June 2015
Volume 56, Issue 7
ARVO Annual Meeting Abstract  |   June 2015
Biochemical and functional characterization of A25T cystatin C, a secreted protease inhibitor associated with exudative macular degeneration
Author Affiliations & Notes
  • John D Hulleman
    Ophthalmology and Pharmacology, University of Texas Southwestern Medical Center, Dallas, TX
  • Annie Nguyen
    Ophthalmology, University of Texas Southwestern Medical Center, Dallas, TX
  • Footnotes
    Commercial Relationships John Hulleman, None; Annie Nguyen, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2015, Vol.56, 807. doi:
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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)

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Purpose: 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.

Methods: 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.

Results: 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.

Conclusions: 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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