Disulfide bond mutations affect the folding of the human chorionic gonadotropin-beta subunit in transfected Chinese hamster ovary cells.
Previous kinetic studies have characterized the intracellular folding pathway of the human chorionic gonadotropin (hCG)-beta subunit in which each of the folding intermediates can be biochemically identified based on the formation of disulfide (S-S) bonds: p beta 1-early--> p beta 1-late--> p beta 2-free--> p beta 2-combined--> native hCG-alpha beta. Based on these data, we postulated that hCG-beta folding coincides with the formation of specific S-S bonds. We have now tested this hypothesis employing Chinese hamster ovary cells transfected with mutated hCG-beta genes in which the Cys residues required for the formation of the final four (of six total) S-S bonds were replaced by Ala. When the Cys residues required for the third hCG-beta S-S linkage to form (bond 9-90) were substituted, folding did not proceed beyond the earliest detectable folding intermediate (p beta 1-early). In the absence of the subsequently formed S-S bond (bond 23-72), p beta 1-early was converted into a second folding intermediate (p beta 1-late), but conversion to the next intermediate (p beta 2-free) was inhibited. When either of the final two S-S bonds (the carboxyl-terminal 93-100 or 26-110 bonds) were removed, conversion of p beta 1-late to p beta 2-free was detected, but conversion of p beta 2-free to the last folding intermediate (p beta 2-combined) was not observed. These data support the hypothesis that individual S-S bonds are involved in discrete steps in the hCG-beta folding pathway.
Bedows
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Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha 68198.
Huth
J R
JR
Suganuma
N
N
Bartels
C F
CF
Boime
I
I
Ruddon
R W
RW
D000409:Alanine; D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D006063:Chorionic Gonadotropin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018997:Chorionic Gonadotropin, beta Subunit, Human; D006224:Cricetinae; D003545:Cysteine / Q000378:metabolism*; D004220:Disulfides / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008961:Models, Structural; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011487:Protein Conformation; D017510:Protein Folding; D014162:Transfection
D006063:Chorionic Gonadotropin; D018997:Chorionic Gonadotropin, beta Subunit, Human; D004220:Disulfides; D010446:Peptide Fragments; D003545:Cysteine; D000409:Alanine