Hormonotoxins: synthesis, characterization and bioefficacy of some defined disulfide linked conjugates of ovine LH with a ribosome inactivating protein, gelonin.

In order to synthesise a bioeffective hormonotoxin for selective targeting to specific cells in the gonads, gelonin, a single chain RIP obtained from an Indian plant, Gelonium multiflorum of Euphorbeaceae family was covalently linked to oLH with the use of N-succinimidyl-3-(2-pyridyldithio)propionate, generating a linkage containing a disulfide bond and a amide bond. The hormonotoxins were separated according to their molecular weight (indirectly according to oLH:gelonin molar ratio) and a complete biochemical analysis was performed. The linkage occurred through the epsilon-NH2 group of alpha oLH as judged from RP-HPLC analysis. The conjugates were devoid of ingredients as determined by SDS-PAGE and RP-HPLC analysis. The hormonotoxins retained substantial receptor binding, steroidogenic activity and immunoreactivity of oLH and gelonin to their antibodies. Hormonotoxins bind to the Leydig tumour cells via oLH part leaving gelonin free as judged by competitive displacement analysis. The hormonotoxin was internalized to the sufficient degree to effectively inhibit protein synthesis. The cytotoxicity of 1:1 molar ratio conjugate was relatively higher than that of others. The cytotoxicity of presently described more defined hormonotoxins exhibited higher receptor binding and cytotoxicity than the hormonotoxins reported earlier [Singh, et al., J Biol Chem, 264 (1989) 3089].     

Hormonotoxins. Preparation and characterization of ovine luteinizing hormone-gelonin conjugate

With the aim of targeting toxins to selected cells in the gonad, we have prepared conjugates of ovine luteinizing hormone (oLH) with a single chain ribosome-inactivating protein called gelonin. The two proteins were thiolated by using N-succinimidyl-3-(2-pyridyldithio)propionate and subsequently reacted under appropriate conditions to form oLH-S-S-gelonin complex. A complete biochemical analysis of thiolated oLH and oLH-gelonin conjugates has been performed. The linkage of the hormone to the toxin probably occurred through a single amino group in the alpha-subunit, with the beta-subunit remaining free. Modification of a single amino group on the alpha-subunit reduced receptor binding and immunological reactivity of the thiolated oLH, but subsequent complexing with the toxin-gelonin did not seriously compromise these activities. oLH and gelonin were calculated to be present in a 1:1 ratio in the hormonotoxin preparation. The conjugate retained significant steroidogenic activity in rat granulosa cells. Upon reaction with mouse tumor Leydig cells (MA-10 cells), the toxin component of the complex became internalized to a sufficient degree to effectively inhibit protein synthesis. The studies provide a rational basis for the design and study of large hormonotoxins.     

Chromatographic separation of the isoforms of the ribosome inactivating protein, gelonin.

The three isoforms of gelonin were separated by affinity chromatography on concanavalin-A Sepharose into discrete components of Mr 31,500, 30,000 and 29,200. Their separation was achieved by apparent differences in interaction with the lectin due to variation in carbohydrate patterns. The Mr 30,000 component representing 67% of the total mixture was the most active in inhibiting protein synthesis in a cell free translation assay using rabbit reticulocyte lysates, although the other two were also active. An antibody prepared against the major fraction (Mr 30,000) reacted well with all three components, demonstrating immunological similarity. This purification may aid the structural elucidation of gelonin and preparation of hormonotoxins and immunotoxins.     

In-vitro selective killing of gonadal cells by a hormonotoxin composed of ovine luteinizing hormone linked by a disulfide bond to the ribosome-inactivating protein, gelonin.

With the aim to synthesize a bioeffective hormonotoxin for selective targeting to specific cells in the gonads, a single chain RIP was conjugated to oLH with the use of SPDP which generated oLH-S-S-gelonin hormonotoxin. Extensive physico-chemical, immunochemical and biochemical characterization reveal that 1:1 oLH-gelonin was linked through the alpha-subunit of the oLH. The hormonotoxin retained substantial receptor binding and steroidogenic activity in the leydig tumor cells. The competitive displacement analysis indicate that the binding occurs via the hormone. The presently described hormonotoxin exhibited higher receptor binding and cytotoxicity to the target cells than that of others reported so far.     

Design of liposome to improve encapsulation efficiency of gelonin and its effect on immunoreactivity and ribosome inactivating property

Gelonin, purified from the seeds of Gelonium multiflorum, using cation-exchange and gel-filtration chromatography was characterised for its purity, homogeneity and molecular weight by reverse-phase HPLC (RP-HPLC) and SDS-PAGE analysis. The HPLC purified gelonin was used for entrapment studies in the liposomes. Liposomes were prepared by reverse phase evaporation (REV) technique using three different types of lipid composition in the same molar ratio. The method resulted in 75–80% entrapment efficiency of gelonin in the liposomes. Entrapped and unentrapped gelonin was characterized for physico-chemical, immunochemical and biological properties. The immunoreactivity of entrapped gelonin was fully preserved but the ribosome-inactivating property was slightly inhibited. The method involved mild conditions, highly reproducible and the liposomes produced appeared to be stable for several months. It has important implications in the development of cell type specific cytotoxic agents where a chemical cross-linking is involved which significantly inhibits both immunoreactivity and ribosome-inactivating ability of the toxin.     

On the contentious sequence and glycosylation motif of the ribosome inactivating plant protein gelonin

The amino acid sequence and the glycosylation motif of the ribosome inactivating protein (RIP) gelonin are identified by Fourier transform ion cyclotron resonance mass spectrometry. Intact gelonin as isolated from the seeds of Gelonium multiflorum consists of at least three different post-translational modified forms: analysis of gelonin peptides as obtained by proteolytic digestion is consistent with the amino acid sequence published by Nolan et al. High resolution mass determination established a glycosylation pattern of GlcNAc2Man(3-5)Xyl. N189 was identified as glycosylation site. The proposed glycan structure is consistent with a standard plant N-glycosylation pattern as found in other RIP. Based on these results we suggest that gelonin is located in the vacuole of Gelonium multiflorum seeds.     

Hormonotoxins. I. Strategy for synthesis of ovine luteinizing hormone--gelonin conjugate bearing the toxin in the beta-subunit

The amino groups in the beta-subunit of ovine luteinizing hormone (oLH) were modified by thiolation using N-succinimidyl-3-(2-pyridyldithio) propionate so that it may be coupled in a disulfide linkage to similarly modified ribosome inactivating protein, gelonin. The modified beta-subunit was able to hybridize with free LH alpha-subunit and the complex retained full biological activity. However, when gelonin was coupled to the beta-subunit, the resulting conformational changes masked or eliminated the sites necessary for intersubunit recognition of the free alpha-subunit. This has important implications for the design in the synthesis of gonadotropin-toxin/drug conjugates.     

Anti-HIV-1 property of trichosanthin correlates with its ribosome inactivating activity

Trichosanthin (TCS) is a type I ribosome inactivating (RI) protein possessing anti-tumor and antiviral activity, including human immunodeficiency virus (HIV). The mechanism of these actions is not entirely clear, but is generally attributed to its RI property. In order to study the relationship between the anti-HIV-1 activity of TCS and its RI activity, three TCS mutants with different RI activities were constructed by using site-directed mutagenesis. The anti-HIV-1 activities of the three mutants were tested in vitro. Results showed that two TCS mutants, namely TCS(M(120-123)), TCS(E160A/E189A), with the greatest decrease in RI activity, lost almost all of the anti-HIV activity and cytopathic effect. Another mutant TCS(R122G), which exhibited a 160-fold decrease in RI activity, retained some anti-HIV activity. The results from this study suggested that RI activity of TCS may have significant contribution to its anti-HIV-1 property.     

Hormonotoxins: the role of positive charge of lysine residue on the immunological,biological and cytotoxic properties of ovine lutropin-S-S-gelonin conjugates

Since the positive charge on the lysine residues plays an important role in the receptor recognition ability of oLH, the hormonotoxin has been synthesised with the use of 2-iminothiolane HC1 (2IT) and N-Succinimidyl-3-(2-pyridyldithio)-propionate (SPDP). The oLH activated with 2IT (oLH-10) was then mixed with SPDP activated gelonin (gelonin-30) in order to obtain a oLH-S-S-gelonin hormonotoxin. The conjugation mixture containing hormonotoxin was purified by gel-filtration chromatography according to the molecular weight and a complete physico-chemical, immunochemical and biochemical analysis were performed. The linkage occured through the -NH₂ groups of -subunit of oLH as judged from RP-HPLC analysis. A 11 (oLH:gelonin) molar ratio was obtained when determined with the use of several techniques. The hormonotoxins retained substantial receptor binding, steroidogenic activity and immunoreactivity. The competitive displacement analysis indicate that the binding occurs via the hormone part leaving the gelonin free which was probed with the gelonin antibodies. The presently described (C150A-02, C160A-02 and C170A-02) hormonotoxins exhibited higher receptor binding and toxicity to the target cells than the hormonotoxins prepared with the use of SPDP only. Therefore it is concluded that higher receptor binding and cytotoxicity may be due to the retention of positive charge on the lysine residues of oLH which was preserved during the conjugation process.Abbreviations BSA Bovine Serum Albumin - CMC Carboxy methyl Cellulose - DTT Dithiothreitol - DMEM Dulbeco's Modified Eagle's Medium - DTNB Ellman's reagent [5,5-dithio-bis-(2-nitrobenzoic acid)] - EDTA Ethylenediaminetetraacetic acid - FPLC Fast Protein Liquid Chromatography - FCA Freund's Complete Adjuvant - FCS Fetal Calf Serum - Gelonin-30 Gelonin modified by SPDP - GnRH Gonadotropin-Releasing Hormone - Gelonin-SPDP SPDP modified derivative of gelonin - HEPES (N-[2-hydroxyethyl] piperazine-N-[-2-ethanesulphonic acid]) - IFA Incomplete Freund's Adjuvant - 2IT 2-Iminothiolane - IODOGEN 1,3,4,6-tetrachloro 3,6-diphenylglycouril - oLH Ovine Luteinizing Hormone - oLH-SPDP SPDP modified derivative of oLH - oLH-10 oLH modified by 2IT - oLH2IT Molar ratio of oLH and 2IT - PDP 2-Pyridyl-dithiopropionate - PAP Pokeweed Antiviral Protein - RIP Ribosome Inactivating Protein - RP-HPLC Reverse-Phase High Performance Liquid Chromatography - RPMI Roswell Park Memorial Institute - RIA Radioimmunoassay - RRA Radioreceptor Assay - SPDP N-Succinimidyl-3(2-pyridyldithio)propionate - SDS-PAGE Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis - TCA Trichloroacetic acid - TFA Trifluroacetic acid     

Hormonotoxins. Effects of modifying the gonadotropin alpha-subunit on the generation of lutropin-toxin conjugates

On the basis of the observation that in the hormonotoxin oLH-gelonin conjugation of the toxin occurs via the alpha-subunit of lutropin, an attempt was made to develop a general method for generation of similar hybrid proteins involving other glycoprotein hormones. In this approach sites suitable for conjugation would be first introduced into the alpha-subunit, hybridized with any native hormone specific beta-subunit of choice (LH-beta, FSH-beta, TSH-beta) following which the toxic component gelonin would be added on in the form of gelonin-S-S-alpha---beta complex. Thus, thiolated lutropin alpha-subunit recombined well with free lutropin beta-subunit, yielding a hybrid which was active in terms of receptor binding, immunoreactivity, and steroidogenic properties. However, subsequent conjugation with thiolated gelonin, a ribosome inactivating protein, resulted in dissociation of beta-subunit from its non-covalent union with the thiolated alpha-subunit. It is concluded that the addition of positively charged gelonin at particular sites on the alpha-subunit led to the destabilization of the lutropin quaternary structure.     

The antileukemic efficacy of an immunotoxin composed of a monoclonal anti-Thy-1 antibody disulfide linked to the ribosome-inactivating protein gelonin

Summary We prepared an immunoconjugate consisting of a monoclonal antibody recognizing the Thy-1 antigen and the ribosome-inactivating protein gelonin linked by a disulfide bond. This immunotoxin preparation was judged to contain less than 5% free antibody or gelonin. It was highly toxic in vitro in an antigen-specific fashion to the Thy-1 expressing RADA leukemia of A/J mice. The IC₅₀ of this preparation on RADA in vitro was 10^–12 M, while the IC₅₀ on the Thy-1 negative S1509a fibrosarcoma of A/J mice was 10^–7 M. The toxicity of this immunoconjugate was also measured in a direct proliferation assay and it was found that a 4-h exposure and a 24-h exposure of RADA cells to a 1 nM concentration of immunotoxin killed 90% and 99.9% of cells, respectively. Furthermore, efficacy in vitro was not due to the intrinsic susceptibility of RADA cells to tis type of immunotoxin, as one prepared with gelonin and an antibody recognizing the TL^a determinant on this leukemia had no efficacy in vitro. Clearance of the anti-Thy-1-gelonin immunoconjugate from the circulation of A/J mice after i.v. injection was rapid, especially during the first 8 h after injection, possibly because of binding to Thy-1 expressing tissue. Delivery of immunoconjugate to ascitic tumor in vivo was substantially better if the immunoconjugate was given by i.p. injection, rather than by the i.v. route. When given either i.v. or i.p. at the time of i.p. tumor inoculation in vivo, the anti-Thy-1-gelonin immunotoxin showed potency in an antigen-specific fashion; while this immunoconjugate prolonged survival and frequently cured RADA-inoculated mice, neither anti-Thy-1 antibody, gelonin, a combination of the two, nor immunotoxin of irrelevant specificity had any significant effect on survival. Anti-Thy-1-gelonin also had no effect on survival of A/J mice inoculated i.p. with S1509a. Furthermore, it was determined that a single i.p. dose of anti-Thy-1-gelonin killed 90% to 99% cells in vivo, and that the immunoconjugate was about as effective in this model as either adriamycin or cytoxan.This work was supported by ImmunoGen Inc. and in part by a grant from the National Institutes of Health, CA-14723     

Fractionation of the ribosome inactivating protein preparations with triazine dyes

Aspergillins are ribosome-inactivating proteins (RIPs), isolated from several strains of Aspergillus. The interaction between Cibacron Blue F3GA and two members of this family, alpha sarcin and mitogillin, and other RIPs of type I, was studied. Alpha sarcin retention depended on pH and ionic strength. By chromatography on Affi-Gel Blue in mild experimental conditions, mitogillin and PAP-I did not interact with the dye, whereas 40% of alpha sarcin and 70-90% of briodin, RTA and gelonin were recovered in the bound fraction. In all cases, the major fraction showed a higher toxicity level in protein synthesis inhibition assays. The unbound alpha sarcin, conjugated with the anti-ovarian carcinoma monoclonal antibody MOv17, showed on OVCA 432 a cytotoxicity which was 900 times higher than that exerted by the alpha sarcin alone.     

Liposome-mediated delivery of ribosome inactivating proteins to cells in vitro

This study describes the liposome-mediated delivery of toxins to a variety of cells in vitro. Gelonin, a potent inhibitor of protein synthesis from Gelonium multiflorum, was delivered to the cytoplasm of TLX5 lymphoma cells most effectively by phosphatidylserine vesicles. These liposomes were also capable of inhibiting protein synthesis in XC (transformed rat fibroblasts) and phytohaemagglutinin-stimulated CBA mouse lymphocytes. Phosphatidylcholine liposomes had no capacity to deliver their contents to the cytoplasm, but the addition of cholesterol to the vesicle membrane resulted in an increased capacity. Delivery events were enhanced further by the addition of mixed bovine brain gangliosides to the membrane in the ratio 5:5:1 phosphatidylcholine/cholesterol/gangliosides. The addition of cholesterol to phosphatidylserine vesicles failed to increase the inhibitory effects of the gelonin liposomes. The A chain of diphtheria toxin encapsulated in phosphatidylserine liposomes had no inhibitory effect on the level of protein synthesis in TLX5 or Daudi cells.     

Flammin and velin: new ribosome inactivating polypeptides from the mushroom Flammulina velutipes

A protein designated flammin and exhibiting a molecular mass of 30kDa, and another protein designated velin and possessing a molecular mass of 19 kDa, were isolated from the fruiting bodies of the edible mushroom Flammulina velutipes. Flammin and velin inhibited translation in a rabbit reticulocyte lysate system with an IC(50) of 1.4 and 2.5 nM, respectively. Flammin demonstrated only a small degree of resemblance in N-terminal sequence to angiosperm type 1 ribosome inactivating proteins (RIPs) such as trichosanthin, alpha-momorcharin and beta-momorcharin but no sequence similarity to other mushroom RIPs. Velin manifested limited sequence homology to the A chain of abrin, a type 2 angiosperm RIP. Neither flammin nor velin showed any ribonuclease or protease activity. Both flammin and velin were unadsorbed on DEAE-cellulose and adsorbed on Affi-gel blue gel and CM-Sepharose. They were separable in gel filtration on Superdex 75 by fast protein liquid chromatography.     

Investigation of ribosome inactivating protein-like activity in tissues of cucurbitaceae plants

• 1.1. Extracts of roots, seeds and fruits of seventeen plant species belonging to Family Cucurbitaceae were examined for the ability to inhibit protein synthesis in rabbit reticulocyte lysate and induce mid-term abortion in mice.
• 2.2. Out of the 22 tissue extracts examined, 16 were found to inhibit protein synthesis by >90%, three caused 65–85% inhibition and 3 caused <25% inhibition.
• 3.3. In general, there was a close correlation between protein synthesis inhibiting activity and mid-term abortifacient activity of the tissue extracts.
• 4.4. SDS-PAGE of the tissue extracts revealed the presence of a Coomassie Blue-stainable band with a mol. wt of ca 30,000. The data suggest that this band is responsible for the protein synthesis inhibiting and mid-term abortifacient activities.

     

Guanidination of ovine luteinizing hormone and effects on activity.

The free amino groups in oLH, oLHalpha and oLHbeta were guanidinated by O-methylisourea. The epsilon-NH2 groups of lysine residues reacted bo substitute these positions in the sequence with the more basic homoarginine residue. The alpha-NH2 groups did not react under the conditions used. Guanidinated oLH or the products of guanidinated oLHalpha + native oLHbeta or guanidinated oLHalpha + guanidinated oLHbeta were inactive in two bioassay systems. Native oLHalpha + guanidinated oLHbeta, however, showed potencies of 39% to 55% of that observed with the native subunit recombinant or native oLH. Possible structural implications for hormone-receptor site interactions are discussed.     

Studies on prolactin. Selective reduction of the disulfide bonds of the ovine hormone.

Methods for selective reduction of the disulfide bonds in ovine prolactin are reported. Cleavage of all three disulfide bonds abolishes biological activity and denatures the hormone. Reduction-carbamidomethylation of one or two of the disulfide bridges does not diminish the biological activities in the pigeon crop-sac and mouse mammary gland bioassays. When compared to the native hormone, monomers of these two partially reduced-carbamidomethylated derivatives also show only modest changes in properties measured by exclusion chromatography, circular dichroism, and immunological cross-reactivities. However, cleavage of cystine-4--11 and cystine-191--199, followed by carbamidomethylation, destroys the biological activity of this derivative in a teleost fish bioassay (Gillichthys urinary bladder). In contrast, reduction of cystine-4--11 actually increased the teleost potency of this derivative compared to the intact hormone. Since teleost prolactin appears to lack a homologue to the cystine-4--11 disulfide bond in the amino-terminal loop of the ovine hormone, selective reduction of this bond in ovine prolactin may produce a derivative whose properties more closely resemble the fish hormone.     

Identification of the catalytic motif of the microbial ribosome inactivating cytotoxin colicin E3

Colicin E3 is a cytotoxic ribonuclease that specifically cleaves 16S rRNA at the ribosomal A-site to abolish protein synthesis in sensitive Escherichia coli cells. We have performed extensive mutagenesis of the 96-residue colicin E3 cytotoxic domain (E3 rRNase), assayed mutant colicins for in vivo cytotoxicity, and tested the corresponding E3 rRNase domains for their ability to inactivate ribosome function in vitro. From 21 alanine mutants, we identified five positions where mutation resulted in a colicin with no measurable cytotoxicity (Y52, D55, H58, E62, and Y64) and four positions (R40, R42, E60, and R90) where mutation caused a significant reduction in cytotoxicity. Mutations that were found to have large in vivo and in vitro effects were tested for structural integrity through circular dichroism and fluorescence spectroscopy using purified rRNase domains. Our data indicate that H58 and E62 likely act as the acid-base pair during catalysis with other residues likely involved in transition state stabilization. Both the Y52 and Y64 mutants were found to be highly destabilized and this is the likely origin of the loss of their cytotoxicity. The identification of important active site residues and sequence alignments of known rRNase homologs has allowed us to identify other proteins containing the putative rRNase active site motif. Proteins that contained this active site motif included three hemagglutinin-type adhesins and we speculate that these have evolved to deliver a cytotoxic rRNase into eukaryotic cells during pathogenesis.