Reicher S, Niv-Spector L, Gertler A, Gootwine E
Institute of Biochemistry, Food Science and Nutrition, Faculty of Agricultural, Food and Environmental Quality Sciences, The Hebrew University of Jerusalem, P.O. Box 12, Rehovot 76100, Israel.
Gen Comp Endocrinol. 2008 Jan 15;155(2):368-77. doi: 10.1016/j.ygcen.2007.07.006. Epub 2007 Aug 6.
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 beta-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.
野生型（WT）GH2-N绵羊生长激素（oGH）和重复的GH2-Z基因在其开放阅读框中有两个非同义替换，预测其产物存在两个氨基酸差异（G9R/G63S）。通过对WT oGH基因进行定点诱变制备了三种重组oGH突变体：G9R、G63S和G9R/G63S，在大肠杆菌中表达，重折叠并纯化为均一性超过98%的单体。对WT oGH和这三种突变体进行的凝胶过滤实验表明，它们与生长激素受体胞外域（oGHR-ECD）形成了1:2复合物。通过表面等离子体共振研究了oGHR-ECD与WT和突变体之间的相互作用。使用双位点模型计算的动力学常数预测，G9R/G63S对oGHR-ECD的亲和力最高，WT oGH最低，G9R和G63S具有中等亲和力。通过放射受体测定进一步研究了这些相对亲和力，G9R/G63S的EC50值最低，WT oGH最高，G9R和G63S为中等。通过用稳定转染兔生长激素受体的FDC-P1-3B9细胞进行增殖测定，确定了WT oGH和oGH突变体的生物活性。用WT、G63S、G9R或G9R/G63S变体处理的培养物中细胞的相对增殖率分别为100%、183%、259%和498%。在转染了oGHR、LHRE-TK-荧光素酶和β-半乳糖苷酶质粒的COS-7细胞中，G9R/G63S的活性比WT oGH高18%（P<0.001）。因此，oGH重复拷贝的产物对生长激素受体具有更高的亲和力和更高的促生长活性。由于GH2-Z基因拷贝在胎盘中表达，oGH位点的等位基因差异可能影响胎儿-胎盘发育。
