Bedows E, Huth J R, Suganuma N, Bartels C F, Boime I, Ruddon R W
Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha 68198.
J Biol Chem. 1993 Jun 5;268(16):11655-62.
Previous kinetic studies have characterized the intracellular folding pathway of the human chorionic gonadotropin (hCG)-beta subunit in which each of the folding intermediates can be biochemically identified based on the formation of disulfide (S-S) bonds: p beta 1-early--> p beta 1-late--> p beta 2-free--> p beta 2-combined--> native hCG-alpha beta. Based on these data, we postulated that hCG-beta folding coincides with the formation of specific S-S bonds. We have now tested this hypothesis employing Chinese hamster ovary cells transfected with mutated hCG-beta genes in which the Cys residues required for the formation of the final four (of six total) S-S bonds were replaced by Ala. When the Cys residues required for the third hCG-beta S-S linkage to form (bond 9-90) were substituted, folding did not proceed beyond the earliest detectable folding intermediate (p beta 1-early). In the absence of the subsequently formed S-S bond (bond 23-72), p beta 1-early was converted into a second folding intermediate (p beta 1-late), but conversion to the next intermediate (p beta 2-free) was inhibited. When either of the final two S-S bonds (the carboxyl-terminal 93-100 or 26-110 bonds) were removed, conversion of p beta 1-late to p beta 2-free was detected, but conversion of p beta 2-free to the last folding intermediate (p beta 2-combined) was not observed. These data support the hypothesis that individual S-S bonds are involved in discrete steps in the hCG-beta folding pathway.
以往的动力学研究已对人绒毛膜促性腺激素（hCG）β亚基的细胞内折叠途径进行了表征，其中每个折叠中间体都可根据二硫键（S-S）的形成进行生化鉴定：pβ1-早期→pβ1-晚期→pβ2-游离态→pβ2-结合态→天然hCG-αβ。基于这些数据，我们推测hCG-β的折叠与特定S-S键的形成相吻合。我们现在通过用突变的hCG-β基因转染的中国仓鼠卵巢细胞来检验这一假设，其中形成最后四个（总共六个）S-S键所需的半胱氨酸（Cys）残基被丙氨酸（Ala）取代。当形成第三个hCG-β S-S键（键9-90）所需的Cys残基被取代时，折叠过程无法超越最早可检测到的折叠中间体（pβ1-早期）。在没有随后形成的S-S键（键23-72）的情况下，pβ1-早期转化为第二个折叠中间体（pβ1-晚期），但向下一个中间体（pβ2-游离态）的转化受到抑制。当去除最后两个S-S键中的任何一个（羧基末端的93-100键或26-110键）时，检测到pβ1-晚期向pβ2-游离态的转化，但未观察到pβ2-游离态向最后一个折叠中间体（pβ2-结合态）的转化。这些数据支持了这样的假设，即单个S-S键参与了hCG-β折叠途径中的离散步骤。
