Mutations of Arg68 of the human chorionic gonadotrophin beta subunit lead to reduced secretion.
The Arg68-Leu69 sequence is invariant in the beta subunits of chorionic gonadotrophin and luteinizing hormone from a variety of species. Using site-directed mutagenesis of the human chorionic gonadotrophin (hCG)-beta cDNA, several replacements of Arg68, an Ala replacement of Leu69, and a multiple replacement with Ala-Ala-Ala-Ala of the tetrapeptide sequence, Arg68-Leu69-Pro70-Gly71, were prepared and characterized. The wild-type and mutant cDNAs were subcloned into a pRSV expression vector and transiently transfected into CHO cells containing a stably integrated gene for bovine a. Concentrations of secreted wild-type and mutant hCG-beta subunit and holoprotein were determined using radioimmunoassays; potencies, i.e. the ratio of biologic to immunologic activity, of several of the mutant heterodimers were measured in vitro via gonadotrophin-mediated steroidogenesis in transformed murine Leydig cells (MA-10). The Leu69-->Ala mutant formed a mutant holoprotein that was essentially equipotent with wild-type hormone in the steroidogenesis assay. The Arg68 replacements with Lys, Ala, and Leu were poorly secreted by the cells, e.g. < 10% that of wild-type hCG; however, sufficient quantities of mutant holoproteins containing Lys68 and Ala68 were obtained for biological assays, and both exhibited greater apparent potencies than wild-type hormone. Likewise, a mutant holoprotein containing the Arg68-Leu69-Pro70-Gly71-->Ala-Ala-Ala-Ala multiple replacement was apparently more potent than wild-type hormone, but it too was secreted at lower levels than wild-type. These results establish that replacements of Arg68 in hCG-beta diminish secretion, but the small amount of holoprotein that is formed and secreted appears to be of somewhat greater potency than wild-type hormone.
Shen
L
L
Dept. of Biochemistry & Molecular Biology, University of Georgia, Athens 30602, USA.
Xia
H
H
Bhowmick
N
N
Narayan
P
P
Puett
D
D
D000818:Animals; D001120:Arginine / Q000235:genetics*; D016466:CHO Cells; D018997:Chorionic Gonadotropin, beta Subunit, Human / Q000235:genetics* / Q000378:metabolism; D006224:Cricetinae; D018076:DNA, Complementary; D006801:Humans; D051379:Mice; D016297:Mutagenesis, Site-Directed
D018997:Chorionic Gonadotropin, beta Subunit, Human; D018076:DNA, Complementary; D001120:Arginine