Expression of a novel follicle stimulating hormone glycoform

Abstract

Heterodimeric pituitary gonadotropins, luteinizing hormone (LH) and follicle-stimulating hormone (FSH), play significant roles in follicular development and maintenance of the female reproductive cycle. FSH functions in the ovary include: stimulation of follicle growth, estradiol synthesis and selection of dominant follicles. FSH exists as two glycoforms, designated di- and tetra-glycosylated FSH. Di-glycosylated FSH contains 2 carbohydrates located only on the α subunit, while tetra-glycosylated FSH has 4 carbohydrates, 2 each on the α and β subunits. Individual pituitary FSH analysis shows that in young, reproductive-age women, di-glycosylated FSH is more abundant than tetra-glycosylated FSH, whereas in postmenopausal women there is more pituitary tetra-glycosylated FSH. In vitro bioassay of di-glycosylated FSH shows that it has significantly greater biological activity than tetra-glycosylated FSH. As the availability of diglycosylated human FSH from natural or mammalian-generated recombinant sources is limited, bacterial expression of recombinant human FSHβ (rec hFSHβ) was explored as an avenue to provide sufficient di-glycosylated hFSH for structural and biological function studies. We chose the E.coli Origami expression system to make non-glycosylated FSHβ because it lacks the ability to N-glycosylate the wild-type protein sequence. The wild-type hFSHβ sequence is desired because mutations to eliminate N-glycosylation are known to reduce expression efficiency. Moreover, mutations to silence the Asn24 glycosylation site, appear to affect protein folding and stability. Bacterially expressed hFSHβ was extracted and separated into soluble and insoluble fractions. We obtained a relatively pure (>90%) hFSHβ preparation derived from the insoluble fraction, which had to be refolded. We also recovered a small quantity of apparently partially folded hFSHβ in the soluble fraction after Affi-Gel Blue purification. The attempts to fold denatured insoluble fraction hFSHβ were unsuccessful, as we could not get the folding reaction product to associate with α subunit and constitute a functional hormone. Furthermore, the soluble fraction, assumed to be folded, because it bound to Affi-Gel Blue via dye intercalation between cystine knot loops 1 and 3, also failed to associate with α subunit. Overall, we could not use a bacterial expression system to make a functional hFSHβ to combine with the α subunit to make a functional intact hormone. Moreover, the electrophoretic mobility of the 12.5 kDa bacterially expressed hFSHβ reflected the formula weight of the primary sequence more closely than that of the 21 kDa non-glycosylated pituitary hFSHβ, suggesting unknown posttranslational modification of the 21 kDa form. However, upon further investigation, the 12.5 kDa band appeared to be a fragment, rather than a full-length subunit. This result rationalizes the failure to fold, as fragments do not fold.