Wilken Jason A, Bedows Elliott
Department of Biochemistry and Molecular Biology and Eppley Cancer Center, University of Nebraska Medical Center, Omaha, Nebraska 68198, USA.
Biochemistry. 2007 Apr 10;46(14):4417-24. doi: 10.1021/bi602449d. Epub 2007 Mar 15.
On the basis of apparent molecular mass heterogeneity following reducing versus nonreducing SDS-PAGE, we determined that the beta-subunit of macaque (Macaca fascicularis) chorionic gonadotropin (mCG-beta) is more conformationally constrained than the beta-subunit of human chorionic gonadotropin (hCG-beta). The amino acid sequences of these two subunits are 81% identical. To determine the conformational variance source, which was not due to glycosylation differences, we generated a series of hCG-beta-mCG-beta chimeras and identified domains that contributed to CG-beta conformational freedom. We discovered that the CG-beta 54-101 domain contained a small subdomain, residues 74-77, that regulated the conformational freedom of the beta-subunit; i.e., when residues 74-77 were of macaque origin (PGVD), the mutated hCG-beta subunit displayed macaque-like conformational rigidity, and when residues 74-77 were of human origin (RGVN), the mutated mCG-beta subunit displayed human-like conformational freedom and microheterogeneity. Additionally, CG-beta N-terminal domain residues (8, 18, 42, and 46-48) were also found to influence CG-beta conformational freedom when residues 74-77 were of human but not macaque origin. The biological significance of the CG-beta conformational variance was tested using a biological assay that showed that the hCG-alpha-hCG-beta heterodimer facilitated human CG receptor-mediated cAMP-driven luciferase reporter gene activity in HEK cells nearly 1 order of magnitude more effectively than the hCG-alpha-mCG-beta chimera. Together, these data demonstrate that two essential amino acid residues within a four-amino acid subdomain regulated CG-beta conformational freedom and that a conformational difference between hCG-beta and mCG-beta was recapitulated in the context of receptor-mediated CG heterodimer signal transduction activation.
基于还原型与非还原型SDS-PAGE后明显的分子质量异质性，我们确定猕猴（食蟹猴）绒毛膜促性腺激素（mCG-β）的β亚基比人绒毛膜促性腺激素（hCG-β）的β亚基具有更多构象限制。这两个亚基的氨基酸序列有81%的同一性。为了确定构象差异的来源（并非由于糖基化差异），我们构建了一系列hCG-β-mCG-β嵌合体，并鉴定了有助于CG-β构象自由度的结构域。我们发现CG-β 54-101结构域包含一个小亚结构域，即74-77位残基，它调节β亚基的构象自由度；也就是说，当74-77位残基来自猕猴（PGVD）时，突变的hCG-β亚基表现出猕猴样的构象刚性，而当74-77位残基来自人类（RGVN）时，突变的mCG-β亚基表现出人类样的构象自由度和微异质性。此外，当74-77位残基来自人类而非猕猴时，还发现CG-β N端结构域残基（8、18、42以及46-48）也会影响CG-β的构象自由度。使用生物测定法测试了CG-β构象差异的生物学意义，结果表明hCG-α-hCG-β异二聚体在HEK细胞中促进人CG受体介导的cAMP驱动的荧光素酶报告基因活性的效率比hCG-α-mCG-β嵌合体高近1个数量级。总之，这些数据表明，一个四氨基酸亚结构域内的两个必需氨基酸残基调节了CG-β的构象自由度，并且在受体介导的CG异二聚体信号转导激活的背景下，hCG-β和mCG-β之间的构象差异得以重现。
