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Completed Project

Phagocytosis, Digestion, and Protease Proteomics in Tetrahymena

Benjamin Hoffman, Vassar College ’16, Fiona Chen, Vassar College ’16 and Prof. J. William Straus
Phagocytosis is a process by which cells capture particulate and soluble materials in membranous vesicles for subsequent breakdown and digestion. In multicellular organisms, phagocytosis plays critical roles in tissue maintenance, wound healing, and both innate and acquired immunity. Tetrahymena thermophila, a single cell ciliate, feeds primarily by phagocytosis and is a well-established model for the study of this process. Three approaches were utilized to better understand the digestive process: fluorescence microscopy, proteomics, and bioinformatics. Proteolytic enzymes constitute over 2% of the T. thermophila genome, dominated by the cysteine proteinase family, but also rich in aspartate and metallo-proteinases. Phylogenetic analysis based on putative protein sequences divides the cysteine proteinases into three groups: papain family, calpain family, and other families. We compared upstream genomic sequences in sub-groupings of the papain and calpain families did not reveal any apparent consensus sequences or regulatory elements, consistent with other Tetrahymena gene families. Cysteine proteases from aqueous cell extracts were confirmed by zymographic analysis, which revealed about 13 proteinases ranging from 20,000 to over 200,000 KDa in size. An affinity chromatography matrix was synthesized by carbodiimide coupling with antipain, a tight-binding inhibitor of many cysteine proteinases. Proteomic analysis by HPLC-tandem mass spectrometry (LC-QTOF) failed to confirm any proteinases in the affinity purified fractions but did yield two validated cysteine proteases in the non-bound fractions, as well as an additional cytosolic peptidase. Fluorescence microscopy against fluorogenic protease substrate revealed digestive activity within seconds of uptake, the activity distributed in small vesicles around and within phagosomes aligned with the oral apparatus. Future bioinformatics work will focus on characterizing similarities in proteinase subfamilies, proteomic efforts will focus on alternative protein purification approaches (such as electrophoresis) for generating less complex samples. Visualization of early phagocytosis will focus on using GFP-recombinant transformants to visualize specific proteins.