Epitope location for two monoclonal antibodies against human cystatin C, representing opposite aggregation inhibitory properties
Epitope location for two monoclonal antibodies against human cystatin C, representing opposite aggregation inhibitory properties
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DOI：
10.1007/s00726-016-2242-z
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发表时间：
2016-07-01
期刊：
影响因子：
3.5
通讯作者：
Czaplewska, Paulina
中科院分区：
文献类型：
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作者：
Behrendt, Izabela;Pradzinska, Martyna;Czaplewska, Paulina
与许多其他淀粉样蛋白一样，人胱抑素 C (hCC) 会二聚化，并可能通过子结构域交换形成聚集体。抑制该过程应抑制导致特定淀粉样变性（遗传性胱抑素 C 淀粉样血管病，HCCAA）的原纤维形成。据报道，针对胱抑素 C 的单克隆抗体等外源性药物能够抑制胱抑素 C 二聚体的形成，并可能控制神经退行性疾病。我们详细研究了两种代表截然不同聚集抑制效力的单克隆抗体 (mAb) Cyst10 和 Cyst28，以找到 hCC 序列中负责免疫复合物形成的结合位点，并为 HCCAA 的可能免疫治疗铺平道路。我们使用表位提取/切除质谱方法，并使用不同的酶，辅以合成 hCC 片段的亲和力研究，作为表位鉴定的基本技术。在 hCC 结构的背景下分析结果，使我们能够讨论两种抗体的结合位点。在 N 末端、环 1 和 2（L1、L2）以及 β（2）和 β（3）链片段中发现了克隆 Cyst28 的表位序列，Cyst28 是一种高效二聚化抑制剂。两种 mAb 的构象表位序列之间的关键区别似乎是 mAb Cyst10 缺乏通过 N 末端和环 1 与 hCC 的相互作用。据推测，mAb 通过 L1 和 β 折叠片段与 hCC 相互作用，使得 hCC 结构刚性且无法进行交换过程。
Human cystatin C (hCC), like many other amyloidogenic proteins, dimerizes and possibly makes aggregates by subdomain swapping. Inhibition of the process should suppress the fibrillogenesis leading to a specific amyloidosis (hereditary cystatin C amyloid angiopathy, HCCAA). It has been reported that exogenous agents like monoclonal antibodies against cystatin C are able to suppress formation of cystatin C dimers and presumably control the neurodegenerative disease. We have studied in detail two monoclonal antibodies (mAbs) representing very different aggregation inhibitory potency, Cyst10 and Cyst28, to find binding sites in hCC sequence responsible for the immunocomplex formation and pave the way for possible immunotherapy of HCCAA. We used the epitope extraction/excision mass spectrometry approach with the use of different enzymes complemented by affinity studies with synthetic hCC fragments as a basic technique for epitope identification. The results were analyzed in the context of hCC structure allowing us to discuss the binding sites for both antibodies. Epitopic sequences for clone Cyst28 which is a highly potent dimerization inhibitor were found in N-terminus, loop 1 and 2 (L1, L2) and fragments of beta(2) and beta(3) strands. The crucial difference between conformational epitope sequences found for both mAbs seems to be the lack of interactions with hCC via N-terminus and the loop 1 in the case of mAb Cyst10. Presumably the interactions of mAbs with hCC via L1 and beta sheet fragments make the hCC structure rigid and unable to undergo the swapping process.