Purification, characterization and molecular cloning of trichoanguin, a novel type I ribosome-inactivating protein from the seeds of Trichosanthes anguina

The seeds of the plant Trichosanthes anguina contain a type I ribosome-inac tivating protein (RIP), designated trichoanguin. which was purified to appa rent homogeneity by the combined use of ion-exchange chromatographies, i.e. first with DE-52 cellulose and then with CM-52 cellulose. The protein was found to be a glycoprotein with a molecular mass of 35 kDa and a pI of 9.1. It strongly inhibits the protein synthesis of rabbit reticulocyte lysate, with an IC50 of 0.08 nM, but only weakly that of HeLa cells, with an IC50 o f 6 mu M. Trichoanguin cleaves at the A4324 site of rat 28 S rRNA by its N- glycosidase activity. The cDNA of trichoanguin consists of 1039 nt and enco des an open reading frame coding for a polypeptide of 294 amino acid residu es. The first 19 residues of this polypeptide encode a signal peptide seque nce and the last 30 residues comprise an extension at its C-terminus. There are four potential glycosylation sites, located at Asn-51, Asn-65, Asn-201 and Asn-226. A comparison of the amino acid sequence of trichoanguin with those of RIPs such as trichosanthin, alpha-momorcharin, ricin A-chain and a brin A-chain reveals 55 %, 48 %, 36 %, and 34 % identity respectively. Mole cular homology modelling of trichoanguin indicates that its tertiary struct ure closely resembles those of trichosanthin and alpha-momorcharin. The lar ge structural similarities might account for their common biological effect s such as an abortifacient, an anti-tumour agent and anti-HIV-1 activities. Trichoanguin contains two cysteine residues, Cys-32 and Cys-155, with the former being likely to be located on the protein surface, which is directly amenable for conjugation with antibodies to form immunoconjugates. It is t herefore conceivable that trichoanguin might be a better type I RIP than an y other so far examined for the preparation of immunotoxins, with a great p otential for application as an effective chemotherapeutic agent for the tre atment of cancer.