J Cancer 2020; 11(3):702-715. doi:10.7150/jca.30813 This issue
1. University of Pittsburgh Cancer Institute, Pittsburgh, PA;
2. Department of Pathology, University of Pittsburgh;
3. Department of Immunology, University of Pittsburgh;
4. Department of Otolaryngology, University of Pittsburgh;
5. Department of Medicine, University of Pittsburgh;
6. Department of Pharmacology and Chemical Biology, University of Pittsburgh, Pittsburgh, PA;
7. VAPHS, Pittsburgh, PA;
8. BioZyme Inc, Apex, NC;
9. Center for Systems Biology, Massachusetts General Hospital and Harvard Medical School, Boston, MA.
Background: Cell-membrane expressing enzymes such as ADAM (a disintegrin and metalloproteinase) superfamily members are thought to be key catalysts of vital cellular functions. To directly measure these enzymes and determine their association with particular cells and functions, individual-cell membrane-bound enzyme activity assays are required, but unavailable.
Methods: We developed two such assays, using a fluorescence resonance energy transfer (FRET) peptide substrate (FPS) and flow cytometry. One assay measured live-cell natural processing of FPS and binding of its fluorescent product onto individual-cell membrane-bound enzymes. The other assay measured processing of specifically-bound and glutaraldehyde-crosslinked FPS, and consequent generation of its coupled fluorescent product onto individual-cell membrane-bound enzymes.
Results: Confocal-microscopy imaging indicated that proteolytic processing of FPS selectively occurred on and labeled cell membrane of individual cells. The new assays measured specific increases of cell-associated FPS fluorescent product in substrate-concentration-, temperature- and time-dependent manners. A large proportion of processed FPS fluorescent products remained cell-associated after cell washing, indicating their binding to cell-membrane expressing enzymes. The assays measured higher levels of cell-associated FPS fluorescent product on wild-type than ADAM10-knockout mouse fibroblasts and on human monocytes than lymphocytes, which correlated with ADAM10 presence and expression levels on cell membrane, respectively. Furthermore, the enzyme activity assays could be combined with fluorescent anti-ADAM10 antibody staining to co-label and more directly associate enzyme activity and ADAM10 protein levels on cell membrane of individual cells.
Conclusions: We report on two novel assays for measuring cell-membrane anchored enzyme activity on individual cells, and their potential use to directly study specific biology of cell-surface-expressing proteases.
Keywords: Single cell, Cell membrane, Flow cytometry, Enzyme activity, Assays, ADAM10.