JBC Advanced Glycation Endproducts

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J Biol Chem, Vol. 275, Issue 20, 15413-15421, May 19, 2000

Cystine Knot Mutations Affect the Folding of the Glycoprotein Hormone alpha -Subunit
DIFFERENTIAL SECRETION AND ASSEMBLY OF PARTIALLY FOLDED INTERMEDIATES*

Ryan J. DarlingDagger §, Raymond W. RuddonDagger §||, Fulvio PeriniDagger §, and Elliott BedowsDagger §||**Dagger Dagger

From the Dagger  Eppley Institute for Research in Cancer and Allied Diseases and the Departments of § Pharmacology, || Biochemistry and Molecular Biology, and ** Obstetrics and Gynecology, University of Nebraska Medical Center, Omaha, Nebraska 68198

The common glycoprotein hormone alpha -subunit (GPH-alpha ) contains five intramolecular disulfide bonds, three of which form a cystine knot motif (10-60, 28-82, and 32-84). By converting each pair of cysteine residues of a given disulfide bond to alanine, we have studied the role of individual disulfide bonds in GPH-alpha folding and have related folding ability to secretion and assembly with the human chorionic gonadotropin beta -subunit (hCG-beta ). Mutation of non-cystine knot disulfide bond 7-31, bond 59-87, or both (leaving only the cystine knot) resulted in an efficiently secreted folding form that was indistinguishable from wild type. Conversely, the cystine knot mutants were inefficiently secreted (<25%). Furthermore, mutation of the cystine knot disulfide bonds resulted in multiple folding intermediates containing 1, 2, or 4 disulfide bonds. High performance liquid chromatographic separation of intracellular and secreted forms of the folding intermediates demonstrated that the most folded forms were preferentially secreted and combined with hCG-beta . From these studies we conclude that: (i) the cystine knot of GPH-alpha is necessary and sufficient for folding and (ii) there is a direct correlation between the extent of GPH-alpha folding, its ability to be secreted, and its ability to heterodimerize with hCG-beta .

* This work is based upon work supported in part by a National Science Foundation Graduate Fellowship (to R. J. D.), by National Institutes of Health Grant CA32949 (to E. B.), and by National Cancer Institute Cancer Center Support Grant P 30 CA36727 to the Eppley Institute.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Present address: Corporate Office of Science and Technology, Johnson & Johnson, 410 George St., New Brunswick, NJ, 08901.

Dagger Dagger To whom correspondence should be addressed: Eppley Inst. for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, 986805 Nebraska Medical Center, Omaha, NE 68198-6805. Tel.: 402-559-6074; Fax: 402-559-4651; E-mail: ebedows@unmc.edu.

Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
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