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Originally published In Press as doi:10.1074/jbc.M010168200 on December 29, 2000

J. Biol. Chem., Vol. 276, Issue 14, 10692-10699, April 6, 2001
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Functional Contributions of Noncysteine Residues within the Cystine Knots of Human Chorionic Gonadotropin Subunits*

Ryan J. DarlingDagger §||||, Jason A. WilkenDagger , Amanda K. Miller-LindholmDagger ||, Teresa M. UrlacherDagger **, Raymond W. RuddonDagger §Dagger Dagger , Simon A. ShermanDagger , and Elliott BedowsDagger §§§¶¶

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

Human chorionic gonadotropin (hCG) is a heterodimeric member of a family of cystine knot-containing proteins that contain the consensus sequences Cys-X1-Gly-X2-Cys and Cys-X3-Cys. Previously, we characterized the contributions that cystine residues of the hCG subunit cystine knots make in folding, assembly, and bioactivity. Here, we determined the contributions that noncysteine residues make in hCG folding, secretion, and assembly. When the X1, X2, and X3 residues of hCG-alpha and -beta were substituted by swapping their respective cystine knot motifs, the resulting chimeras appeared to fold correctly and were efficiently secreted. However, assembly of the chimeras with their wild type partner was almost completely abrogated. No single amino acid substitution completely accounted for the assembly inhibition, although the X2 residue made the greatest individual contribution. Analysis by tryptic mapping, high performance liquid chromatography, and SDS-polyacrylamide gel electrophoresis revealed that substitution of the central Gly in the Cys-X1-Gly-X2-Cys sequence of either the alpha - or beta -subunit cystine knot resulted in non-native disulfide bond formation and subunit misfolding. This occurred even when the most conservative change possible (Gly right-arrow Ala) was made. From these studies we conclude that all three "X" residues within the hCG cystine knots are collectively, but not individually, required for the formation of assembly-competent hCG subunits and that the invariant Gly residue is required for efficient cystine knot formation and subunit folding.

* This work was supported in part by National Institutes of Health Grant CA32949 (to E. B.), NCI, National Institutes of Health Cancer Center Support Grant P 30 CA36727 to the Eppley Institute, a National Science Foundation Graduate Fellowship (to R. J. D.), and an Emley Fellowship (to A. K. M.-L.).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.

|| Current address: Transgenomic Inc., 12325 Emmet St., Omaha, NE 68164.

** Current address: LI-COR Inc., 4308 Progressive Avenue, Lincoln, NE 68504.

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

|||| Current address: Eli Lilly and Co., Lilly Corporate Center, Indianapolis, IN 46295.

¶¶ To whom correspondence should be addressed: Eppley Institute 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 © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
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