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.     

Role of positive charge of lysine residue on ribosome-inactivating property of gelonin

The report that gelonin cross-linked with monoclonal antibodies with the use of 2-iminothiolane (2-IT) exhibited higher cytotoxicity than the conjugates prepared with the use of N-succinimidyl-3-(2-pyridylthio) propionate (SPDP) alone, has prompted us to investigate the effect of epsilon-NH2 group modification with 2-IT on the ribosome-inactivating property (RIP) of gelonin. The purified gelonin was modified with 2-IT at a different molar ratio and their effects on immunoreactivity and ribosome-inactivating property were compared with those of N-succinimidyl 6-[3-(2-pyridyldithio) propionamido] hexanoate (long chain-SPDP) and SPDP modified gelonin derivatives. Modification of single amino group with 2-IT results in about 25-50% inhibition of immunoreactivity and 60-70% loss of protein synthesis inhibition activity. Modification of 2-3 amino groups further hampers both immunoreactivity and protein synthesis inhibition property of gelonin. Both the long chain-SPDP with SPDP modifications showed more pronounced effects on immunoreactivity and RIP activity as compared to the similar ratio of 2-IT modification(s). It may, therefore, be concluded that the positive charge plays an important role in the immunological as well as the protein synthesis inhibitory effect of gelonin.     

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. 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.     

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     

Effect of thiolation of amino groups of gelonin on its protein-biosynthesis inhibitor activity.

Gelonin was purified from the dry seeds of Gelonium multiflorum by ammonium sulfate fractionation followed by cation-exchange and gel-filtration chromatography in order to minimize extraction of non-proteineous material. Gelonin was characterized for its purity, homogeneity and molecular weight determination by RP-HPLC and SDS-PAGE analysis respectively. The amino groups of pure gelonin were thiolated by a hererobifunctional cross-linking agent, SPDP which is used in the design of cytotoxic hybrid molecules. Therefore, an attempt has been made to study the effect of thiolation on the ribosome inactivating property of gelonin. Thiolation of one amino group resulted in the loss of about 90% protein synthesis inhibition activity. Further modification of 2-3 amino groups further hampered the bioactivity (greater than 95-99.5%) of gelonin, suggesting that a 1:1 molar ratio of carrier-toxin conjugate would be highly active against the target cells.     

Modification of ovine luteinizing hormone subunits with SMPT and its effect on subunit recombination, immunological activity, receptor binding and steroidogenic activity

The increasing use of heterobifunctional cross-linking agents in the design of defined conjugates for selective targeting and inducing immune response has prompted us to study the role of epsilon-NH2 group modification of oLH subunits, their recombination and effect on immunoreactivity, receptor binding and biological activity. The epsilon-NH2 groups of alpha oLH and beta oLH subunits were separately modified by using SMPT. The alpha oLH-SMPT modified derivatives hybridize to beta oLH. Similarly, the beta oLH-SMPT derivatives recombined with alpha oLH. The recombination was judged by gel filtration chromatography and RP-HPLC analysis. The sequential modification of subunits led to progressive reduction in immunoreactivity and receptor binding activity. The modification of six or more epsilon-NH2 groups in alpha oLH although recombine fully with native beta oLH but failed to react to anti-oLH antibody. Moreover, the steroidogenic activity was also abolished. Introduction upto four SMPT groups in alpha oLH compromised immunological and biological activities but further addition of two or more SMPT groups completely abolished antibody reactivity, receptor binding and steroidogenic activity indicating the importance of later two amino groups in the receptor binding and steroidogenic activity. The present investigation clearly demonstrate that only 1:2-3 molar ratio of oLH subunits:SMPT could generate the site(s) in the subunits of the oLH that retained reasonable immunological, receptor binding and biological activity of the hormone. Therefore, this molar ratio may be used in future for the design and synthesis of bioeffective hormonotoxins.     

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.     

Effects of thiolation on the immunoreactivity of the ribosome-inactivating protein gelonin.

Gelonin purified from the seeds of Gelonium multiflorum using cation-exchange and gel-filtration chromatography was characterized for its purity, homogeneity and Mr by reverse-phase h.p.l.c. and SDS/polyacrylamide-gel electrophoresis analysis and judged to be 98% pure. As the cross-linking agent N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP) has been used for linking gelonin via its epsilon-NH2 group to its carrier antibodies or hormones for immunotoxin or hormonotoxin respectively, an attempt was made to study the effect of this modification of gelonin on its immunoreactivity. A radioimmunoassay was developed for this purpose. By sequential modification, four categories of amino group modifications on immunoreactivity were observed. Even one or two modifications, representing one-twentieth to one-tenth of available epsilon-NH2 groups in the protein caused about 75% loss in immunoreactivity, with additional reactions contributing to further deteriorations. By using a gelonin radioimmunoassay, the immunoreactivity of gelonin in three hormonotoxins was determined with gelonin and modified gelonin as standards. The gelonin equivalent in our hormonotoxins was in agreement with the values determined by spectrophotometric and gel-electrophoresis methods. As the immunoreactivity of gelonin-SPDP was not further altered after conjugation to its carrier protein ovine lutropin, a specific radioimmunoassay of gelonin could be used to evaluate the molar ratio of the conjugates prepared by using SPDP as cross-linker and gelonin-SPDP as a standard.     

Purification,characterization and chemical modification studies on a translation inhibitor protein from Luffa cylindrica

A ribosome-inactivating protein (RIP), luffin has been isolated from the seeds of Luffa cylindrica of Cucurbitaceae family by ammonium sulfate fractionation followed by cation exchange and gel-filtration chromatography. Extensive physico-chemical, immunological and biological characterizations were carried out on luffin and compared with that of gelonin. The molecular mass of luffin was -28 kDa as determined by gel-filtration chromatography and SDS-PAGE. The epsilon-NH2 group(s) of luffin were sequentially modified by N-succinimidyl 6-[3-(2-pyridyldithio) propionamido] hexanoate (LC-SPDP), N-succinimidyl-3-(2-pyridylthio)propionate (SPDP) and 2-iminothiolane (2IT) and their effect on immunoreactivity and ribosome inactivating property was evaluated. Modification of single amino group resulted in about 80% inhibition of immunoreactivity and more than 90% loss of protein synthesis inhibition activity. Modification of 2-3 amino groups further hampered both immunoreactivity and protein-synthesis inhibition property LC-SPDP modification played more pronounced effects on immunoreactivity and RIP activity than that of SPDP. However, 2IT modification retained both the immunoreactivity and RIP activity of luffin-LC-SPDP substantially. SPDP showed more pronounced effect on immunoreactivity and RIP activity as compared to 2IT. Therefore, it seems that the positive charge on lysine residues plays an important role in immunological as well as protein synthesis inhibitory effect of luffin.     

Cytotoxicity of gelonin and its conjugates with antibodies is determined by the extent of their endocytosis

Conjugates of the single-chain ribosome-inactivating protein gelonin with ligands that bind to cell surface molecules vary greatly in their cytotoxicity. Conjugates that are not endocytosed after binding to cells exhibit low cytotoxicity similar to that of free gelonin, while conjugates that are endocytosed demonstrate enhanced cytotoxicity relative to free gelonin. However, the number of internalized gelonin molecules needed to intoxicate cells to the same degree has been found to be similar for all conjugates and for free gelonin. The intracellular concentration of gelonin has to be between 2,000-10,000 molecules/cells to achieve a surviving fraction of 0.37. Our studies revealed the presence of three distinct categories of cell surface molecules, those that are efficient in mediating endocytosis of immunotoxins, those that are only moderately efficient, and those that seem not to cause internalization of bound immunotoxins.     

Purification and characterisation of gelonin from seeds of Gelonium multiflorum

Gelonin, a ribosome-inactivating protein has been isolated from the seeds of Gelonium multifluorum of Euphorbiaceae family by two methods and the results are compared. In method-I conventional aqueous extraction, cation-exchange and gel-filtration chromatography has been used. In method-II S-Sepharose fast flow gel has been used to purify the proteins from the seed extract, followed by ammonium sulfate fractionation, cation-exchange and gel-filtration chromatography. Extensive physico-chemical and immunological characterizations show that molecular weight of gelonin as determined by gel-filtration chromatography and SDS-PAGE is approximately 30 kDa. The non-proteinous material which binds to CMC-gel in association with gelonin in method-I is substantially removed when gelonin is purified by method-II. Cation exchange, G-100 chromatography, RP-HPLC and SDS-PAGE show that method-II yields 50% more purified gelonin when compared to the yield by method-I. The immunoreactivity of gelonin obtained by methods I and II vary from 22-26% and 50-66% respectively and the ribosome-inactivating property vary from 46-56% and 70-87% respectively.     

Release of gelonin from endosomes and lysosomes to cytosol by photochemical internalization

Gelonin, a type I ribosome-inactivating plant toxin, executes N-glycosidase activity on eukaryotic ribosomes. However, on intact cells, gelonin is relatively non-toxic, due to an incapability to penetrate cell membranes. Recently, a novel method, photochemical internalization (PCI), was invented for the translocation of membrane-impermeable molecules including gelonin to the cytosol [K. Berg et al., Cancer Res. 59 (1999) 1180-1183]. The combination of gelonin and photoactivation of endosomal and lysosomal localizing photosensitizers gives strong synergistic cytotoxic effects. In this study, we have evaluated the intracellular transport and stability of gelonin. By fluorescence microscopy, it was shown that gelonin co-localizes with the endosomal and lysosomal localizing photosensitizer, aluminum phthalocyanine with two sulfonate groups on adjacent phenyl rings, and both molecules re-localized to cytosol subsequently to light exposure. Gelonin accumulated in endosomal compartments by incubation at 18 degrees C was released to cytosol by PCI with concomitant inhibition of protein synthesis indicating that PCI can be executed through rupture of endosomal vesicles. The cathepsin inhibitor L-trans-epoxysuccinyl-leucyl amido(4-guanido)butane increased the cytotoxic effect of gelonin after PCI when gelonin was provided as a 2 h pulse followed by 4 h chase before PCI. Thus, although gelonin can enter the cytosol from lysosomes, lysosomal degradation is a limiting factor for the outcome of PCI of gelonin.     

Trichosanthin, alpha-momorcharin and beta-momorcharin: identity of abortifacient and ribosome-inactivating proteins

The abortifacient proteins trichosanthin, alpha-momorcharin and beta-momorcharin at nM concentrations inhibit cell-free protein synthesis. The momorcharins and the ribosome-inactivating proteins isolated from Momordica charantia seeds cross-react with the respective antisera. The ribosome-inactivating proteins saporins, pokeweed antiviral protein (PAP) and, to a lesser extent, gelonin have abortifacient activity on pregnant mice.     

Disulfide linked αoLH-gelonin conjugate failed to recombine with βoLH subunit to generate bioeffective hormonotoxin

Since, linking of ovine luteinizing hormone (oLH) to ribosome inactivating protein gelonin (in oLH-gelonin conjugate) occur via the alpha-subunit, oLH, an attempt has been made to develop a universal hormonotoxin for selective targeting to specific cells in the gonads. Four different molar ratios of oLH and N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP) were used to activate the epsilon amino (-NH₂) groups of oLH. The oLH-SPDP derivatives recombine to native beta subunit of oLH (oLH) and the purified recombinants retained substantial receptor binding, steroidogenic activity and immunoreactivity to native oLH. The disulfide linked oLH-S-S-gelonin conjugates prepared by SPDP method were purified by gel filtration chromatography and analysed by reverse-phase high performance liquid chromatography (RP-HPLC). In order to obtain specificity and bioeffectivity, the oLH-S-S-gelonin conjugates were allowed to recombine to native oLH and the recombination mixture was further purified by gel-filtration chromatography. The RP-HPLC analysis of these recombinants indicated that oLH-S-S-gelonin did not recombine to oLH. The failure of recombination may be due to the reasons. (i) The site of -NH₂ activation by SPDP may be different in the oLH than the native oLH. (ii) The activation site may be in close proximity to the annealing site which facilitates the recombination of -subunit but failured to reassociate to oLH-S-S-gelonin conjugate. (iii) The introduction of gelonin (30 kDa basic protein) might have induced some steric hinderence for oLH to recombine to the oLH site which might have been masked in oLH-S-S-gelonin conjugates. (Mol Cell Biochem120: 95–102, 1993)Abbreviations oLH ovine Luteinizing Hormone - oLH alpha subunit of oLH - oLH beta subunit of oLH - BSA Bovine Serum Albumin - DTT Dithiothreitol - RP-HPLC Reverse Phase High Performance Liquid Chromatography - TSH Thyroid Stimulating Hormone - FSH Follicle Stimulating Hormone - LH Luteinizing Hormone - eCG equine Chorionic Gonadotropin - DMEM Dulbecco's Modified Eagles Medium - HEPES 4-(2-Hydroxyethyl)-1 Piperazine Ethane Sulfonic acid - PAP Pokeweed Antiviral Protein - RIA Radioimmunoassay - hCG human Chorionic Gonadotropin - TRH Thyrotropin Releasing Hormone - CRF Corticotropin Releasing Factor - hPL human Placental Lactogen - TFA Trifluroacetic Acid - oLH-SPDP SPDP activated derivative of oLH     

Quantitative analysis of protein synthesis inhibition and recovery in CRM107 immunotoxin-treated HeLa cells

Previously a mathematical model was proposed that quantitatively related protein synthesis inhibition kinetics of antitransferrin receptor-gelonin immunotoxins to the cellular trafficking of the targeting agent. That work is here extended to describe protein synthesis inhibition kinetics of immunotoxins containing the diphtheria toxin mutant CRM107. CRM107 differs from gelonin in both translocation and ribosomal inactivation mechanisms. Targeting agents used were antitransferrin monoclonal antibodies 5E9 and OKT9, OKT9Fab, and transferrin. CRM107 conjugates inhibited protein synthesis at substantially lower concentrations than gelonin conjugates; this effect was attributed to substantially higher translocation rates for CRM107. However, under certain conditions, CRM107 immunotoxin-treated cells were able to recover completely; this behavior was never observed with gelonin immunotoxins. To quantitatively capture this phenomenon, extracellular and cytosolic degradation of the toxin as well as growth-related recovery from toxin-induced damage were incorporated into the mathematical model. Translocation and cytosolic degradation rate constants were determined for each immunotoxin. Unlike the gelonin conjugates, the translocation rate of CRM107 conjugates depended on the targeting molecule. This provided indirect evidence that CRM107 remains disulfide linked to the targeting agent for at least part of the translocation process. Although the CRM107 conjugates all had higher translocation rates and inhibited protein synthesis at lower concentrations than the gelonin conjugates, the cells' ability to recover from protein synthesis inhibition at low immunotoxin concentrations limits the utility of CRM107 conjugates for targeted cell killing.     

Cytotoxicity acquired by ribosome-inactivating proteins carried by reconstituted Sendai virus envelopes

Association of the ribosome-inactivating proteins (RIPs): pokeweed antiviral protein (PAP), gelonin, Momordica charantia inhibitor (MCI), with reconstituted Sendai virus envelopes (RSVE) was obtained without detectable loss of activities either of RIPs or of viral envelope glycoproteins. RIPs are inactive towards intact cells, but, once encapsulated in RSVE, they become cytotoxic. The concentration of RSVE-associated PAP, which causes 50% inhibition of protein synthesis by Friend erythroleukemic cells, is 0.5 ng/ml. Substances capable to inhibit the viral activities block the acquired cytotoxicity of RIPs associated to RSVE.     

Uptake and intracellular fate of gelonin,a ribosome-inactivating protein,in rat liver

Gelonin, a type 1 ribosome-inactivating protein, has been used as toxin conjugate for several therapeutic purposes. We have investigated the endocytosis of gelonin by rat liver in vivo. Subcellular distribution of [125I]gelonin was established after differential and isopycnic centrifugation. Fractions were analyzed for acid-soluble and acid-precipitable radioactivity. Results show that gelonin is rapidly cleared from the blood and within 15min reaches a peak (25% of total injected) in the liver. With time, radioactivity associated with the liver markedly decreases. Two important observations are made: (a) Radioactivity associated with all fractions, at any time point, is greater than 80% acid precipitable. (b) Even at 5min, a significant amount of intact gelonin is present in the cytosolic fraction. Our work suggests that, though gelonin is rapidly cleared from the blood, there are still intact molecules that have entered the cytosol where they could exert their toxic effect.     

Cofactor requirement of ribosome-inactivating proteins from plants

A large number of type 1 ribosome-inactivating proteins (RIPs)from plants (families of Caryophyllaceae, Cucurbitaceae, Euphorbiaceae,Phytolaccaceae, and Poaceae) were examined for their requirementfor ATP and supernatant factors for full activity. A markedrequirement was observed with agrostin among Caryophyllaceae,gelonin among Euphorbiaceae, and with both barley RIP and tritin-Samong Poaceae. The distribution of cofactor requirement in Phytolaccaceaediscriminates leaf forms (cofactor-independent) from seed androot forms (cofactor-dependent). The results are discussed onthe basis of the present knowledge on the tissue localizationof RIPs and on the sensitivity of ribosomes to conspecific RIPs. Key words: Cofactors, ribosome-inactivating proteins, RNA-N-glycosidase, up-regulation