Department of Biochemistry, University of Oulu, Oulu, Finland.
PLoS One. 2011;6(5):e19915. doi: 10.1371/journal.pone.0019915. Epub 2011 May 25.
As an essential structural protein required for tight compaction of the central nervous system myelin sheath, myelin basic protein (MBP) is one of the candidate autoantigens of the human inflammatory demyelinating disease multiple sclerosis, which is characterized by the active degradation of the myelin sheath. In this work, recombinant murine analogues of the natural C1 and C8 charge components (rmC1 and rmC8), two isoforms of the classic 18.5-kDa MBP, were used as model proteins to get insights into the structure and function of the charge isomers. Various biochemical and biophysical methods such as size exclusion chromatography, calorimetry, surface plasmon resonance, small angle X-ray and neutron scattering, Raman and fluorescence spectroscopy, and conventional as well as synchrotron radiation circular dichroism were used to investigate differences between these two isoforms, both from the structural point of view, and regarding interactions with ligands, including calmodulin (CaM), various detergents, nucleotide analogues, and lipids. Overall, our results provide further proof that rmC8 is deficient both in structure and especially in function, when compared to rmC1. While the CaM binding properties of the two forms are very similar, their interactions with membrane mimics are different. CaM can be used to remove MBP from immobilized lipid monolayers made of synthetic lipids--a phenomenon, which may be of relevance for MBP function and its regulation. Furthermore, using fluorescently labelled nucleotides, we observed binding of ATP and GTP, but not AMP, by MBP; the binding of nucleoside triphosphates was inhibited by the presence of CaM. Together, our results provide important further data on the interactions between MBP and its ligands, and on the differences in the structure and function between MBP charge isomers.
作为中枢神经系统髓鞘紧密压缩所必需的结构蛋白，髓鞘碱性蛋白（MBP）是人类炎症性脱髓鞘疾病多发性硬化症的候选自身抗原之一，其特征是髓鞘的主动降解。在这项工作中，天然 C1 和 C8 电荷成分（rmC1 和 rmC8）的重组鼠类似物，经典的 18.5kDa MBP 的两种同工型，被用作模型蛋白，以深入了解电荷异构体的结构和功能。各种生化和生物物理方法，如尺寸排阻色谱、量热法、表面等离子体共振、小角 X 射线和中子散射、拉曼和荧光光谱以及常规和同步辐射圆二色性，用于研究这两种同工型之间的差异，从结构角度来看，以及与配体的相互作用，包括钙调蛋白（CaM）、各种去污剂、核苷酸类似物和脂质。总的来说，我们的结果进一步证明，与 rmC1 相比，rmC8 在结构上特别是在功能上都存在缺陷。虽然两种形式的 CaM 结合特性非常相似，但它们与膜类似物的相互作用不同。CaM 可用于从由合成脂质制成的固定化脂质单层中去除 MBP--这一现象可能与 MBP 功能及其调节有关。此外，使用荧光标记的核苷酸，我们观察到 MBP 与 ATP 和 GTP 的结合，但 AMP 没有结合；CaM 的存在抑制了核苷三磷酸的结合。总之，我们的结果为 MBP 与其配体之间的相互作用以及 MBP 电荷异构体的结构和功能差异提供了重要的进一步数据。
