Misfolded human chorionic gonadotropin beta subunits are secreted from transfected Chinese hamster ovary cells.
There are six intramolecular disulfide (S-S) bonds that form during intracellular folding of the human chorionic gonadotropin (hCG)-beta subunit. Site-directed mutagenesis of every pair of Cys residues involved in the formation of each S-S bond was used to examine the roles that S-S bonds play in beta subunit folding and secretion. Tryptic maps of secreted hCG-beta showed that only one S-S bond formed in all S-S bond mutants that failed to fold from the earliest detectable beta folding intermediate, p beta 1, into a second major intermediate, p beta 2 (C34A-C88A, C38A-C57A or C9A-C90A mutants), whereas all 5 remaining S-S bonds formed in mutants when p beta 1-->p beta 2 conversion occurred (C23A-C72A, C93A-C100A, or C26A-C110A mutants). Nonreducing SDS-polyacrylamide gel electrophoresis showed that beta multimers were secreted from cells expressing S-S bond mutations where the folding of p beta 1-->p beta 2 was blocked. However, for mutations where p beta 1-->p beta 2 conversion was efficient, beta monomers rather than multimers were secreted. For all cell lines studied, secreted hCG-beta migrated as monomeric beta during reducing SDS-polyacrylamide gel electrophoresis, indicating that hCG-beta multimers formed via intermolecular cross-linking of unpaired thiols. Tryptic maps of hCG-beta isolated from mutants lacking the 34-88 bond, where > 80% turnover occurs, showed that only the 38-57 S-S bond formed. beta Subunits lacking the 9-90 linkage also have only S-S bond 38-57 formed, but < 10% turnover of C9A-C90A hCG-beta occurs. Thus, subtle conformational differences between partially folded or misfolded beta subunits may determine whether hCG-beta is degraded, or undergoes intracellular translocation and secretion.
Bedows
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Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha 68198.
Norton
S E
SE
Huth
J R
JR
Suganuma
N
N
Boime
I
I
Ruddon
R W
RW
D000818:Animals; D016466:CHO Cells; D006063:Chorionic Gonadotropin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D006224:Cricetinae; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D016297:Mutagenesis, Site-Directed; D010449:Peptide Mapping; D017510:Protein Folding; D014162:Transfection; D014357:Trypsin
D006063:Chorionic Gonadotropin; D014357:Trypsin