Boulassel M R, Tomasi J P, Deggouj N, Gersdorff M
Laboratory of Autoimmunity, University of Louvain Medical School, Brussels, Belgium.
Otol Neurotol. 2001 Sep;22(5):614-8. doi: 10.1097/00129492-200109000-00009.
This study was designed to identify the 58-kDa inner ear protein against which the sera of some patients with idiopathic, progressive sensorineural hearing loss or Ménière's disease strongly react.
We and other groups have previously demonstrated that a 58-kDa protein extracted from guinea pig or bovine inner ear tissue is a target of antibodies in serum samples from some patients with autoimmune inner ear diseases.
After separation of inner ear proteins by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis, the bands corresponding to 58 kDa were localized and excised from the gel. The concentrated protein was then digested with trypsin, and the peptide fragments were separated by high-pressure liquid chromatography. Three fractions were subjected to amino acid sequencing by the classic Edman degradation.
The sequence of a stretch of 14 amino acids of the first fragment was identical to that of amino acids 526 to 539 of the COCH5B2 protein. The sequences of 11 and 10 amino acids of the second and third fragments, respectively, also were identical to residues 417 to 427 and 396 to 405 of the COCH5B2 protein. These data, together with two-dimensional gel electrophoresis followed by Western blot experiments, confirmed that the 58-kDa inner ear protein is the COCH5B2 protein.
These findings indicate that the 58-kDa target protein of antibodies in serum samples of patients with autoimmune inner ear diseases is the COCH5B2 protein, a molecule that is highly and specifically expressed in the cochlea and vestibule.
本研究旨在鉴定一种58 kDa的内耳蛋白，一些特发性、进行性感音神经性听力损失或梅尼埃病患者的血清会与其发生强烈反应。
我们和其他研究小组之前已经证明，从豚鼠或牛内耳组织中提取的一种58 kDa蛋白是一些自身免疫性内耳疾病患者血清样本中抗体的靶标。
通过10%十二烷基硫酸钠聚丙烯酰胺凝胶电泳分离内耳蛋白后，定位并从凝胶中切下对应58 kDa的条带。然后用胰蛋白酶消化浓缩后的蛋白，肽片段通过高压液相色谱分离。三个组分通过经典的埃德曼降解法进行氨基酸测序。
第一个片段的一段14个氨基酸的序列与COCH5B2蛋白的第526至539位氨基酸序列相同。第二个和第三个片段分别为11个和10个氨基酸的序列，也与COCH5B2蛋白的第417至427位和第396至405位残基相同。这些数据，连同二维凝胶电泳随后的蛋白质印迹实验，证实58 kDa的内耳蛋白是COCH5B2蛋白。
这些发现表明，自身免疫性内耳疾病患者血清样本中抗体的58 kDa靶蛋白是COCH5B2蛋白，这是一种在耳蜗和前庭中高度特异性表达的分子。
