ABSTRACT
Trichosanthin is a ribosome-inactivating protein that possesses antitumor and antiviral activities. Clinical trials of trichosanthin on AIDS patients, however, elicit anaphylactic reactions. To reduce the antigenicity of trichosanthin as a drug while preserving its biological activity, the C-terminal domain (residues 203 to 247), which contains a putative antigenic site, was systemically deleted. We have found that the minimum length of trichosanthin that can fold into an active conformation is residue 1 to 240. The mini-trichosanthin (C7) generated by deleting the last seven C-terminal amino acid residues has 2.7-fold decrease in antigenicity, 10-fold reduction in in vitro ribosome-inactivation activity, and in vivo cytotoxicity toward K562 cells, and 2-fold reduction in abortificient activity. Structural analyses of C7 indicate decrease in the helix content, increased exposure of Trp192, and lower thermodynamic stability. The deletion of the C-terminal residues (Leu241 to Ala247) probably perturbs local structure of the C-terminal antigenic epitope that results in the decrease in antigenicity and activities of C7.
Subject(s)
Abortifacient Agents, Nonsteroidal/immunology , Anti-HIV Agents/immunology , Antineoplastic Agents, Phytogenic/immunology , Trichosanthin/immunology , Amino Acid Sequence , Circular Dichroism , Guanidine/pharmacology , Models, Molecular , Molecular Sequence Data , Protein Denaturation , Protein Engineering , Protein Structure, Secondary , Ribosomes/drug effects , Sequence Deletion , Trichosanthin/geneticsSubject(s)
Alzheimer Disease/metabolism , Brain/metabolism , Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Phosphoproteins/metabolism , tau Proteins/metabolism , Blotting, Western , Electrophoresis, Polyacrylamide Gel , Glycogen Synthase Kinases , Humans , Molecular Weight , Phosphoproteins/isolation & purification , Phosphorylation , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , tau Proteins/isolation & purificationABSTRACT
Extensive in vitro phosphorylation of a purified preparation of control human brain tau consistently produces four rather than, as previously believed, three tau species on SDS-PAGE. The species thus generated are shifted on SDS-PAGE to positions that match those of PHF-tau isolated from Alzheimer's disease brain. A mixture of recombinant human tau isoforms phosphorylated by GSK-3 beta gave similar results to those obtained with control human brain tau. In vitro phosphorylation of the individual recombinant isoforms by GSK-3 beta showed that the four bands of PHF-tau are likely to consist of isoforms 3R,0 alone; 4R,0 with 3R,29; 4R,29 with 3R,58 and 4R,58 alone.
