Localization and Purification of Phagosome Associated Proteases in Tetrahymena Thermophila

Tripta Kaur, Vassar College ’10 and Profs. J. William Straus and Jay Carreon

Phagocytosis is a process by which cells internalize and digest food and other materials. The single-cell ciliate Tetrahymena thermophila forms phagosomes at the oral cavity approximately every minute. Phagosome maturation involves sequential introduction of various oxidases and hydrolases, including proteases, that degrade encapsulated materials. Past utilization of fluorogenic protease substrates in our laboratory indicated that protease activity is usually associated with small vesicle like structures at the phagosome surface and occasionally in the lumen. Our current focus is to localize and purify phagosome associated cysteine proteases in T. thermophila using fluorescent and affinity-tagged conjugates of an irreversible inhibitor DCG-04 (lys(Boc)-6-aminohexanoic acid-tyr-leu-ethyl (2S, 3S)-oxirane-2, 3-dicarboxylate after Greenbaum et al, 2000). Biotinylated DCG-04 was used to isolate cysteine proteases by affinity chromatography and gel electrophoresis. Two irreversible active-site directed fluorescent cysteine protease inhibitors (6 FAM, SE DCG-04 and BODIPY 588/616-DCG-04) were synthesized using a combination of solution phase and standard F-moc solid phase peptide chemistry. The masses of the products were confirmed with MALDI-TOF mass spectrometry and they were purified by reverse phase HPLC. The binding of 6 FAM, SE DCG-04 to formaldehyde fixed, detergent permeabilized cells was assessed by fluorescence microscopy in the presence and absence of a 100 fold excess of E-64, a competing irreversible cysteine protease inhibitor. There was extensive punctate binding of the fluorescent inhibitor only in the absence of E-64, in patterns that were consistent with fluorogenic substrate localization. 6 FAM, SE DCG-04 was taken up by live cells and following fixation, the fluor was observed to co-localize with fluorogenic protease substrate. For enzyme purification, clarified extracts of actively growing cells were prepared by detergent lysis and centrifugation. Endogenous biotin was blocked with avidin, extracts were incubated with biotinylated DCG-04, and protease-inhibitor complexes were isolated by affinity chromatography against monomeric avidin. One-dimensional electrophoresis revealed at least 21 distinct proteins ranging in size from about 18 – 200 KDa. Future efforts will focus on proteomic analysis of these isolated proteins.