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Dissociation of Early Folding Events from Assembly of the Human Lutropin ß-Subunit

Departments of Molecular Biology and Pharmacology and Obstetrics and Gynecology (M.M., I.B.) Washington University School of Medicine St Louis, Missouri 63110

The human LH of the anterior pituitary is a member of the glycoprotein hormone family that includes FSH, TSH, and placental CG. All are noncovalently bound heterodimers that share a common -subunit and ß-subunits that confer biological specificity. LHß and CGß share more than 80% amino acid sequence identity; however, in transfected Chinese hamster ovary (CHO) cells, LHß assembles with the -subunit more slowly than does hCGß, and only a fraction of the LHß synthesized is secreted, whereas CGß is secreted efficiently. To understand why the assembly and secretion of these related ß-subunits differ, we studied the folding of LHß in CHO cells transfected with either the LHß gene alone, or in cells cotransfected with the gene expressing the common -subunit, and compared our findings to those previously seen for CG. We found that the rate of conversion of the earliest detectable folding intermediate of LH, pß1, to the second major folding form, pß2, did not differ significantly from the pß1-to-pß2 conversion of CGß, suggesting that variations between the intracellular fates of the two ß-subunits cannot be explained by differences in the rates of their early folding steps. Rather, we discovered that unlike CGß, where the folding to pß2 results in an assembly-competent product, apparently greater than 90% of the LH pß2 recovered from LHß-transfected CHO cells was assembly incompetent, accounting for inefficient LHß assembly with the -subunit. Using the formation of disulfide (S-S) bonds as an index, we observed that, in contrast to CGß, all 12 LHß cysteine residues formed S-S linkages as soon as pß2 was detected. Attempts to facilitate LH assembly with protein disulfide isomerase in vitro using LH pß2 and excess urinary -subunit as substrate were unsuccessful, although protein disulfide isomerase did facilitate CG assembly in this assay. Moreover, unlike CGß, LHß homodimers were recovered from transfected CHO cells. Taken together, these data suggest that differences seen in the rate and extent of LH assembly and secretion, as compared to those of CG, reflect conformational differences between the folding intermediates of the respective ß-subunits.

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