Calpain-3 (CAPN3), a 94-kDa member of the calpain protease family, is abundant in skeletal muscle. Mutations in the CAPN3 gene cause limb girdle muscular dystrophy type 2A, indicating that CAPN3 plays important roles in muscle physiology. CAPN3 has several unique features. A crystallographic study revealed that its C-terminal penta-EF-hand domains form a homodimer, suggesting that CAPN3 functions as a homodimeric protease. To analyze complex formation of CAPN3 in a more convenient manner, we performed blue native polyacrylamide gel electrophoresis and found that the observed molecular weight of native CAPN3, as well as recombinant CAPN3, was larger than 240 kDa. Further analysis by cross-linking and sequential immunoprecipitation revealed that CAPN3 in fact forms a homotrimer. Trimer formation was abolished by the deletion of the PEF domain, but not the CAPN3-specific insertion sequences NS, IS1, and IS2. The PEF domain alone formed a homodimer, as reported, but addition of the adjacent CBSW domain to its N-terminus reinforced the trimer-forming property. Collectively, these results suggest that CAPN3 forms a homotrimer in which the PEF domain's dimer-forming ability is influenced by other domains.

中文翻译：

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肌肉特异性钙蛋白酶CAPN3形成同源三聚体。

Calpain-3（CAPN3）是钙蛋白酶蛋白酶家族的94 kDa成员，在骨骼肌中含量很高。CAPN3基因的突变会导致2A型腰带型肌营养不良，表明CAPN3在肌肉生理中起重要作用。CAPN3具有几个独特的功能。晶体学研究表明，其C端五-EF-手结构域形成同型二聚体，表明CAPN3作为同型二聚体蛋白酶起作用。为了以更方便的方式分析CAPN3的复杂形成，我们进行了蓝色天然聚丙烯酰胺凝胶电泳，发现观察到的天然CAPN3以及重组CAPN3的分子量均大于240 kDa。通过交联和顺序免疫沉淀的进一步分析表明，CAPN3实际上形成了同源三聚体。PEF结构域的删除取消了三聚体的形成，但不是CAPN3特定的插入序列NS，IS1和IS2。如所报道的，仅PEF结构域形成同源二聚体，但是在其N末端添加相邻的CBSW结构域增强了三聚体形成性质。总的来说，这些结果表明CAPN3形成同三聚体，其中PEF结构域的二聚体形成能力受其他结构域影响。