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-inactivating protein (RIP), designated trichoanguin. which was purified to apparent 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 of 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 encodes an open reading frame coding for a polypeptide of 294 amino acid residues. The first 19 residues of this polypeptide encode a signal peptide sequence 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 abrin A-chain reveals 55 %, 48 %, 36 %, and 34 % identity respectively. Molecular homology modelling of trichoanguin indicates that its tertiary structure closely resembles those of trichosanthin and alpha-momorcharin. The large structural similarities might account for their common biological effects 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 therefore conceivable that trichoanguin might be a better type I RIP than any other so far examined for the preparation of immunotoxins, with a great potential for application as an effective chemotherapeutic agent for the treatment of cancer.