Department of Medicinal Chemistry, University of Gdansk, Sobieskiego 18, 80-952 Gdansk, Poland.
J Mol Recognit. 2011 Jul-Aug;24(4):687-99. doi: 10.1002/jmr.1100.
Human cystatin C (hCC), like many other amyloidogenic proteins, has been shown to form dimers by exchange of subdomains of the monomeric protein. Considering the model of hCC fibrillogenesis by propagated domain swapping, it seems possible that inhibition of this process should also suppress the entire process of dimerization and fibrillogenesis which leads to specific amyloidosis (hereditary cystatin C amyloid angiopathy (HCCAA)). It was reported that exogenous agents like monoclonal antibody against cystatin C are able to suppress formation of cystatin C dimers. In the effort to find a way of controlling the cystatin fibrillization process, the interactions between monoclonal antibody Cyst-13 and cystatin C were studied in detail. The present work describes the determination of the epitope of hCC to a monoclonal antibody raised against cystatin C, Cyst-13, by MALDI mass spectrometry, using proteolytic excision of the immune complex. The shortest epitope sequence was determined as hCC(107-114). Affinity studies of synthetic peptides revealed that the octapeptide with epitope sequence does not have binding ability to Cyst-13, whereas its longer counterpart, hCC(105-114), binds the studied antibody. The secondary structure of the peptides with epitope sequence was studied using circular dichroism and NMR spectroscopy.
人半胱氨酸蛋白酶抑制剂 C（hCC）与许多其他淀粉样蛋白一样，已经被证明可以通过单体蛋白亚结构域的交换形成二聚体。考虑到 hCC 纤维发生的传播结构域交换模型，似乎有可能抑制这个过程也应该抑制导致特定淀粉样变性（遗传性半胱氨酸蛋白酶抑制剂血管淀粉样变性（HCCAA））的整个二聚化和纤维发生过程。据报道，像针对半胱氨酸蛋白酶抑制剂的单克隆抗体这样的外源性试剂能够抑制半胱氨酸蛋白酶抑制剂二聚体的形成。为了寻找控制半胱氨酸纤维形成过程的方法，详细研究了单克隆抗体 Cyst-13 与半胱氨酸蛋白酶抑制剂 C 之间的相互作用。本工作通过使用酶切免疫复合物的 MALDI 质谱法，确定了针对半胱氨酸蛋白酶抑制剂 C 的单克隆抗体 Cyst-13 的 hCC 表位，使用 MALDI 质谱法，确定了针对半胱氨酸蛋白酶抑制剂 C 的单克隆抗体 Cyst-13 的 hCC 表位，使用 MALDI 质谱法，确定了针对半胱氨酸蛋白酶抑制剂 C 的单克隆抗体 Cyst-13 的 hCC 表位。使用酶切免疫复合物的 MALDI 质谱法，确定了针对半胱氨酸蛋白酶抑制剂 C 的单克隆抗体 Cyst-13 的 hCC 表位。使用酶切免疫复合物的 MALDI 质谱法，确定了针对半胱氨酸蛋白酶抑制剂 C 的单克隆抗体 Cyst-13 的 hCC 表位。使用酶切免疫复合物的 MALDI 质谱法，确定了针对半胱氨酸蛋白酶抑制剂 C 的单克隆抗体 Cyst-13 的 hCC 表位。该最短表位序列被确定为 hCC(107-114)。合成肽的亲和研究表明，具有表位序列的八肽没有与 Cyst-13 结合能力，而其较长的对应物 hCC(105-114)则与研究中的抗体结合。使用圆二色性和 NMR 光谱研究了具有表位序列的肽的二级结构。
