Importance of the glu 160 and glu 189 residues to the various biological activities of the ribosome inactivating protein trichosanthin |
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| Institution: | 1. Department of Biochemistry, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong;2. Department of Anatomy, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong;1. Department of Natural Sciences, University of South Carolina Beaufort, 1 University Boulevard, Bluffton, SC 29909, USA;2. NMRService LLC, Raleigh, NC 27612, USA;3. Department of Molecular and Structural Biochemistry, North Carolina State University, 128 Polk Hall, Raleigh, NC 27695, USA;1. Department of Natural Sciences, University of South Carolina Beaufort, 1 University Boulevard, Bluffton, South Carolina 29909, USA;2. NE-CAT, Department of Chemistry and Chemical Biology, Cornell University, Building 436E, Argonne National Laboratory, 9700 S. Cass Avenue, Argonne, Illinois 60439, USA;3. Department of Molecular and Structural Biochemistry, North Carolina State University, 128 Polk Hall, Raleigh, North Carolina 27695, USA;4. Department of Microbiology, New York University School of Medicine, 550 First Avenue, New York, New York 10016, USA;1. Brazilian Center for Research in Energy and Materials, Brazilian Biosciences National Laboratory, Campinas, SP 13083-970, Brazil;2. Department of Natural Sciences, University of South Carolina Beaufort, 1 University Boulevard, Bluffton, SC 29909, USA;3. Department of Microbiology, New York University School of Medicine, 430 East 29th Street, New York, NY 10016, USA |
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| Abstract: | Site-directed mutagenesis of trichosanthin (TCS), a ribosome inactivating protein with a broad spectrum of biological activities, was carried out to ascertain the importance of the Glu 160 and Glu 189 residues to the protein synthesis-inhibitory, antiproliferative, immunosuppressive and embryotoxic activities of TCS. Replacement of Glu 160 with alanine and with aspartate produced muteins, designated E160A] and E160D] respectively, with considerably attenuated protein synthesis-inhibitory, antiproliferative, immunosuppressive and embryotoxic activities, indicating that Glu 160 in TCS plays a role in its biological activity. E160A] was, however, more potent than E160D] because in the former mutein, Glu 189 constitutes a back-up of the carboxylate group but in the latter mutein, the negative charge from Asp is at a suboptimal position. The mutein E160A, E189A] formed by mutation of both Glu 160 and Glu 189 retained considerable embryotoxic activity, suggesting that other amino acids in the active site were able to partially replace the role of Glu 160 and Glu 189. The TCS muteins also exhibit higher toxicity toward cultured embryos than cultured cells (spleen cells and tumor cells). |
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