The intracellular and in vitro folding and assembly pathway of human chorionic gonadotropin.

Abstract

We have investigated the intracellular and in vitro folding pathways of the $\beta$ subunit of human chorionic gonadotropin (hCG), a heterodimeric glycoprotein hormone, and the mechanisms of assembly of hCG-$\beta$ with hCG-$\alpha$ to address the following questions: (1) What is the rate-limiting event in the folding pathway of hCG? (2) Can the folding pathway of hCG-$\beta$ be explained by a simple sequential model? (3) Is additional information for this folding and assembly pathway provided by cellular folding factors as well as by the amino acid sequences of the hCG subunits? Five folding intermediates were purified from metabolically labeled choriocarcinoma cells that had been lysed in the presence of iodoacetate. The identification of the alkylated ($\sp{35}$S) cysteines in each hCG-$\beta$ folding intermediate was used to determine the intracellular order of disulfide bond formation, an indication of the conformational changes during protein folding. Only native disulfide bonds seemed to be significantly populated. Four disulfide bonds formed sequentially and posttranslationally: 34-88 (t$\sb{1/2}$ = 1-2 min), 38-57 (t$\sb{1/2}$ = 2-3 min), and 9-90 and 23-72 (t$\sb{1/2}$ = 4 min). Upon completion of formation of the bonds 9-90 and 23-72, the rate-limiting folding events, hCG-$\beta$ adopted an assembly-competent conformation. The disulfide bonds 93-100 and 26-110 formed at the same time as the other four S-S bonds, indicating that hCG-$\beta$ does not fold via a simple sequential pathway. Subunit assembly occurred before complete formation of the 26-110 disulfide bond. The same pathway was observed in vitro, although the t$\sb{1/2}$ of the rate-limiting event was $\ge$80 min compared to 4 min in a cell. Protein disulfide isomerase (PDI) increased the in vitro rate of this event (t$\sb{1/2}$ = 25 min) without changing the order of disulfide bond formation. PDI also catalyzed the in vitro rate of hCG subunit assembly. The mechanism of this catalysis involved reduction of the 26-110 disulfide bond of hCG-$\beta$, indicating that, both in intact cells and in vitro, partially unfolded hCG-$\beta$ is more assembly-competent than is fully folded hCG-$\beta$. Our results indicate that cellular factor(s) increase the rate of folding and assembly of hCG but do not change the steps in the pathway.