Analysis of hCG and LH active site by using molecular biological method

人绒毛膜促性腺激素(HCG)、促黄体生成素(LH)、促甲状腺激素(FSH)和卵泡刺激素(FSH)是由两个非共价连接的亚基组成，即α和β亚基。β亚基对每种激素都是特定的，并负责生物特异性，但不同激素的β亚基在结构上显示出一定程度的同源性。CAGY(半胱氨酸-丙氨酸-甘氨酸-酪氨酸)区是不同β亚基中同源的氨基酸序列之一，在物种间高度保守。本研究采用体外定点突变的方法改变hCG-β亚基cagy区中心的三个核苷酸，并通过体外翻译的方法研究这些突变对非洲爪哇卵母细胞hCG产量的影响。制备了3个突变的cDNA：MI(Ala-&Gt；Ser)、M2(Gly-&Gt；Arg)和M3(Gly-&Gt；Asp)。用Nor-…混合物处理卵母细胞更多的正常α和突变βmRNAs(M2和M3)产生与阴性对照一样少的hCG，而非NALαmRNA和m1突变mRNA处理的卵母细胞产生与阳性对照相同数量的hCG。这些结果表明，CAGY区域，特别是β亚基36位的甘氨酸残基是产生免疫活性FICG所必需的。这一发现与之前的研究一致，该区域对人类TSH的生物学活动是必要的。阿尔法亚单位与所有四种激素都是共同的。为了研究cagy区域在α和β亚基相互作用中的作用，我们进一步分析了hCG分子的立体凝血作用。通过对这些激素的三维分析，推测了它们的重要区域：^&lt；28&gt；Cys-^&lt；29&gt；Met-^&lt；30&gt；Gly-^&lt；31&gt；Cys-^&lt；32&gt；Phe-^&lt；34&gt；Ser-^&lt；35&gt；Arg-^&lt；36&gt；Ala-^&lt；37&gt；Tyr。因此，我们利用聚合酶链式反应的方法在天然αCDNA中引入了几个点突变。结果产生了6个α亚基突变体：MI(^&lt；28&gt；Cys-&gt；Tyr)、M2(^&lt；29&gt；Met-&gt；Arg)、M3(^&lt；30；Gly-gt；Arg)、M4(^&lt；30&gt；GIY-&gt；Ala)、M5(^&lt；30&gt；Gly-&gt；Asp)和M6(^&lt；37&gt；Tyr-&Gt；Asp)。在体外转录突变或天然α亚基CDNA的信使RNA，并将其与天然β亚基的mRNA共同注射到卵母细胞中。产生的突变hCG分子的特征目前正在进行中。较少

The heterodimeric glycoprotein hormones, human chorionic gonadotropin(hCG), lutenizing hormone(LH), thyroid stimulating hormone(FSH)and follicles stimulating hormone(FSH), consist of two non-covalently linked subunits, the alpha and beta subunits. The beta subunit is specific for each hormone and is responsible for the biological specificity, but the beta subunits of different hormones show some degree of structural homology. The CAGY(cysteine-alanine-glycine-tyrosine)region is one of the amino acid sequences that is homologous in different beta subunits and is highly conserved between species. In the present study, site-specific in-vitro mutagenesis was used to change three individual nucleotides in the center of the CAGY region of the hCG-beta subunit, and the effect of these mutations on hCG production was determined by in-vitro translation in Xenopus laevis oocytes. Three mutated cDNAs, MI(Ala->Ser), M2(Gly->Arg), and M3(Gly->Asp)were prepared. Oocytes treated with a mixture of nor … More mal alpha and mutant beta mRNAs(M2 and M3)produced as little hCG as negative control whereas oocytes treated with non-nal alpha MRNA and Ml mutant MRNA produced the same amount of hCG as the positive control. These results indicate that the CAGY region, particularly the glycine residue at position 36 in the beta subunit, is essential for the production of immunoreactive fiCG. This finding is consistent with previous studies showing that this region is necessary for the biological activity of human TSH.The alpha subunit is shared commonly with the all four hormones. To investigate the role of CAGY region on the interaction between alpha and beta subunits, we further analyzed the stereo-coaguration of hCG molecule. By the three dimensional analyses of these hormones, the important regions Of ^<28>Cys- ^<29>Met- ^<30>Gly- ^<31>Cys- ^<32>Cys- ^<33>phe- ^<34>Ser- ^<35>Arg- ^<36>Ala- ^<37>Tyr were presumed. We therefore introduced several point mutations into native alpha CDNA by using polymerase chain reaction method. As a result, 6 mutants of alpha subunit, MI( ^<28>Cys->Tyr), M2( ^<29>Met->Arg), M3(^<30>Gly->Arg), M4( ^<30>GIy->Ala), M5( ^<30>Gly->Asp), and M6( ^<37>Tyr->Asp)were generated. Messenger RNAs of mutated or native alpha subunit CDNA were transcribed in vitro and were co-injected into oocytes with MRNA of native beta subunit. The characterizations of the produced mutated hCG molecules were currently underway. Less