Molecular Characterization of Human MUC16 (CA125) in Breast Cancer
Abstract
This study was designed to understand the role and implications of MUC16 cytoplasmic tail in breast cancer pathogenesis. We would like to update our findings with respect to it since our last report submission. One important question towards this end was whether MUC16 indeed undergoes cleavage, which was addressed using a dual-epitope tagging (N-ter FLAG and C-ter HA). We demonstrated that cleavage of MUC16 could be taking place in the membrane proximal region (twelve amino acids), however deletion of these twelve amino acids partially abrogated the cleavage. Further, Circular Dichroism (CD spectra) analysis using the bacterially purified protein showed an increased alpha helical nature of the protein at acidic pH (5.8). In addition, we demonstrate that the cellular location for the cleavage is Golgi apparatus and the acidic pH in the Golgi is critical for the cleavage. We therefore believe that structural changes brought about by the acidic pH in the Golgi is probably the major reason for the cleavage of MUC16 C-ter and this could be auto-proteolytic in nature. Besides, we verified our preliminary observation of N-glycosylation using site directed mutagenesis approach and we demonstrate that MUC16 C-ter undergoes N-glycosylation and this is critical for the stability of the protein. Here we have demonstrated that, MUC16 C-ter undergoes pH dependent cleavage in the Golgi apparatus and it takes place in the membrane proximal region of the protein. It undergoes Ubiquitylation and N-glycosylation, which are required for its stability. We are furthering our studies to understand the functional and mechanistic insight into its role in cancer pathogenesis.
Document Details
- Document Type
- Technical Report
- Publication Date
- Feb 01, 2014
- Accession Number
- ADA601644
Entities
People
- Srustidhar Das
Organizations
- University of Nebraska Omaha