Engineering of a mini-trichosanthin that has lower antigenicity by deleting its C-terminal amino acid residues.

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.

Kinetics of IgE antibody response to trichosanthin alpha-momorcharin and beta-momorcharin in mice.

Allergenicity of three plant abortifacient proteins, trichosanthin (TCS), alpha-momorcharin (alpha-MMC) and beta-momorcharin (beta-MMC) and the kinetics of IgE antibody response against these proteins were studied in C57BL/6N and BALB/cAn mice. These proteins were purified from Chinese medicinal herbs, characterized by biochemical and immunological procedures and found to be homogeneous by PAGE, SDS-PAGE and immunoelectrophoresis. The results obtained were summarized as follows: 1) different levels of IgE antibody responses against TCS, alpha-MMC and beta-MMC were induced when they were injected i.p. as antigen adsorbed onto AI(OH)3 gel into BALB/cAn or C57BL/6N mice on day 0 and day 28; 2) the allergenicity of the proteins was in the order of beta-MMC greater than alpha-MMC greater than TCS; 3) two injections of 10 micrograms or 50 micrograms of TCS or alpha-MMC given at 4 weeks interval without adjuvant elicited a low or undetectable PCA titer, while the same dose given weekly for 5 weeks resulted in high levels of IgE production; and 4) on the basis of PCA observations, no cross immunological reactivity among these three proteins was found, it seemed to support that TCS, alpha-MMC and beta-MMC may be alternately used for inducing abortion.

Antigenic determinant of trichosanthin peptide fragments.

Purified plant protein, Trichosanthin (TCS), was partially digested with chymotrypsin to produce peptide fragments. Two large fragments, I and II, have been isolated and identified as C-terminal peptides located at sequence positions between 79-234 and 107-234. Their molecular weights determined by electrophoretic mobility on SDS gel were 16,000 and 14,000 daltons. Studies using fluorescence quenching measurements by titrating anti-TCS Fab with TCS and fragment I and II showed that four epitopes were present in intact TCS and one each epitope in fragment I and II. One of the epitopes is therefore located between sequence positions 107 and 234.

Isolation and characterization of an immunosuppressive protein from Trichosanthes kirilowii root tubers.

An immunosuppressive protein was isolated from Trichosanthes kirilowii root tubers by a procedure involving acetone fractionation and ion exchange chromatography on CM-Sepharose. Homogeneity of the protein was demonstrated in immunoelectrophoresis, SDS-polyacrylamide gel electrophoresis, gel filtration, high performance liquid chromatography and a single NH2-terminal sequence. The protein had a molecular weight of 26,000, aspartic acid as the NH2-terminal amino acid and no cysteine or carbohydrates in its molecule. It inhibited ConA-induced transformation in lymphocytes isolated from spleens of CBA mice. The protein was also potent in inducing mid-term abortion in mice.

Application of monoclonal antibodies for antigen mapping of male and female generative tissue.

With the single experiment of nature hindering the rejection of the fetus in the mother, new basic mechanisms of this immunological phenomenon can be studied. The technique of monoclonal antibodies provides a helpful tool for the study of membrane structural and cytoplasmic molecules. Hybridoma clones offer for the first time the possibility of obtaining unlimited amounts of almost pure antibodies or very high titers and of defined characteristics. The most common application with reproductive tract monoclonals are found as follows: 1) Immuno-assays; 2) Immuno-diagnostics; 3) Imaging of tumors; 4) Immuno-therapy; 5) Induction of anti-idio-types; 6) Fertility control; 7) Application in basic studies and mechanisms. The following interesting results were obtained in this area: 1) The biological role of hormones can be critically analysed by the use of highly specific non-cross-reactive monoclonal antibodies. 2) A synthetic decapeptide earlier shown by our group to be reactive with naturally occurring human iso and autosperm antibodies demonstrated to be reactive with the monoclonal antibodies Ki VII-5 presenting potentials for an investigation of sperm as target of immunological contraception for possible diagnostic and therapeutic use in immunologic infertility. 3) A specific post fertilization-development influencing antigen was identified by Menge using monoclonal antibodies. 4) Five hybridoma clones producing monoclonals to porcine zona pellucida were established, and showed a characteristic staining pattern of oocytes from human eggs, hamsters, rabbits and mice by immunofluorescence.