The common α subunit of bovine glycoprotein hormones: Limited formation of native structure by the totally nonglycosylated polypeptide chain

Abstract

The folding of the bovine glycoprotein hormone alpha subunit, synthesized in bacteria following insertion of the nucleotide sequence coding for this polypeptide, has been studied to determine the effect that a complete lack of carbohydrate has on this process. The bacterially derived alpha polypeptide (bac-alpha), extracted from E. coli in the presence of reductant and denaturant, had an estimated 0.2% native structure as determined by a conformationally sensitive radioimmunoassay. Upon reduction of disulfide bonds and reoxidation in air, the amount of native structure increased about 18-fold. Approximately 2% of the refolded bac-alpha preparation combines with the beta subunit of human chorionic gonadotropin (hCG beta) to form a complex that binds to the gonadotropin receptor and elicits a biological response. Since the correct folding (by immunological criteria) of bac-alpha (ca 3%) is significantly greater than expected from a random formation of disulfide bonds (0.1%), it appears that correct folding of alpha subunit can occur in the complete absence of carbohydrate, though in very low yield. Native bovine lutropin alpha subunit (LH alpha) and chemically deglycosylated LH alpha (which retains two asparagine-linked N-acetyl glucosamine residues per alpha oligosaccharide) were subjected to the same reduction/reoxidation regimen as the bacterially produced alpha subunit. As has been reported previously [Giudice LC, Pierce, JG, J Biol Chem 251: 6392, 1976] intact LH alpha fully regained its native structure. The partially deglycosylated LH alpha also refolds to a native-like structure in high yield as assessed by immunological assays and by its ability to combine with HCG beta to form a biologically active complex. The data show that carbohydrate, while not obligatory for correct folding, greatly facilitates the formation of functional alpha subunit.

Characterization of the Equine Glycoprotein Hormone Alpha-Subunit Gene Reveals Divergence in the Mechanism of Pituitary and Placental Expression1

“…However, the lengths of the introns differ somewhat. The first intron is 14 kbp, similar in size to that of the bovine gene (15 kbp) [43], larger than the first intron of the human gene (7 kbp) [42], and smaller than that of the rhesus monkey (17 kbp) [38]. Since the human and monkey ot-subunit genes are expressed in placenta while that of cattle is not, there is no correlation between the size of this intron and placental expression.…”

NMR Studies of the Free α Subunit of Human Chorionic Gonadotropin

“…Furthermore, ahCG expressed in Escherichia coli, which lacks carbohydrates at both Asn sites, docs not fold to its correct conformation (Strickland et al, 1985). In contrast, reduced exoglycosidasetreated IxhCG, which bears at least one GlcNAc residue at each Asn site, is able to refold correctly (Goverman et al, 1982).…”

Expression of biologically active heterodimeric bovine follicle-stimulating hormone in milk of transgenic mice.

“…The a and FSHI subunits are posttranslationally modified, and the nature and extent of such modifications can exert a profound effect on subunit assembly, secretion, and stability (4)(5)(6). Only heterodimers with appropriately glycosylated subunits exhibit significant biological and receptor-binding activity (5,7,8). Targeting FSH to the mammary gland of transgenic animals would, therefore, serve as a model system in which to study glycoprotein processing and secretion as well as a means to produce large quantities of FSH.…”