Das Srustidhar, Majhi Prabin D, Al-Mugotir Mona H, Rachagani Satyanarayana, Sorgen Paul, Batra Surinder K
Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE 68198, USA.
1] Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE 68198, USA [2] Department of Pathology, University of Nebraska Medical Center, Omaha, NE 68198, USA [3] Buffett Cancer Center, Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, NE 68198, USA.
Sci Rep. 2015 Jun 5;5:9759. doi: 10.1038/srep09759.
MUC16, precursor of the most widely used ovarian cancer biomarker CA125, is up regulated in multiple malignancies and is associated with poor prognosis. While the pro-tumorigenic and metastatic roles of MUC16 are ascribed to the cell-associated carboxyl-terminal MUC16 (MUC16-Cter), the exact biochemical nature of MUC16 cleavage generating MUC16-Cter has remained unknown. Using different lengths of dual-epitope (N-terminal FLAG- and C-terminal HA-Tag) tagged C-terminal MUC16 fragments, we demonstrate that MUC16 cleavage takes place in the juxta-membrane ectodomain stretch of twelve amino acids that generates a ~17 kDa cleaved product and is distinct from the predicted sites. This was further corroborated by domain swapping experiment. Further, the cleavage of MUC16 was found to take place in the Golgi/post-Golgi compartments and is dependent on the acidic pH in the secretory pathway. A similar pattern of ~17 kDa cleaved MUC16 was observed in multiple cell types eliminating the possibility of cell type specific phenomenon. MUC16-Cter translocates to the nucleus in a cleavage dependent manner and binds to the chromatin suggesting its involvement in regulation of gene expression. Taken together, we demonstrate for the first time the oft-predicted cleavage of MUC16 that is critical in designing successful therapeutic interventions based on MUC16.
MUC16是最常用的卵巢癌生物标志物CA125的前体，在多种恶性肿瘤中上调，且与预后不良相关。虽然MUC16的促肿瘤发生和转移作用归因于细胞相关的羧基末端MUC16（MUC16-Cter），但产生MUC16-Cter的MUC16裂解的确切生化性质仍不清楚。使用不同长度的双表位（N端FLAG和C端HA标签）标记的C端MUC16片段，我们证明MUC16裂解发生在由12个氨基酸组成的近膜胞外区域，产生一个约17 kDa的裂解产物，且与预测位点不同。结构域交换实验进一步证实了这一点。此外，发现MUC16的裂解发生在高尔基体/高尔基体后区室，并且依赖于分泌途径中的酸性pH。在多种细胞类型中观察到类似的约17 kDa的裂解MUC16模式，排除了细胞类型特异性现象的可能性。MUC16-Cter以裂解依赖的方式转运到细胞核并与染色质结合，表明其参与基因表达调控。综上所述，我们首次证明了MUC16的裂解，这对于基于MUC16设计成功的治疗干预措施至关重要。
