title: Recombinant TAT-gelonin fusion toxin: synthesis and characterization of heparin/protamine-regulated cell transduction.
abstract: Protein toxins, such as gelonin, are highly desirable anti-cancer drug candidates due to their unparalleled potency and repetitive reaction mechanism in inhibiting protein translation. However, for its potential application in cancer therapy, there remains the cell membrane barrier that allows permeation of only small molecules, which must be overcome. To address this challenge, we conjugated gelonin with a protein transduction domain (PTD), the TAT peptide, via genetic recombination. The chimeric TAT-gelonin fusion protein (TAT-Gel) retained equipotent N-glycosidase activity yet displayed greater cell uptake than unmodified recombinant gelonin (rGel), thereby yielding a significantly augmented cytotoxic activity. Remarkably, TAT-Gel displayed up to 177-fold lower IC₅₀ (avg. 54.3 nM) than rGel (avg. IC₅₀ : 3640 nM) in tested cell lines. This enhanced cytotoxicity, however, also raised potential toxicity concerns due to the non-selectivity of PTD in its mediated cell transduction. To solve this problem, we investigated the plausibility of regulating the cell transduction of TAT-Gel via a reversible masking using heparin and protamine. Here, we demonstrated, both in vitro and in vivo, that the cell transduction of TAT-Gel can be completely curbed with heparin and yet this heparin block can be efficiently reversed by the addition of protamine. This reversible tight regulation of the cell transduction of TAT-Gel by heparin and protamine sheds light of possible application of TAT-Gel in achieving a highly effective yet safe drug therapy for the treatment of tumors.
authors.affiliation: Tianjin Key Laboratory on Technologies Enabling Development of Clinical Therapeutics and Diagnosis, School of Pharmacy, Tianjin Medical University, Tianjin 300070, China.|Department of Pharmaceutical Sciences, College of Pharmacy, University of Michigan, 428 Church St., Ann Arbor, MI 48109, USA.
authors.forename: Meong Cheol
authors.initials: MC
authors.lastname: Shin
authors.affiliation: Department of Molecular and Cellular Pharmacology, School of Pharmaceutical Science and Technology, Tianjin University, Tianjin 300072, China.
authors.forename: Jingwen
authors.initials: J
authors.lastname: Zhao
authors.affiliation: Biomedical Polymers Laboratory, and Jiangsu Key Laboratory of Advanced Functional Polymer Design and Application, Department of Polymer Science and Engineering, College of Chemistry, Chemical Engineering and Materials Science, Soochow University, Suzhou, 215123, China.
authors.forename: Jian
authors.initials: J
authors.lastname: Zhang
authors.affiliation: Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 501 Hai-ke Rd, Shanghai 201203, China.
authors.forename: Yongzhuo
authors.initials: Y
authors.lastname: Huang
authors.affiliation: Tianjin Key Laboratory on Technologies Enabling Development of Clinical Therapeutics and Diagnosis, School of Pharmacy, Tianjin Medical University, Tianjin 300070, China.
authors.forename: Huining
authors.initials: H
authors.lastname: He
authors.affiliation: College of Pharmacy, Xinjiang Medical University, 393 Xinyi Road, Urumqi 830011, China.
authors.forename: Mei
authors.initials: M
authors.lastname: Wang
authors.affiliation: Department of Pharmaceutical Sciences, College of Pharmacy, University of Michigan, 428 Church St., Ann Arbor, MI 48109, USA.
authors.forename: Kyoung Ah
authors.initials: KA
authors.lastname: Min
authors.affiliation: Tianjin Key Laboratory on Technologies Enabling Development of Clinical Therapeutics and Diagnosis, School of Pharmacy, Tianjin Medical University, Tianjin 300070, China.|Department of Pharmaceutical Sciences, College of Pharmacy, University of Michigan, 428 Church St., Ann Arbor, MI 48109, USA.
authors.forename: Victor C
authors.initials: VC
authors.lastname: Yang
mesh_terms: D000818:Animals; D002460:Cell Line; D015696:Gene Products, tat / Q000737:chemistry*; D006493:Heparin / Q000737:chemistry*; D006801:Humans; D011479:Protamines / Q000737:chemistry*; D011993:Recombinant Fusion Proteins / Q000737:chemistry*; D054789:Ribosome Inactivating Proteins, Type 1 / Q000737:chemistry*; D014161:Transduction, Genetic
chemical_list: D015696:Gene Products, tat; D011479:Protamines; D011993:Recombinant Fusion Proteins; D054789:Ribosome Inactivating Proteins, Type 1; C025674:GEL protein, Gelonium multiflorum; D006493:Heparin
keywords: TAT; gelonin; heparin; protamine; tumor
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