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.     

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     

Nonspecific deadenylation on sarcin/ricin domain RNA catalyzed by gelonin under acidic conditions

Gelonin is a single-chain ribosome-inactivating protein that can hydrolyze the glycosidic bond of a highly conserved adenosine residue in the sarcin/ricin domain (SRD) of the largest RNA in ribosome and thus irreversibly inhibit protein synthesis. Recently, the specificity in substrate recognition was challenged by the fact that gelonin could remove adenines from some other oligoribonucleotide substrates. However, the site specificity of gelonin to deadenylate various substrates were unknown. Hereby, the effect of pH values upon site specificity of the deadenylation activity of gelonin was studied using the synthetic oligoribonucleotide (named SRD RNA) that mimicked the ribosomal SRD. Interestingly, gelonin gradually acquired the ability to nonspecifically remove adenines from SRD RNA when pH values changed from neutral to acidic conditions. Another two SRD RNA mutants, either with the conserved adenosine deleted or with the tetraloop converted, showed very similar cleavage style to wild-type SRD RNA, underscoring the important role of pH value in site specificity of recognition by gelonin. Furthermore, the RNA N-glycosidase activity of gelonin was also enhanced with the decreasing of pH values. In addition, no obvious change was observed in the molecular conformation of gelonin at various pH values. Taken together, our data implied that the protonation of adenosines in SRD RNA was potentially an important factor for the nonspecific deadenlyation by gelonin.     

截短型细胞毒素Gelonin的分子构象和生物活性

为了探索免疫络合物中具杀伤靶细胞的毒素,gelonin的结构与功能的关系,根据化学合成的gelonin基因序列和3维分子构象设计了N端区Gly,Leu,Asp和/或C端区Asp,Lys,Asp,Pro,Lys缺失的gelonin. 以重组质粒pE gel为模板,在相应引物存在下,用PCR法获得5′端区和/或3′端区碱基序列缺失的gelonin基因片段. 经克隆、表达和纯化得到3种截短型gelonin（G-N3、G-C5、G-N3C5）. CD谱和荧光谱表明,完整型gelonin（G-O）与截短型gelonin的分子构象有明显的差异.它们的构象变化与类DNase活性和抑制肿瘤细胞生长的能力均为G-O≥ G-N3＞G C5＞G-N3C5. 结果再一次证明了具有α+β型结构蛋白,gelonin的构象与生物活性的一致性.     

Interaction of alpha-sarcin and gelonin with Cibacron blue

Alpha-sarcin and gelonin, two proteins which inactivate the 60S ribosomal subunit, interact with Cibacron blue and bind to blue dextran-Sepharose, from which they are partially desorbed by nucleoside triphosphates and, more efficiently, by homopolynucleotides. It is further shown that the two proteins bind to poly(U)-Sepharose and that homopolynucleotides protect dilute solutions of both inhibitors from inactivation. It is suggested that the presence of a polynucleotide site on alphasarcin is related to its ribonuclease activity. The existence of a similar site on gelonin might be a clue to its yet unknown mechanism of action.