title: A cytotoxic type-2 ribosome inactivating protein (from leafless mistletoe) lacking sugar binding activity.
abstract: Articulatin-D, a 66 kDa ribosome inactivating protein (RIP) comprised of 29 kDa A-chain linked to 35 kDa B-chain, is purified from leafless mistletoe (Viscum articulatum) parasitic on Dalbergia sp. from Western Ghats (India). N-terminal sequence and LC-MS/MS analyses of A- and B-chain confirmed that articulatin-D is a type-2 RIP having high homology with other mistletoe lectins. Translation inhibition and diagnostic N-glycosidase activity of articulatin-D illustrate the presence of catalytically active A-chain. Its inability to: (i) bind to acid treated Sepharose CL-6B column, (ii) agglutinate trypsin-treated and untreated RBCs of human (A, B, O, AB), mice, rat, rabbit, buffalo, porcine, pigeon, cock, fish, sheep and goat even with 10mg/ml of purified articulatin-D, (iii) show change in circular dichroism spectra after addition of sugar to the native protein, (iv) bind to different sugars (galactose, lactose, gal-NAc, rhamnose, arabinose, fucose and mannose) immobilized on Sepharose 4B matrix, and (v) show change in enthalpy during titration with galactose confirm that the B-chain of articulatin-D lacks sugar binding activity. Despite this, articulatin-D is highly toxic as characterized with low IC(50) against different cancer cell lines (Jurkat: 0.31 ± 0.02 nM, MOLT-4: 0.51 ± 0.03 nM, U-937: 0.64 ± 0.07 nM, HL-60: 0.79 ± 0.11 nM, Raji: 1.45 ± 0.09 nM). Toxicity of RIPs has been ascribed to the absence/presence of B-chain with sugar binding activity. Identification of articulatin-D, the first cytotoxic RIP with B-chain lacking sugar binding activity opens new vistas in understanding cytotoxic action of RIPs.
authors.affiliation: Bioresources and Environmental Biotechnology Laboratory, Department of Environmental Biology, University of Delhi, Delhi 110007, India.
authors.forename: Mrinal Kumar
authors.initials: MK
authors.lastname: Das
authors.forename: Radhey Shyam
authors.initials: RS
authors.lastname: Sharma
authors.forename: Vandana
authors.initials: V
authors.lastname: Mishra
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D045744:Cell Line, Tumor; D002942:Circular Dichroism; D004912:Erythrocytes / Q000187:drug effects; D006384:Hemagglutination / Q000187:drug effects; D006801:Humans; D007194:India; D020128:Inhibitory Concentration 50; D008969:Molecular Sequence Data; D009005:Monosaccharides / Q000737:chemistry / Q000378:metabolism; D009369:Neoplasms / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D028321:Plant Preparations / Q000737:chemistry* / Q000302:isolation & purification / Q000494:pharmacology; D011485:Protein Binding; D021122:Protein Subunits / Q000737:chemistry* / Q000302:isolation & purification / Q000494:pharmacology; D054790:Ribosome Inactivating Proteins, Type 2 / Q000737:chemistry* / Q000302:isolation & purification / Q000494:pharmacology; D012685:Sepharose / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D053719:Tandem Mass Spectrometry; D014118:Toxins, Biological / Q000737:chemistry* / Q000302:isolation & purification / Q000494:pharmacology; D028181:Viscum / Q000737:chemistry*
chemical_list: D009005:Monosaccharides; D028321:Plant Preparations; D021122:Protein Subunits; D054790:Ribosome Inactivating Proteins, Type 2; D014118:Toxins, Biological; C047264:sepharose CL 6B; D012685:Sepharose