the way to study functional differences between them both in vivo and in vitro.

Hypoglycosylated hFSH Has Greater Bioactivity Than Fully Glycosylated Recombinant hFSH in Human Granulosa Cells

CONTEXT Previous studies suggest that aging in women is associated with a reduction in hypoglycosylated forms of FSH. OBJECTIVE Experiments were performed to determine whether glycosylation of the FSHβ subunit modulates the biological activity of FSH in human granulosa cells. DESIGN AND SETTING Recombinant human FSH (hFSH) derived from GH3 pituitary cells was purified into fractions containing hypoglycosylated hFSH(21/18) and fully glycosylated hFSH(24). The response to FSH glycoforms was evaluated using the well-characterized, FSH-responsive human granulosa cell line, KGN at an academic medical center. INTERVENTIONS Granulosa cells were treated with increasing concentrations of fully- or hypoglycosylated FSH glycoforms for periods up to 48 hours. MAIN OUTCOME MEASURE(S) The main outcomes were indices of cAMP-dependent cell signaling and estrogen and progesterone synthesis. RESULTS We observed that hypoglycosylated FSH(21/18) was significantly more effective than fully glycosylated FSH(24) at stimulating cAMP accumulation, protein kinase A (PKA) activity, and cAMP response element binding protein (CREB) (S133) phosphorylation. FSH(21/18) was also much more effective than hFSH(24) on the stimulation CREB-response element-mediated transcription, expression of aromatase and STAR proteins, and synthesis of estrogen and progesterone. Adenoviral-mediated expression of the endogenous inhibitor of PKA, inhibited FSH(21/18)- and FSH(24)-stimulated CREB phosphorylation, and steroidogenesis. CONCLUSIONS Hypoglycosylated FSH(21/18) has greater bioactivity than fully glycosylated hFSH(24), suggesting that age-dependent decreases in hypoglycosylated hFSH contribute to reduced ovarian responsiveness. Hypoglycosylated FSH may be useful in follicle stimulation protocols for older patients using assisted reproduction technologies.

Glycosylated equine prolactin and its carbohydrate moiety

Glycosylated equine prolactin (G-ePRL) and nonglycosylated ePRL were purified to homogeneity from side fractions obtained during isolation of LH/FSH from horse pituitaries. Both PRL forms were isolated together in high yield by the isolation procedure used for glycosylated porcine PRL/(G-pPRL) and pPRL, involving acetone extraction/precipitation, NaCl and isoelectric precipitation, and gel filtration. Purification of G-ePRL required additional Con A chromatography. The N-terminal amino acid sequencing for 32 cycles of G-ePRL and ePRL resulted in sequences identical to the known primary structure of ePRL. Based on MALDI mass spectrometry analysis and SDS-PAGE mobilities,G-ePRL and ePRL had estimated molecular weights of 25,000 and 23,000 Da, respectively. G-ePRL displayed only 60% of the immunoreactivity of ePRL in homologous radioimmunoassay. Using the Nb2 lymphoma cell bioassay, ePRL was found to have about l/30th the mitogenic activity of bovine PRL; G-ePRL was approximately l/10th as active as ePRL. Glycosylation of G-ePRL at Asn31 was confirmed by isolation and sequence analysis of an enzymatically derived G-ePRL glycopeptide spanning residues 29–37. Monosaccharide compositions of intact G-ePRL and this glycopeptide were very similar (Man3, GlcNAc2, GalNAc1, Fuc0.6, Gal0.2, NeuAc0.15) and resembled that of G-pPRL. The glycopeptide contained one sulfate residue as determined by ion chromatography after acid hydrolysis, indicating the presence of a sulfated monosaccharide. Comparative carbohydrate analysis of G-ePRL and other G-PRL preparations suggests that the functionally significant Asn31 carbohydrate unit is a fucosylated complex mono- and/or biantennary oligosaccharide terminating with a sulfated GalNAc residue and two or three Man residues.