Approach to the direct intramolecular localization of antigenic determinants in Androctonus australis hemocyanin with monoclonal antibodies by molecular immunoelectron microscopy.
Approach to the direct intramolecular localization of antigenic determinants in Androctonus australis hemocyanin with monoclonal antibodies by molecular immunoelectron microscopy.
复制标题
通过分子免疫电子显微镜用单克隆抗体直接分子内定位Androctonus australis血蓝蛋白中的抗原决定簇。
DOI：
10.1021/bi00341a038
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发表时间：
1985
期刊：
影响因子：
2.9
通讯作者：
Motta,G
中科院分区：
文献类型：
--
作者：
Lamy,J;Lamy,J;Billiald,P;Sizaret,PY;Cavé,G;Frank,J;Motta,G
采用分子免疫电镜（MIEM）技术，利用单克隆抗体（mAb）对南雄蝎子（Androctonus australis）血青素（He）亚基进行直接定位。使用了四种mAb。首先，mAb 6302是一种对a2亚基高度特异性的IgG克隆，由天然血青素长链产生，该长链由侧视图和45视图中的血青素分子组成。在较低浓度下，得到由两个He分子和一个单克隆免疫球蛋白G （IgG）分子组成的“降落伞”和“蝴蝶”结构。从mAb 6302制备的Fab片段精确地结合在分子的上下边缘。第二种类型的mAb(6003)，针对亚基Aa 2，与自由亚基产生良好的免疫复合物，但与天然低聚物没有反应。提示由于空间位阻或构象变化，表位在天然分子中是不可接近的。第三个单抗属于IgM类，在Aa 2区与He结合。然而，由于难以将免疫复合物从残留的单抗中分离出来以及IgM分子的多态性，单克隆IgM不再用于MIEM。最后一种mAb（5701）对a6亚基具有高亲和力和高特异性。它与天然He产生两种类型的免疫复合物。这两种类型的不同之处在于围绕Fab臂的一个旋转180度。这些复合物支持Wrigley等人[Wrigley， n.g, Brown， EB, & Skehel, JJ (1983) J. Mol. Biol. 169, 771-774]和Roux [Roux， KH (1983) Eur.]的最新研究结果。J. Immunol. 14, 459-464]，表明单克隆IgG在Fab臂周围具有高度的旋转灵活性。单克隆抗体5701恰好结合在已知亚基Aa 6所在区域的分子角上。表位定位的MIEM方法要求结构模型和四元结构模型非常精确。因此，Gaykema et al.[Gaykema， W. P. J, Hoi， J. M, Vereijken， J. M, Soeter， N. M, Bak， H. J, & Beintema, JJ (1984) Nature (London) 309, 23-29]和Van Heel et al.[Van Heel， M., Keegstra， W., Schutter， W., & Van Bruggen, EF J.(1983) Life Chem.]的最新发现。代表,增刊。[Ser. 1, 69-73]导致了对以前模型的重新检查。具体来说，十二聚体的左对映体被右对映体取代，并引入了n端和c端结构域之间的区别。该模型与MIEM图的比较表明，mAb 6302和5701的表位分别位于亚基Aa 2和Aa 6的c端结构域。讨论了（4 × 6）-mer和（8 × 6）-mer He的四元结构的结构模型重新检验的结果。血青素是节肢动物和软体动物中含有蓝色铜的氧载体。本文讨论的节肢动物血青素是含有6个亚基倍数的低聚物，分别称为（1 × 6）-聚体或六聚体、（2 × 6）-聚体或十二聚体、（4 × 6）-聚体和（8 × 6）-聚体。除了这些众所周知的形式，Mangum等人（1984）最近的研究结果表明，蜈蚣中存在(6 X 6)-meric结构。血青素的聚集水平和亚基异质性取决于其生理起源。例如，六聚体血青素
Monoclonal antibodies (mAb) directed vs. subunits from hemocyanin (He) of the scorpion Androctonus australis were used in molecular immunoelectron microscopy (MIEM) to directly localize the epitopes within the subunits. Four types of mAb were used. First, mAb 6302, an IgG clone highly specific for subunit Aa 2, produced with native hemocyaninlong strings composed of hemocyanin molecules in the side view and in the 45 view. At lower concentration,“parachute” and “butterfly” structures composed of two He molecules and one monoclonal immunoglobin G (IgG) molecule were obtained. Fab fragments prepared from mAb 6302 bound exactly on the top and bottom edges of the molecule. The second type of mAb (6003), directed vs. subunit Aa 2, produced nice immunocomplexes with the free subunit but nothing with the native oligomer. It is suggested that due to steric hindrance or to conformational changes the epitope is not accessible in the native molecule. The third mAb belonged to the IgM class and apparently bound He in the Aa 2 area. However, because of the difficulty of separating the immunocomplexes from the residual mAb and the polymorphism of the IgM molecules, monoclonal IgM are no longer used for MIEM. The last type of mAb (5701) had a high affinity and a high specificity for subunit Aa 6. It produced two types of immunocomplexes with native He. The two types differed by a 180 rotation around one of the Fab arms. These complexes, which support recent results of Wrigley et al.[Wrigley, N. G., Brown, EB, & Skehel, JJ (1983) J. Mol. Biol. 169, 771-774] and of Roux [Roux, KH (1983) Eur. J. Immunol. 14, 459-464], indicate that monoclonal IgG have a high degree of rotational flexibility around the Fab arm. Monoclonal antibody 5701 bound exactly at the corner of the molecule in the area where subunit Aa 6 is known to be located. The MIEM approach of the location of the epitope requires the model of the architecture and of the quaternary structure to be very precise. Thus, recent findings of Gaykema et al.[Gaykema, W. P. J., Hoi, J. M., Vereijken, J. M., Soeter, N. M., Bak, H. J., & Beintema, JJ (1984) Nature (London) 309, 23-29] and of Van Heel et al.[Van Heel, M., Keegstra, W., Schutter, W., & Van Bruggen, EF J.(1983) Life Chem. Rep., Suppl. Ser. 1, 69-73] led to a reexamination of previous models. Specifically, the left enantiomorph of the dodecamer was substituted with the right enantiomorph and a distinction between the N-and C-terminal domains was introduced. A comparison of this model to the MIEM picture suggests that the epitopes of mAb 6302 and 5701 are both located in the C-terminal domains of subunits Aa 2 and Aa 6, respectively. The consequences of the reexamination of the architecture model of the quaternary structure of (4 X 6)-mer and (8X 6)-mer He are discussed.Hemocyanins are blue copper-containing oxygen carriers occurring in arthropods and molluscs. Arthropodan hemocyanins, the matter of this paper, are oligomers containing multiples of six subunits and called (1 X 6)-mer or hexamer,(2 X 6)-mer or dodecamer,(4 X 6)-mer and (8 X 6)-mer, respectively. In addition to these well-known forms, recent results of Mangum et al.(1984) indicatethat a (6 X 6)-meric structure occurs in centipedes. The aggregation level and the subunit heterogeneity of hemocyanins depend on their phy-logenic origin. For example, the hexameric hemocyanin of