Isolation and enzymatic characterization of lamjapin, the first ribosome-inactivating protein from cryptogamic algal plant (Laminaria japonica A). (25/139)

Lamjapin, a novel type Iota ribosome-inactivating protein, has been isolated from kelp (Laminaria japonica A), a marine alga. This protein has been extensively purified through multiple chromatography columns. With a molecular mass of approximately 36 kDa, lamjapin is slightly larger than the other known single-chain ribosome-inactivating proteins from the higher plants. Lamjapin can inhibit protein synthesis in rabbit reticulocyte lysate with an IC50 of 0.69 nm. It can depurinate at multiple sites of RNA in rat ribosome and produce the diagnostic R-fragment and three additional larger fragments after the aniline reaction. Lamjapin can deadenylate specifically at the site A20 of the synthetic oligoribonucleotide (35-mer) substrate that mimics the sarcin/ricin domain (SRD) of rat ribosomal 28S RNA. However, it cannot hydrolyze the N-C glycosidic bond of guanosine, cytidine or uridine at the corresponding site of the A20 of three mutant SRD RNAs. Lamjapin exhibits the same base and position requirement as the ribosome-inactivating proteins from higher plants. We conclude that lamjapin is an RNA N-glycosidase that belongs to the ribosome-inactivating protein family. This study reports for the first time that ribosome-inactivating protein exists in the lower cryptogamic algal plant.  (+info)

Interaction of glycophorin A with lectins as measured by surface plasmon resonance (SPR). (26/139)

Glycophorin A (GPA), the major sialoglycoprotein of the human erythrocyte membrane, was isolated from erythrocytes of healthy individuals of blood groups A, B and O using phenol-water extraction of erythrocyte membranes. Interaction of individual GPA samples with three lectins (Psathyrella velutina lectin, PVL; Triticum vulgaris lectin, WGA and Sambucus nigra I agglutinin SNA-I) was analyzed using a BIAcore biosensor equipped with a surface plasmon resonance (SPR) detector. The experiments showed no substantial differences in the interaction between native and desialylated GPA samples originating from erythrocytes of either blood group and each of the lectins. Desialylated samples reacted weaker than the native ones with all three lectins. PVL reacted about 50-fold more strongly than WGA which, similar to PVL, recognizes GlcNAc and Neu5Ac residues. SNA-I lectin, recognizing alpha2-6 linked Neu5Ac residues, showed relatively weak reaction with native and only residual reaction with desialylated GPA samples. The data obtained show that SPR is a valuable method to determine interaction of glycoproteins with lectins, which potentially can be used to detect differences in the carbohydrate moiety of individual glycoprotein samples.  (+info)

The cytotoxic activity of ribosome-inactivating protein saporin-6 is attributed to its rRNA N-glycosidase and internucleosomal DNA fragmentation activities. (27/139)

Saporin-6 produced by the plant Saponaria officinalis belongs to the family of single chain ribosome-inactivating proteins. It potently inhibits protein synthesis in eukaryotic cells, by cleaving the N-glycosidic bond of a specific adenine in 28 S rRNA, which results in the cell death. Saporin-6 has also been shown to be active on DNA and induces apoptosis. In the current study, we have investigated the roles of rRNA depurination and the activity of saporin-6 on genomic DNA in its cytotoxic activity. The role of putative active site residues, Tyr(72), Tyr(120), Glu(176), Arg(179), and Trp(208), and two invariant residues, Tyr(16) and Arg(24), proposed to be important for structural stability of saporin-6, has been investigated in its catalytic and cytotoxic activity. These residues were mutated to alanine to generate seven mutants, Y16A, R24A, Y72A, Y120A, E176A, R179A, and W208A. We show that for the RNA N-glycosidase activity of saporin-6, residues Tyr(16), Tyr(72), and Arg(179) are absolutely critical; Tyr(120) and Glu(176) can be partially dispensed with, whereas Trp(208) and Arg(24) do not appear to be involved in this activity. The residues Tyr(72), Tyr(120), Glu(176), Arg(179), and Trp(208) were found to be essential for the genomic DNA fragmentation activity, whereas residues Tyr(16) and Arg(24) do not appear to be required for the DNA fragmentation. The study shows that saporin-6 possesses two catalytic activities, namely RNA N-glycosidase and genomic DNA fragmentation activity, and for its complete cytotoxic activity both activities are required.  (+info)

Antitumor effects of curcin from seeds of Jatropha curcas. (28/139)

AIM: To study the antitumor effects of curcin from Jatropha curcas. METHODS: Antitumor activity of curcin was tested by MTT assay. The N-glycosidase activity of curcin was determined by characterization of R-fragment in gel. A cell-free system, rabbit reticulocyte lysate, was introduced to quantify the inhibitory activity of curcin on protein biosynthesis. RESULTS: The curcin had a powerful inhibitory action upon protein synthesis in reticulocyte lysate with an IC50 (95 % confidence limits) value of 0.19 (0.11-0.27) nmol/L. The IC50 (95 % confidence limits) of curcin on SGC-7901, Sp2/0, and human hepatoma was 0.23 (0.15-0.32) mg/L, 0.66 (0.35-0.97) mg/L, 3.16 (2.74-3.58) mg/L, respectively. Curcin was found to have no toxic to Hela cells and normal cells (MRC). After the rRNA of ribosome was treated with curcin and aniline at acidic condition, a cleaved R-fragment of approximately 450 nt appeared, but this fragment did not occur after treatment with curcin only. A comparison of the amino acid sequences of curcin, ricin A-chain and trichosanthin revealed that there were relatively high similarities among them. The percentages of homology between curcin and ricin A chain, between curcin and trichosanthin were found to be 54 % and 57 % respectively. Especially, the conserved residues forming the active sites of the A chain of ricin and trichosanthin occurred in curcin. CONCLUSION: Curcin has an obvious antitumor effect and its mechanisms are related to the N-glycosidase activity.  (+info)

Both N- and C-terminal regions are essential for cinnamomin A-chain to deadenylate ribosomal RNA and supercoiled double-stranded DNA. (29/139)

Cinnamomin is a type II ribosome-inactivating protein and its A-chain exhibits RNA N-glycosidase activity to remove an adenine in the conserved sarcin/ricin loop of the largest RNA in ribosome, arresting protein synthesis at the elongation step. In this report, deadenylation of both rRNA and supercoiled DNA by native and recombinant cinnamomin A-chain expressed in Escherichia coli was demonstrated. However, the mutants of cinnamomin A-chain devoid of N-terminal 52 or/and C-terminal 51 amino acid residues lost both the activity of RNA N-glycosidase and the ability to release adenines from supercoiled DNA. Additionally, supercoiled DNA could not be cleaved into nicked and linear forms by these mutants. These results indicate that both N- and C-terminal regions are essential for the cinnamomin A-chain to deadenylate rRNA and supercoiled DNA. It was suggested that phosphodiester bonds in the extensively deadenylated region of supercoiled DNA would become fragile and liable to be broken spontaneously owing to the existence of tension in the supercoiled DNA.  (+info)

Preparation of a stable subunit of Japanese elderberry (Sambucus sieboldiana) bark lectin and its application for the study of cell surface carbohydrates by flow cytometry. (30/139)

A stable subunit of Sambucus sieboldiana bark lectin (MSSA) was prepared by selective reduction of disulfide bridges between the subunits and alkylation with 4-vinylpyridine. Amino acid analysis of MSSA revealed that 1.4 cysteine residues per subunit were selectively modified. MSSA failed to agglutinate rabbit erythrocytes and precipitate fetuin. However, MSSA retained the ability to bind to fetuin, as detected by ELISA. Neu5Ac alpha 2-6lactose inhibited the binding to fetuin of both SSA and MSSA. Flow cytometric analysis showed that human histocytic lymphoma U937 cells were clearly stained with FITC-labeled MSSA (FITC-MSSA) without any detectable agglutination and that this staining was almost completely inhibited by the addition of Neu5Ac alpha 2-6lactose (2 mM). Treatment of U937 cells with native FITC-SSA at the sub-agglutinating concentration (0.3 microgram/ml) showed much poorer fluorescence intensity than that of MSSA, suggesting that MSSA is an invaluable tool for the detection of cell surface glycoconjugates containing NeuAc alpha 2-6Gal/GalNAc sequences by flow cytometry.  (+info)

The interaction of elderberry (Sambucus sieboldiana) bark lectin and sialyloligosaccharides as detected by 1H-NMR. (31/139)

The interaction of Japanese elderberry bark lectin (Sambucus sieboldiana agglutinin, SSA) with carbohydrate was investigated by 1H-NMR. When a low affinity ligand, methyl beta-D-galactoside (beta MeGal), was mixed with SSA, each proton signal of beta MeGal was broadened. The signal of H-4 was markedly broad, while those of H-1, OCH3, and H-2 of beta MeGal were rather sharp. The specific broadening of Gal H-4 was more evident when SSA was mixed with methyl-beta-D-lactoside (beta MeLac). Position-dependent signal broadening suggests that beta MeGal binds to SSA such that H-4 is closely involved in the contact region, but H-1, OCH3, and H-2 are far from this region. In the case of a high affinity ligand, Neu5Ac(alpha 2-6)Gal(beta 1-4)Glc(= N6L), ligand signals of the SSA-N6L mixture did not change at all. But when a small amount of N6L was added to the SSA-beta MeGal mixture, the broad signals of bound beta MeGal became dramatically sharp. This indicates that the added N6L molecules liberated the bound beta MeGal from SSA. On the other hand, the sialyllactose with the alpha(2-3)-linkage(= N3L) could not substitute for bound beta MeGal because of its lower affinity. This demonstrates that the competitive binding experiment between two ligands is a useful technique to detect the interaction of lectins with high affinity ligands which could not be observed directly by NMR signal broadening and/or chemical shift change.  (+info)

Purification and characterization of Moschatin, a novel type I ribosome-inactivating protein from the mature seeds of pumpkin (Cucurbita moschata), and preparation of its immunotoxin against human melanoma cells. (32/139)

A novel ribosome-inactivating protein designated Moschatin from the mature seeds of pumpkin (Cucurbita moschata) has been successively purified to homogeneity, using ammonium sulfate precipitation, CM-cellulose 52 column chromatography, Blue Sepharose CL-6B Affinity column chromatography and FPLC size-exclusion column chromatography. Moschatin is a type 1 RIP with a pI of 9.4 and molecular weight of approximately 29 kD. It is a rRNA N-glycosidase and potently blocked the protein synthesis in the rabbit reticulocyte lysate with a IC50 of 0.26 nM. Using the anti-human melanoma McAb Ng76, a novel immunotoxin Moschatin-Ng76 was prepared successfully and it efficiently inhibited the growth of targeted melanoma cells M21 with a IC50 of 0.04 nM, 1500 times lower than that of free Moschatin. The results implied that Moschatin could be used as a new potential anticancer agent.  (+info)