Functional Contributions of Noncysteine Residues within the Cystine Knots of Human Chorionic Gonadotropin Subunits

Ryan J Darling,Jason A Wilken,Amanda K Miller-Lindholm,Teresa M Urlacher,Raymond W Ruddon,Simon A Sherman,Elliott Bedows

doi:10.1074/jbc.m010168200

Abstract

Human chorionic gonadotropin (hCG) is a heterodimeric member of a family of cystine knot-containing proteins that contain the consensus sequences Cys-X(1)-Gly-X(2)-Cys and Cys-X(3)-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 X(1), X(2), and X(3) 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 X(2) 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-X(1)-Gly-X(2)-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 --> 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.

Highlights

The cystine knot motif defines a superfamily of dimeric proteins and appears to function as a structural scaffold that stabilizes the 3-loop structures of the individual subunits (1)
Swapping of human chorionic gonadotropin (hCG) cystine knot motifs was accomplished by site-directed mutagenesis at the X1, X2, and X3 positions to match the residue(s) of the other subunit
For the 8-residue rings, such as those found in GPH-␣ and hCG-␤, the two sides of the ring contain 5- and 3-residues and are linked by two S-S bridges

Summary

The abbreviations used are

S-S, disulfide; hCG, human chorionic gonadotropin; GPH-␣, glycoprotein hormone ␣-subunit; TSH, thyroidstimulating hormone; DTT, dithiothreitol; NEM, N-ethylmaleimide; PAGE, polyacrylamide gel electrophoresis; WT, wild type; HPLC, high performance liquid chromatography. The residues at the X1, X2, and X3 positions vary among cystine knot-containing proteins and their functional importance is largely unknown. We report that: (i) there is a subunit-specific complement of three “X” residues, all of which are needed for efficient assembly and (ii) the presence of the central invariant Gly is an absolute requirement for efficient folding, cystine knot formation, and hCG assembly

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION