Which Plant Proteins Are Involved in Antiviral Defense? Review on In Vivo and In Vitro Activities of Selected Plant Proteins against Viruses.

Plants have evolved a variety of defense mechanisms to tackle virus attack. Endogenous plant proteins can function as virus suppressors. Different types of proteins mediate defense responses against plant viruses. Pathogenesis-related (PR) proteins are activated upon pathogen infections or in different stress situations and their production is one of many components in plant defense. Ribosome-inactivating proteins (RIPs) suppress translation by enzymatically damaging ribosomes and they have been found to have antiviral activity. RNA-binding proteins (RBPs) bind to target RNAs via specialized RNA-binding domain and can directly or indirectly function in plant defense system against RNA viruses. Proteins involved in silencing machinery, namely Dicer-like (DCL) proteins, Argonaute (AGO) proteins, and RNA-dependent RNA polymerases (RDRs) confer innate antiviral defense in plants as they are able to degrade foreign RNA of viral origin. This review aims to provide a comprehensive and up-to-date picture of plant proteins participating in antiviral defense. As a result we discuss proteins conferring plant antiviral resistance and their potential future applications in different fields of life including agriculture and medicine.

Increased Avidity of the Sambucus nigra Lectin-Reactive Antibodies to the Thomsen-Friedenreich Antigen as a Potential Biomarker for Gastric Cancer.

AIM: To determine whether the naturally occurring Thomsen-Friedenreich (TF) antigen-specific antibodies differ in avidity between cancer patients and controls to find a novel biomarker for stomach cancer. METHODS: Serum samples were taken from patients with cancer and controls. The level of TF-specific antibodies and their sialylation were determined using ELISA with synthetic TF-polyacrylamide conjugate as antigen and sialic acid-specific Sambucus nigra agglutinin (SNA). The avidity was determined using ammonium thiocyanate as a chaotrope. RESULTS: A significantly higher SNA lectin binding to anti-TF antibodies was found in cancer patients irrespective of disease stage. The avidity of only IgM TF-specific antibodies was significantly higher in cancer patients compared to controls. The SNA-positive anti-TF antibodies of cancer patients showed a significantly higher avidity, P < 0.001. The sensitivity and specificity of this increase for gastric cancer were 73.53% and 73.08%, respectively, with a 73.2% diagnostic accuracy. The higher avidity of SNA-reactive anti-TF antibodies was associated with a benefit in survival of stage 3 cancer patients. CONCLUSION: The SNA-reactive TF-specific antibodies display a significantly higher avidity in gastric cancer patients compared to controls, which can be used as a potential serologic biomarker for gastric cancer. It appears that IgM is the main target responsible for the above changes.

Differential neutralizing activities of a single domain camelid antibody (VHH) specific for ricin toxin's binding subunit (RTB).

Ricin, a member of the A-B family of ribosome-inactivating proteins, is classified as a Select Toxin by the Centers for Disease Control and Prevention because of its potential use as a biothreat agent. In an effort to engineer therapeutics for ricin, we recently produced a collection of alpaca-derived, heavy-chain only antibody VH domains (VHH or "nanobody") specific for ricin's enzymatic (RTA) and binding (RTB) subunits. We reported that one particular RTB-specific VHH, RTB-B7, when covalently linked via a peptide spacer to different RTA-specific VHHs, resulted in heterodimers like VHH D10/B7 that were capable of passively protecting mice against a lethal dose challenge with ricin. However, RTB-B7 itself, when mixed with ricin at a 1 ∶ 10 toxin:antibody ratio did not afford any protection in vivo, even though it had demonstrable toxin-neutralizing activity in vitro. To better define the specific attributes of antibodies associated with ricin neutralization in vitro and in vivo, we undertook a more thorough characterization of RTB-B7. We report that RTB-B7, even at 100-fold molar excess (toxin:antibody) was unable to alter the toxicity of ricin in a mouse model. On the other hand, in two well-established cytotoxicity assays, RTB-B7 neutralized ricin with a 50% inhibitory concentration (IC50) that was equivalent to that of 24B11, a well-characterized and potent RTB-specific murine monoclonal antibody. In fact, RTB-B7 and 24B11 were virtually identical when compared across a series of in vitro assays, including adherence to and neutralization of ricin after the toxin was pre-bound to cell surface receptors. RTB-B7 differed from both 24B11 and VHH D10/B7 in that it was relatively less effective at blocking ricin attachment to receptors on host cells and was not able to form high molecular weight toxin:antibody complexes in solution. Whether either of these activities is important in ricin toxin neutralizing activity in vivo remains to be determined.

Lectin-dependent inhibition of antigen-antibody reaction: application for measuring α2,6-sialylated glycoforms of transferrin.

We developed a high-throughput Enzyme-linked immunosorbent assay (ELISA) for measuring α2,6-sialylated transferrin (Tf), based on inhibition of anti-Tf antibody binding to α2,6-sialylated Tf by a lectin, Sambucus sieboldiana Agglutinin (SSA). The inhibition was not observed with other glycoforms, such as periodate-treated, sialidase-treated and sialidase/galactosidase-treated Tf, suggesting that the assay was glycoform specific. This finding was applied to an automated latex-agglutination immunoassay, using SSA lectin as an inhibitor (SSA-ALI). The concentration of α2,6-sialylated Tf measured by SSA-ALI in human cerebrospinal fluid was correlated with that of ELISA (r2 = 0.8554), previously developed for measuring α2,6-sialylated Tf.

Enhanced pest resistance of maize leaves expressing monocot crop plant-derived ribosome-inactivating protein and agglutinin.

Although many insect resistance genes have been identified, the number of studies examining their effects in combination using transgenic systems is limited. This study introduced a construct into maize containing the coding sequence for maize ribosome-inactivating protein (MRIP) and wheat germ agglutinin (WGA). Many transformants produced both the MRIP and WGA in leaves. Mature leaves expressing higher levels of these two proteins were more resistant to feeding by first-instar larvae of fall armyworms (Spodoptera frugiperda) and corn earworms (Helicoverpa zea), and the level of resistance was correlated with levels of MRIP and WGA. There was also some indication that resistance to Fusarium verticillioides was increased in the transgenic plant leaves. No statistically significant synergism or antagonism occurred between the activities of the two proteins. MRIP and WGA represent compatible class examples of food plant-derived proteins for multigene resistance to insects.

Transient injury-dependent up-regulation of CD105 and its specific targeting with an anti-vascular anti-mouse endoglin-nigrin b immunotoxin.

Endoglin (CD105), a cell-surface co-receptor for transforming growth factor-beta (TGF-ß) superfamily members, is over-expressed in tumor neovasculature and can be targeted with anti-endoglin antibodies, thus becoming an important tool for anti-tumoral therapy. Injury of the mouse tail induced the transient expression of endoglin, this peaking at three days after injury and disappearing six days later. An immunotoxin containing the anti-mouse endoglin rat monoclonal antibody MJ7/18 and the non-toxic ribosome-inactivating protein nigrin b (Ngb) was found to be very active in targeting mouse endoglin in the L929 fibroblast cell line (IC(50) of 4 x 10(-11) M). At that concentration, the immunotoxin lacked unspecific activity. Upon induction of endoglin after injury, the MJ7-Ngb immunotoxin strongly attacked and deranged the injured tail, inducing tissue damage. Such effects were dependent on the age of the animals and were evident in six-week-old mice, but not in eight-month-old mice. Our results indicate that endoglin is up-regulated in newly formed vessels upon injury and can be targeted by the MJ7-Ngb immunotoxin; thus, it could be a useful tool for tumor ablation research.

Preparation of an antitumor and antivirus agent: chemical modification of α-MMC and MAP30 from Momordica Charantia L. with covalent conjugation of polyethyelene glycol.

BACKGROUND: Alpha-momorcharin (α-MMC) and momordica anti-HIV protein (MAP30) derived from Momordica charantia L. have been confirmed to possess antitumor and antivirus activities due to their RNA-N-glycosidase activity. However, strong immunogenicity and short plasma half-life limit their clinical application. To solve this problem, the two proteins were modified with (mPEG)(2)-Lys-NHS (20 kDa). METHODOLOGY/PRINCIPAL FINDINGS: In this article, a novel purification strategy for the two main type I ribosome-inactivating proteins (RIPs), α-MMC and MAP30, was successfully developed for laboratory-scale preparation. Using this dramatic method, 200 mg of α-MMC and about 120 mg of MAP30 was obtained in only one purification process from 200 g of Momordica charantia seeds. The homogeneity and some other properties of the two proteins were assessed by gradient SDS-PAGE, electrospray ionization quadruple mass spectrometry, and N-terminal sequence analysis as well as Western blot. Two polyethylene glycol (PEG)ylated proteins were synthesized and purified. Homogeneous mono-, di-, or tri-PEGylated proteins were characterized by matrix-assisted laser desorption ionization-time of flight mass spectrometry. The analysis of antitumor and antivirus activities indicated that the serial PEGylated RIPs preserved moderate activities on JAR choriocarcinoma cells and herpes simplex virus-1. Furthermore, both PEGylated proteins showed about 60%-70% antitumor and antivirus activities, and at the same time decreased 50%-70% immunogenicity when compared with their unmodified counterparts. CONCLUSION/SIGNIFICANCE: α-MMC and MAP30 obtained from this novel purification strategy can meet the requirement of a large amount of samples for research. Their chemical modification can solve the problem of strong immunogenicity and meanwhile preserve moderate activities. All these findings suggest the potential application of PEGylated α-MMC and PEGylated MAP30 as antitumor and antivirus agents. According to these results, PEGylated RIPs can be constructed with nanomaterials to be a targeting drug that can further decrease immunogenicity and side effects. Through nanotechnology we can make them low-release drugs, which can further prolong their half-life period in the human body.

Alpha-momorcharin possessing high immunogenicity, immunotoxicity and hepatotoxicity in SD rats.

UNLABELLED: Momordica charantia L., a genus of Momordica Linn. of the family Cucurbitaceae, commonly known as bitter melon, has been widely planted in China, Southeast Asia, Turkey and other areas, and has been used as a medicine for a long time. Alpha-momorcharin (α-MMC) extracted and purified from bitter melon seeds has significant anti-tumor and anti-virus effects, and has potential toxicity as well, especially when taken overdose. However, up to date studies on its safety evaluation are still insufficient. AIMS OF THE STUDY: The immunogenicity, immunotoxicity and general toxicity of α-MMC were investigated in rats and guinea-pigs, and the potential toxic effects of the agent on the body were also examined. MATERIALS AND METHODS: The major ribosome-inactivating protein was isolated by column chromatographies from the protein extracted from bitter melon seeds, and was verified as α-MMC. After rats were immunized by α-MMC, titers of specific antibody to α-MMC in immunized rats serum were detected by indirect ELISA. Guinea-pigs and rats immunized with α-MMC were used to evaluate the active systemic anaphylaxis and passive cutaneous anaphylaxis induced by α-MMC relatively. α-MMC of 6.25 mg/kg, 2.08 mg/kg and 0.70 mg/kg was administered to rats every 2 days. Five weeks later, animals were sacrificed, and then, biochemical examination, analysis of bone marrow and peripheral blood cells, and histopathologic examination were performed. RESULTS: The ribosome-inactivating protein isolated and purified from bitter melon seeds was identified as α-MMC. It induced high titer (1:46.4) of specific IgG and high positive results of the active systemic anaphylaxis and passive cutaneous anaphylaxis tests in animals. With the time of the α-MMC administration increasing, the body weights of the animals administered with α-MMC of 6.25 mg/kg decreased significantly, and point necrosis was also observed in liver cells, along with abnormal findings in serum chemistry, hematology and bone marrow histopathology test. The toxic effect lessened with the decrease of the dose of α-MMC and further reduced after the convalescence stage. CONCLUSIONS: The results of the study show that α-MMC has high immunogenicity and immunotoxicity, and can cause obvious organic liver lesion.

[Preparation and identification of monoclonal antibody against alpha-momorcharins].

Three BALB/c mice were immunized four times with alpha-momorcharins (alpha-MMC). Using polyethylene glycol (PEG) method, the immunized splenocytes were fused with SP2/0 cells. One strain of hybridoma cells was obtained which secrete antibodies against alpha-MMC. To get ascites, the hybridoma cells were injected into the abdominal cavity of mice. The antibodies were purified from ascites. Indirect enzyme linked immunosorbent assay (ELISA) and Western blot assay were applied to determine the specifity of the monoclonal antibody (McAb). The results showed that the McAb was specific to alpha-MMC without detectable cross-activity with MAP30. The McAb provided detecting method for further research of the structure and function of alpha-MMC.

PEGylation of alpha-momorcharin: synthesis and characterization of novel anti-tumor conjugates with therapeutic potential.

Alpha-momorcharin (alpha-MMC) is a ribosome-inactivating protein (RIP) with excellent cytotoxicity to tumor cells. However, its strong immunogenicity and short plasma half-life limit its clinical applications. To overcome this, we have to PEGylated alpha-MMC using a branched 20 kDa (mPEG) (2)-Lys-NHS. Homogeneous mono-, di- and tri-PEGylated alpha-MMCs were synthesized, purified and characterized. In vitro and in vivo analysis indicated that the serial PEG-conjugates preserved moderate anti-tumor activity with 36% acute toxicity and at most 66% immunogenicity decrease. These results suggested the potential application of alpha-MMC-PEG conjugates as an anti-tumor agent.

Ribosome-inactivating proteins: from plant defense to tumor attack.

Ribosome-inactivating proteins (RIPs) are EC3.2.32.22 N-glycosidases that recognize a universally conserved stem-loop structure in 23S/25S/28S rRNA, depurinating a single adenine (A4324 in rat) and irreversibly blocking protein translation, leading finally to cell death of intoxicated mammalian cells. Ricin, the plant RIP prototype that comprises a catalytic A subunit linked to a galactose-binding lectin B subunit to allow cell surface binding and toxin entry in most mammalian cells, shows a potency in the picomolar range. The most promising way to exploit plant RIPs as weapons against cancer cells is either by designing molecules in which the toxic domains are linked to selective tumor targeting domains or directly delivered as suicide genes for cancer gene therapy. Here, we will provide a comprehensive picture of plant RIPs and discuss successful designs and features of chimeric molecules having therapeutic potential.

A close look at human IgG sialylation and subclass distribution after lectin fractionation.

Polyspecific human IgG preparations are indicated for the treatment of primary immunodeficiency disorders associated with defects in humoral immunity. In addition, intraveneous IgG (IVIG) is used to treat patients with autoimmune and systemic inflammatory diseases. Lectin chromatography on Sambucus nigra agglutinin stood at the cradle of the hypothesis that the anti-inflammatory properties depend on sialylation of the N-glycans in the Fc region of IgG. A detailed analysis of fractions obtained by lectin chromatography revealed that binding of IVIG is essentially mediated by Fab glycosylation. Moreover, experiments with a monoclonal antibody from a human cell line and IVIG Fc fragments indicated that at least two sialic acids in the Fc region of an antibody are required for lectin binding. Such glycoforms contain either two monosialylated glycans or a disialylated glycan and constitute 1% or less of the total human IgG. Arguably this small proportion holds the entire anti-inflammatory potency. A new mass spectrometric quantification method of IgG subclass ratio revealed that the IVIG Fc preparation essentially consists of IgG1. This observation may be relevant when studying the effect of human Fc in murine models of inflammation because mouse IgG subclasses differ substantially in their interaction with receptors.

Effect of site-directed PEGylation of trichosanthin on its biological activity, immunogenicity, and pharmacokinetics.

Trichosanthin (TCS) is a type I ribosome-inactivating protein (RIP) with multiple biological and pharmacological activities. It has been approved effective in the clinical treatment of AIDS and tumor, but its strong immunogenicity and short plasma half-life have limited the clinical administration. To reduce the immunogenicity and prolong the plasma half-life of this compound, three TCS muteins (M(1), M(2) and M(3)) and two PEGylated TCS muteins (PM(1) and PM(2)) were constructed by site-directed mutagenesis and PEGylation, respectively. Compared with the unmodified TCS, both PEGylated TCS showed a 3- to 4-fold decrease in immunogenicity, a 0.5- to 0.8-fold decrease in non-specific toxicity, and a 4.5- to 6-fold increase in plasma half-life. But there is a problem of activity reduction. The increased circulating half-life in vivo may compensate for the reduced activity. Together with the other benefits of PEGylation such as reduced immunogenicity and toxicity, it is worthwhile to further explore the potential application of the PEGylated TCS as a better therapeutic agent for AIDS and tumor.

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.