The Cathepsin B of Toxoplasma gondii,Toxopain-1, Is Critical for Parasite Invasion and Rhoptry Protein Processing

• Published in: The Journal of biological chemistry 2002-07, Vol.277 (28), p.25791-25797
• Main Authors: Que, Xuchu, Ngô, Huân, Lawton, Jeffrey, Gray, Mary, Liu, Qing, Engel, Juan, Brinen, Linda, Ghosh, Partho, Joiner, Keith A., Reed, Sharon L.
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Base Sequence; Cloning, Molecular; Cysteine Endopeptidases - chemistry; Cysteine Endopeptidases - genetics; Cysteine Endopeptidases - metabolism; Cysteine Proteinase Inhibitors - pharmacology; DNA Primers; Membrane Proteins - chemistry; Membrane Proteins - metabolism; Microscopy, Immunoelectron; Molecular Sequence Data; Protein Conformation; Protein Processing, Post-Translational; Protozoan Proteins - chemistry; Protozoan Proteins - metabolism; Sequence Homology, Amino Acid; Toxoplasma - enzymology; Toxoplasma - physiology
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M202659200

Cysteine proteinases play a major role in invasion and intracellular survival of a number of pathogenic parasites. We cloned a single copy gene, tgcp1, fromToxoplasma gondii and refolded recombinant enzyme to yield active proteinase. Substrate specificity of the enzyme and homology modeling identified the proteinase as a cathepsin B. Specific cysteine proteinase inhibitors interrupted invasion by tachyzoites. The T. gondii cathepsin B localized to rhoptries, secretory organelles required for parasite invasion into cells. Processing of the pro-rhoptry protein 2 to mature rhoptry proteins was delayed by incubation of extracellular parasites with a cathepsin B inhibitor prior to pulse-chase immunoprecipitation. Delivery of cathepsin B to mature rhoptries was impaired in organisms with disruptions in rhoptry formation by expression of a dominant negative μ1-adaptin. Similar disruption of rhoptry formation was observed when infected fibroblasts were treated with a specific inhibitor of cathepsin B, generating small and poorly developed rhoptries. This first evidence for localization of a cysteine proteinase to the unusual rhoptry secretory organelle of an apicomplexan parasite suggests that the rhoptries may be a prototype of a lysosome-related organelle and provides a critical link between cysteine proteinases and parasite invasion for this class of organism.