ISSN:
0730-2312
Keywords:
protein folding
;
carbohydrate deglycosylation
;
Life and Medical Sciences
;
Cell & Developmental Biology
Source:
Wiley InterScience Backfile Collection 1832-2000
Topics:
Biology
,
Chemistry and Pharmacology
,
Medicine
Notes:
The folding of the bovine glycoprotein hormone α 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 α polypeptide (bac-α), 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-α preparation combines with the β subunit of human chorionic gonadotropin (hCGβ) to form a complex that binds to the gonadotropin receptor and elicits a biological response. Since the correct folding (by immunological criteria) of bac-α (ca 3%) is significantly greater than expected from a random formation of disulfide bonds (0.1 %), it appears that correct folding of α subunit can occur in the complete absence of carbohydrate, though in very low yield. Native bovine lutropin α subunit (LHα) and chemically deglycosylated LHα (which retains two asparagine-linked N-acetyl glucosamine residues per α oligosaccharide) were subjected to the same reduction/reoxidation regimen as the bacterially produced a subunit. As has been reported previously [Giudice LC, Pierce, JG, J Biol Chem 251: 6392, 1976] intact LHα fully regained its native structure. The partially deglycosylated LHα also refolds to a native-like structure in high yield as assessed by immunological assays and by its ability to combine with HGCβ to form a biologically active complex. The data show that carbohydrate, while not obligatory for correct folding, greatly facilitates the formation of functional α subunit.
Additional Material:
5 Ill.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1002/jcb.240290307
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