Functional expression of yoked human chorionic gonadotropin in baculovirus-infected insect cells.
hCG is a glycoprotein hormone composed of an alpha-subunit, common to all gonadotropins and to TSH, and a hormone-specific beta-subunit. The non-covalent association of the two subunits is an obligatory step for the formation of biologically active hormones. The correct assembly of the heterodimer is also important for efficient secretion of the hormone, receptor binding, and signal transduction. Herein, we have demonstrated that expression of the two subunits from independent promoters present in a single recombinant baculovirus resulted in subunit association and secretion of biologically active holoprotein by the insect cells. To determine whether the active conformation of heterodimer could be achieved when the two subunits were synthesized in tandem on a single polypeptide chain, two single chain or yoked hCG1, the C-terminus of the complete beta-subunit (145 amino acid residues) was conjoined to the N-terminus of the alpha-subunit. Yoked hCG2 was similar, except that it contained the N-terminal 123 amino acid residues of the beta-subunit. Both yoked hCG molecules bound LH/CG receptor with high affinity and stimulated adenylate cyclase and progesterone levels in transformed mouse Leydig (MA-10) cells. Therefore, the alpha- and beta-subunits are able to fold into a biologically active conformation when covalently linked. Interestingly, when compared with urinary hCG, the hormone expressed in baculovirus-infected insect cells binds to the LH/CG receptor with higher affinity, but exhibits diminished signaling, thus providing another example of a partial dissociation between receptor binding and activation.
Narayan
P
P
Department of Biochemistry and Molecular Biology, University of Georgia, Athens 30602, USA.
Wu
C
C
Puett
D
D
D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D001483:Base Sequence; D001667:Binding, Competitive; D006063:Chorionic Gonadotropin / Q000737:chemistry* / Q000235:genetics*; D015870:Gene Expression; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D006801:Humans; D046911:Macromolecular Substances; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011487:Protein Conformation; D011994:Recombinant Proteins; D018411:Spodoptera / Q000378:metabolism*
D006063:Chorionic Gonadotropin; D046911:Macromolecular Substances; D011994:Recombinant Proteins