Zhu Jeff X, Aswad Dana W
Department of Molecular Biology and Biochemistry, University of California Irvine, Irvine, CA, 92697-3900, USA.
Shanghai Reinovax Biologics Co. LTD, 5th Floor, Building I, 367 Sheng Rong Road, Pudong New District, Shanghai, 201210, China.
Amino Acids. 2024 Dec 24;57(1):5. doi: 10.1007/s00726-024-03435-0.
Collapsin response mediator protein 2 (CRMP2) functions in the genesis and activity of neuronal connections in mammalian brain. We previously reported that a protein coincident with CRMP2 on 2D-gels undergoes marked accumulation of abnormal L-isoaspartyl sites in brain extracts of mice missing the repair enzyme, protein L-isoaspartyl methyltransferase (PIMT). To confirm and explore the significance of isoaspartyl damage in CRMP2, we expressed and purified recombinant mouse CRMP2 (rCRMP2). A polyclonal antibody made against the recombinant protein precipitated CRMP2 from brain extracts of PIMT-KO mice, but not from WT mice, suggesting that (1) the rCRMP2 antigen underwent significant isoAsp formation in the process of antibody production and (2) the isoAsp form of CRMP2 is considerably more immunogenic than the native protein. In vitro aging of rCRMP2 at pH 7.4, 37 °C for 0-28 days led to robust accumulation of isoAsp sites that were repairable by PIMT, and also induced a progressive accumulation of apparent dimers and higher-mass oligomers as judged by SDS-PAGE. A similar pattern of CRMP2 aggregation was observed in mice, with levels increasing throughout the lifespan. We conclude that CRMP2 is indeed a major target of PIMT-mediated protein repair in the brain; that isoAsp forms of CRMP2 are highly immunogenic; and that CRMP2 dysfunction makes a significant contribution to neuropathology in the PIMT-KO mouse.
塌陷反应介导蛋白2（CRMP2）在哺乳动物大脑神经元连接的形成和活动中发挥作用。我们之前报道过，在二维凝胶上与CRMP2一致的一种蛋白质，在缺失修复酶——蛋白质L-异天冬氨酰甲基转移酶（PIMT）的小鼠脑提取物中，异常L-异天冬氨酰位点显著积累。为了证实并探究CRMP2中异天冬氨酰损伤的意义，我们表达并纯化了重组小鼠CRMP2（rCRMP2）。一种针对重组蛋白制备的多克隆抗体，能从PIMT基因敲除小鼠的脑提取物中沉淀出CRMP2，但不能从小鼠野生型脑提取物中沉淀出CRMP2，这表明（1）rCRMP2抗原在抗体产生过程中发生了显著的异天冬氨酸形成，并且（2）CRMP2的异天冬氨酸形式比天然蛋白具有更强的免疫原性。在pH 7.4、37℃条件下对rCRMP2进行0 - 28天的体外老化处理，导致可被PIMT修复的异天冬氨酰位点大量积累，同时通过SDS-PAGE判断，还诱导了明显的二聚体和更高质量寡聚体的逐渐积累。在小鼠中也观察到了类似的CRMP2聚集模式，其水平在整个生命周期中都在增加。我们得出结论，CRMP2确实是大脑中PIMT介导的蛋白质修复的主要靶点；CRMP2的异天冬氨酸形式具有高度免疫原性；并且CRMP2功能障碍对PIMT基因敲除小鼠的神经病理学有重大影响。
