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John D Hulleman, Annie Nguyen; Biochemical and functional characterization of A25T cystatin C, a secreted protease inhibitor associated with exudative macular degeneration. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):807.
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© ARVO (1962-2015); The Authors (2016-present)
An Ala25Thr (A25T) mutation in the secreted protease inhibitor, cystatin C, has been associated with both late-onset Alzheimer's disease as well as exudative macular degeneration. The purpose of this project was to assess the impact that the A25T mutation has on cystatin C signal sequence cleavage, secretion, post-translational modifications and function. Ultimately, our findings will be related to how this mutation may compromise cystatin C function and cause disease.
In order to easily track the fate of cystatin C, while minimizing the impact of introducing protein tags that potentially disrupt normal cystatin C folding, we tagged WT and A25T cystatin C with either FLAG or hemagglutinin epitopes. Additionally, we have generated GFP-tagged cystatin C variants similar to previously used constructs. Cystatin C variants were transfected into ARPE-19 cells and their secretion into conditioned media was measured by western blotting. To determine whether secreted cystatin C was glycosylated, we used lectin binding and enzymatic assays. Differential cystatin C signal sequence cleavage sites were evaluated by using engineered constructs expressing an N-terminal FLAG tag. Cystatin C functionality was assessed by using activity-based protein probes.
Surprisingly, we found that A25T tagged with a variety of different epitopes (FLAG, HA, or GFP) are all secreted as efficiently from ARPE-19 cells as their WT counterparts. Preliminary experiments indicate that cystatin C can be glycosylated. Additionally, we have found that WT cystatin C can be secreted following cleavage after residue 20 or 26 of the signal sequence. Furthermore, we have also determined that A25T cystatin C does traverse the conventional endoplasmic reticulum (ER)-to-Golgi pathway, just like WT cystatin C. Interestingly, A25T expression does not appear to activate the unfolded protein response, another indication that A25T cystatin C behaves similarly to WT cystatin C.
Our results suggest that the A25T mutation may not cause disease simply by reducing protein secretion. Rather, we speculate that the A25T mutation may reduce cystatin C protease function by either shifting the signal sequence cleavage site and/or by altering cystatin C post-translational modifications.
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