The production of TNFα by human endometrial cells in culture

Abstract

The wild-type (WT) GH2-N ovine growth hormone (oGH) and duplicated GH2-Z genes differ in their open reading frame by two nonsynonymous substitutions, predicting a two-amino-acid difference in their product (G9R/G63S). Three recombinant oGH muteins: G9R, G63S and G9R/G63S, were prepared by site-directed mutagenesis of the WT oGH gene, expressed in E. coli, refolded and purified as monomers with over 98% homogeneity. Gel-filtration experiments with WT oGH and the three muteins indicated formation of 1:2 complexes with oGH receptor extracellular domain (oGHR-ECD). Interactions of oGHR-ECD with the WT and the muteins were studied by surface plasmon resonance. Kinetics constants calculated using a two-site model predicted that G9R/G63S has the highest affinity to oGHR-ECD, WT oGH the lowest, and G9R and G63S have intermediate affinities. These relative affinities were further investigated by radioreceptor assay with EC50 values were the lowest for G9R/G63S, highest for WT oGH, and intermediate for G9R and G63S. Bioactivity of the WT oGH and oGH muteins was determined by proliferation assay with FDC-P1-3B9 cells stably transfected with rabbit GHR. Relative proliferation rates of cells in cultures treated with the WT, G63S, G9R or G9R/G63S variants were 100%, 183%, 259% and 498%, respectively. In COS-7 transfected with oGHR, LHRE-TK-luciferase and β-galactosidase plasmids G9R/G63S showed 18% higher activity than WT oGH (P < 0.001). Thus the product of the oGH duplicated copy has higher affinity for GHR and higher somatogenic activity. As the GH2-Z gene copy is expressed in the placenta, allelic differences at the oGH locus may influence feto-placental development.