Department of Neuroscience, California Pacific Medical Center Research Institute, 475 Brannan Street, San Francisco, CA 94107, USA; E-Mail:
Int J Mol Sci. 2010 Oct 20;11(10):4051-62. doi: 10.3390/ijms11104051.
We recently reported the presence of a novel 32 kDa protein immunoreactive to a copper, zinc superoxide dismutase (SOD1) antibody within the spinal cord of patients with amyotrophic lateral sclerosis (ALS). This unique protein species was generated by biotinylation of spinal cord tissue extracts to detect conformational changes of SOD1 specific to ALS patients. To further characterize this protein, we enriched the protein by column chromatography and determined its protein identity by mass spectrometry. The protein that gave rise to the 32 kDa species upon biotinylation was identified as carbonic anhydrase I (CA I). Biotinylation of CA I from ALS spinal cord resulted in the generation of a novel epitope recognized by the SOD1 antibody. This epitope could also be generated by biotinylation of extracts from cultured cells expressing human CA I. Peptide competition assays identified the amino acid sequence in carbonic anhydrase I responsible for binding the SOD1 antibody. We conclude that chemical modifications used to identify pathogenic protein conformations can lead to the identification of unanticipated proteins that may participate in disease pathogenesis.
我们最近报道了在肌萎缩侧索硬化症（ALS）患者的脊髓中存在一种新型的 32 kDa 蛋白，该蛋白对铜锌超氧化物歧化酶（SOD1）抗体具有免疫反应性。这种独特的蛋白种类是通过生物素化脊髓组织提取物来检测 ALS 患者特异性的 SOD1 构象变化而产生的。为了进一步表征这种蛋白，我们通过柱层析对其进行了富集，并通过质谱确定了其蛋白身份。在生物素化后产生 32 kDa 产物的蛋白被鉴定为碳酸酐酶 I（CA I）。ALS 脊髓中的 CA I 生物素化导致产生了一种新的表位，该表位被 SOD1 抗体识别。通过培养细胞表达的人 CA I 提取物的生物素化也可以产生这种表位。肽竞争测定鉴定出负责与 SOD1 抗体结合的碳酸酐酶 I 中的氨基酸序列。我们得出结论，用于识别致病蛋白构象的化学修饰方法可能会导致鉴定出可能参与疾病发病机制的意外蛋白。
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