The maximum amount of cAMP produced at high gonadotropin concentrations is less in the expressed forms than in urinary hCG, and yoked hCG appears more potent than wild-type hCG. The differences between expressed wild-type hCG and urinary hCG are attributed primarily to differences in the carbohydrate moieties. When expressed in mammalian (CHO) cells, the full-length hCG^-a yoked (or tethered) hormone is several-fold less potent than wild-type hCG in binding and several-fold more potent in cAMP stimulation when assayed in HEK 293 cells expressing LHR. Of considerable interest was the observation that the singlechain, full-length mammalian cell-expressed hCG was more potent than wild-type hCG in an in vivo assay.
These findings document unequivocally that recombinant biologically active single-chain forms of hCG can be prepared. Moreover, full-length hCG(3 is not required, nor is full-length a. As recently shown, yoked hCGs are proving useful in addressing structure-function studies on the gonadotropin that are difficult or impossible to achieve with separate subunits. buy asthma inhaler
The study using protein engineering to introduce unnatural intersubunit disulfides represents a clever approach to achieve increased thermal stability without interfering with the chain termini as required in the single-chain constructs. The a-Gln5-»Cys/|3-Arg8—»Cys derivative is interesting in that it exhibits somewhat reduced affinity for LHR compared to wild-type hCG, yet it is as potent as wild-type hCG in stimulating cAMP production. Analogous to the mammalian cell-expressed full-length single-chain hCG, this disulfide-linked form of еф appears to bind to LHR with reduced affinity relative to wild-type hCG; but once complex formation has occurred, the altered gonadotropin is more efficacious in promoting signaling.