Targeting Aminoglycoside Acetyltransferase Activity of Mycobacterium tuberculosis (H37Rv) Derived Eis (Enhanced Intracellular Survival) Protein with Quercetin.

Eis (Enhanced intracellular survival) protein is an aminoglycoside acetyltransferase enzyme classified under the family - GNAT (GCN5-related family of N-acetyltransferases) secreted by Mycobacterium tuberculosis (Mtb). The enzymatic activity of Eis results in the acetylation of kanamycin, thereby impairing the drug's action. In this study, we expressed and purified recombinant Eis (rEis) to determine the enzymatic activity of Eis and its potential inhibitor. Glide-enhanced precision docking was used to perform molecular docking with chosen ligands. Quercetin was found to interact Eis with a maximum binding affinity of -8.379 kcal/mol as compared to other ligands. Quercetin shows a specific interaction between the positively charged amino acid arginine in Eis and the aromatic ring of quercetin through π-cation interaction. Further, the effect of rEis was studied on the antibiotic activity of kanamycin A in the presence and absence of quercetin. It was observed that the activity of rEis aminoglycoside acetyltransferase decreased with increasing quercetin concentration. The results from the disk diffusion assay confirmed that increasing the concentration of quercetin inhibits the rEis protein activity. In conclusion, quercetin may act as a potential Eis inhibitor.

Antitumor Activity of Taxol Engross Taxol-Caveolin-1 Interaction via Lipid Raft Structure-"Caveolae".

Taxol is one of the most widely used natural antitumor drugs that have shown considerable success in treating cancers of different lineage. However, the development of resistance to taxol is still a significant issue. Caveolae, the cave-like structures found on the surface of many cancerous cells, are enriched in cholesterol and are known to play a pivotal role in drug uptake. Caveolin-1 (Cav-1), the principal structural proteins of the caveolae, interacts with signaling molecules through a scaffolding domain. In the present study, we observed that Cav-1-GFP clusters were instantly recruited to the cell membrane. Interestingly, Caveolae formation followed by internalization was observed after the treatment with time. The recruitment and the formation of the Cav-1-GFP clusters are provided in supplementary video 2 (SV2). The results obtained from molecular docking indicate favorable taxol-Cav-1 interaction. To further confirm the influence of Cav-1 proteins in the uptake and effects of taxol, the cells were treated with beta-cyclodextrin (ß-CD), cholesterol, and taxol combinations. The result suggests that the depletion of cholesterol in HeLa cells makes them less susceptible to taxol at a lower concentration. These observations provide evidence of the interaction between Cav-1 and taxol. Further studies that may elucidate the molecular mechanism of uptake of taxol through caveolae/Cav-1 will help to determine if Cav-1 can be used to increase the uptake of taxol by cancer cells and sensitize the drug-resistant cancer cells to taxol.

Organic osmolyte betaine mitigates the deleterious effects of Diclofenac in vivo in wistar albino rats

Abstract Diclofenac sodium (DF) is a non-steroidal anti-inflammatory drug (NSAID) that possesses antipyretic, analgesic, antinociceptive and anti-inflammatory activities. Like other NSAIDs, DF is known to be associated with renal, cardiovascular, and gastrointestinal complications. The present study was carried out to evaluate the adverse effects of DF in vivo in wistar albino rats and to assess if oral administration of the organic osmolyte betaine mitigates the adverse effect of DF. Eighteen male Wistar rats were divided into three groups, one group of animals was fed orally with 20 mg/kg of DF once/day, and the other group received a combination of 20 mg/kg of DF and 30 mg/kg of betaine, once/day. Apart from the hematological and biochemical parameters, histopathological changes in the liver, lungs, brain, heart and kidney were also investigated. Histopathological alterations that were found in the liver, kidney, and lungs of DF-treated animals were found to be minimal or absent in DF + betaine-treated animals, as compared to untreated control. The results showed that betaine mitigates the adverse effects associated with DF treatment.

Effect of pH on Diclofenac-Lysozyme Interaction: Structural and Functional Aspect.

As a nonsteroidal antiinflammatory drug, diclofenac (DCF) is used in the treatment of a variety of human ailments. It has already been reported that the use of this class of drugs for a longer duration is associated with numerous side effects such as cardiovascular implications, reno-medullary complications, etc. In the present study, the effect of DCF on the structure, stability, and function of lysozyme was studied. The study was designed to examine the effect of DCF only at various pH values. Heat-induced denaturation of lysozyme was analyzed in the presence and absence of various molar concentrations of DCF at different pH values. The values of thermodynamic parameters, the midpoint of denaturation (T m), enthalpy change at T m (ΔH m), constant pressure heat capacity change (ΔC p), and Gibbs energy change at 25°C (ΔG D o), thus obtained under a given set of conditions (pH and molar concentration of DCF), demonstrated the following 1) DCF destabilized lysozyme with respect of T m and ΔG D o at all the pH values, 2) the magnitude of protein destabilization is lesser at acidic pH than at physiological pH, 3) structural changes in lysozyme are less projecting at pH 2.0 than at pH 7.0, and 4) quenching is observed at both pH values. Furthermore, the process of protein destabilization in the presence of DCF is entropically driven.

Immunomodulatory Effects of Jacalin, a Dietary Plant Lectin on the Peripheral Blood Mononuclear Cells (PBMCs).

The tumor microenvironment that refers to the tumor's surroundings is a key modulator of tumor growth and invasion. The tumor-derived signals are known to downregulate the anti-tumor effects of the effector cells present in the TME. Thus, the cross-talk between the tumor cells with the surrounding immune cells helps in evading the tumor surveillance as well as aiding in tumor growth and proliferation. Hence, knowledge regarding the effects of drugs/compound on the tumor-stromal interactions is gaining importance. In the present study, the effects of jacalin, a dietary lectin on the proliferation and cytokine production of peripheral blood mononuclear cells (PBMCs), are investigated. Jacalin was shown to act as a mitogen of PBMCs, the key cytokine secreting immune cells. Also, jacalin initially induced increased mRNA expression of pro-inflammatory cytokine IFN-γ; however, prolonged stimulation of PBMCs resulted in increased expression of anti-inflammatory cytokine, mainly TGF-ß. Furthermore, 6 h jacalin prestimulated PBMCs (Jac-PBMCs) were shown to inhibit HeLa cell proliferation while 24 h Jac-PBMCs were found to favor tumor growth. Thus, it may be postulated that while jacalin initially polarizes the PBMCs to hinder the tumor growth, after a stipulated time point, interaction of jacalin with PBMCs can lead to an immunosuppressive TME that may probably assist in tumor growth and progression.

Green synthesis of silver nanoparticles from plant latex and their antibacterial and photocatalytic studies.

The present work describes a facile synthesis of silver nanoparticles from calotropis procera (CP-AgNPs). The CP-AgNPs were well characterized by many methods. The synthesized CP-AgNPs are stable for more than 5 months. Then we have used CP-AgNPs as photo catalysts for the degradation of methyl orange (MO) dye. The photocatalytic degradation efficiency was 0.0076. Moreover, we also have studied the antibacterial activity against pseudomonas aeruginosa (PA), klebsiella pneumonia (KP), staphylococcus aureus (SA) and bacillus subtilis (BS) bacteria. Interestingly, all four different bacteria causing biofilm were inhibited by CP-AgNPs by 80%. To the best of our knowledge, this is the first report for the synthesis of silver nanoparticles from calotropis procera plant latex. Furthermore, CP-AgNPs effectively were applied as photo catalysts for the degradation of MO dye and also as anti-biofilm agents.

Designing of a Chimeric Vaccine Using EIS (Rv2416c) Protein Against Mycobacterium tuberculosis H37Rv: an Immunoinformatics Approach.

Mycobacterium tuberculosis (Mtb) is a respiratory pathogen that causes tuberculosis (TB). There are a large number of proteins that are involved in the pathogenesis of TB. Stimulating the immune response against TB is very important to clear the pathogens from host. In the present study, an immunoinformatics conduit is used for designing an epitope based chimeric vaccine against TB. Enhanced intracellular survival (EIS) protein from Mtb is used for designing the chimeric vaccine. One B cell epitope, 8 cytotoxic T lymphocyte (CTL), and 6 helper T lymphocyte (HTL) epitopes were predicted based on the MHC allele binding, immunogenicity, antigenicity, allergenicity, toxicity and IFN epitopes. The selected epitopes were used for chimeric vaccine designing. Furthermore, 3D structure elucidation, structural refinement and validation of the designed chimeric vaccine were carried out. The 3D structure was used for protein-protein docking studies with Toll-like receptor 4 (TLR-4), followed by molecular dynamic simulation (MDS) and the interaction between the chimeric vaccine and TLR-4 complex was verified.

Increased ERK phosphorylation and caveolin-1 expression on K562 human chronic myelogenous leukemia cells by jacalin, a dietary plant lectin.

The potential antitumor effects of jacalin, the plant lectin that specifically recognizes the tumor-associated Thomsen-Friedenreich antigen has been extensively studied. We had earlier reported jacalin to be mitogenic to K562, the Bcr-Abl expressing erythroleukemia cell line. The dearth of studies highlighting the proliferative effects of jacalin and other lectins motivated us to unveil the mechanism underlying the mitogenic effects of jacalin. Caveolin-1 (cav-1) is an integral membrane protein, known to play a crucial role in cell signaling, lipid transport, and membrane trafficking. The role of cav-1 in tumorigenesis is considered to be controversial as it can suppress as well as promote tumor growth, depending on the cellular context. In the present study, we propose that cav-1 plays the central role in the mitogenic effects of jacalin on the K562 cells. In accordance, the mRNA, as well as protein expression of cav-1 was found to be upregulated in the jacalin-treated K562 cells as compared to the untreated control. Further, jacalin stimulation also increased the phosphorylation of ERK and Akt. The rationale that leads to the initial conjecture about cav-1 was that the sequence of jacalin possesses a cav-1-binding site.

Mechanistic insights into the urea-induced denaturation of a non-seleno thiol specific antioxidant human peroxiredoxin 6.

Peroxiredoxin 6 (Prdx6) is a unique enzyme among mammalian peroxiredoxins as it lacks resolving cysteine. It is found to be involved in number of different diseases including tumours and its expression level is highest in lungs as compared to other organs. It has been found that Prdx6 plays a significant role different metabolic diseases, ocular damage, neurodegeneration and male infertility. It is a bifunctional protein having phospholipase A2 and peroxidase (also has the ability to reduce phospholipid hydroperoxides) activities. In order to complete the peroxidise reaction cycle it requires glutathione catalyzed by glutathione S-transferase. Equilibrium unfolding and conformational stability of Prdx6 was studied by using urea as a chemical denaturant to understand the changes it goes under cellular stress conditions. Three different spectroscopic methods were employed to monitor urea-induced denaturation. From the results obtained, it was found that the urea denaturation of Prdx6 follows a variable two state process due to non-coincidence of the normalized transition curves obtained from different optical probes. The different denaturation curves were normalized and thermodynamic parameters, ΔGDo, Gibbs free energy change related to the urea-induced denaturation, midpoint of denaturation (Cm), and m = (δΔGD / [urea]) were obtained. The structural information of Prdx6 were further analysed by several parameters obtained by 100 ns MD simulation. The results of MD simulation clearly favour the outcome of spectroscopic studies.

Emerging Role of Mitophagy in Inflammatory Diseases: Cellular and Molecular Episodes.

Mitochondria are the crucial regulators for the major source of ATP for different cellular events. Due to damage episodes, mitochondria have been established for a plethora ofalarming signals of stress that lead to cellular deterioration, thereby causing programmed cell death. Defects in mitochondria play a key role in arbitrating pathophysiological machinery with recent evince delineating a constructive role in mitophagy mediated mitochondrial injury. Mitophagy has been known for the eradication of damaged mitochondria via the autophagy process. Mitophagy has been investigated as an evolutionarily conserved mechanism for mitochondrial quality control and homeostasis. Impaired mitophagy has been critically linked with the pathogenesis of inflammatory diseases. Nevertheless, the exact mechanism is not quite revealed, and it is still debatable. The purpose of this review was to investigate the possible role of mitophagy and its associated mechanism in inflammation-mediated diseases at both the cellular and molecular levels.

Increased Expression of TGF-ß and IFN-γ in Peripheral Blood Mononuclear Cells (PBMCs) Cultured in Conditioned Medium (CM) of K562 Cell Culture.

In the present study, we investigated the effects of conditioned media (CM) collected from the cancer cell lines (K562, MCF-7, and HeLa) on peripheral blood mononuclear cells (PBMCs) isolated from the healthy human blood. The soluble factors in the CM are probably responsible for the differential mRNA expressions of Foxp3, Helios, Neuropilin- 1 (NRP-1), and glycoprotein A repetitions predominant (GARP), along with IFN-γ and TGF-ß in PBMCs cultured with cancer cells CM. The PBMCs cultured with CM of K562 showed increased expression of Foxp3, Helios, NRP-1, GARP, IFN-γ, and TGF-ß compared to PBMCs cultured with CM of MCF-7 and HeLa cells. In addition, the intracellular staining on PBMCs cultured with CM from cell lines were also evaluated for CD4, CD25, Foxp3, Helios, and NRP-1 by multicolor flow cytometry. The expression of CD4+CD25+Foxp3+, CD4+Helios+Foxp3+ and CD+NRP-1+Foxp3+ showed retarded cell population compared to control PBMCs. Our data suggest that soluble factors in CM of cancer cells may trigger the immune response in PBMCs resulting in a systematic response. Further research could lead to the identification of specific soluble factors that are involved in trafficking of cells into the immune cascades, which could be a safe and promising strategy for targeting human cancers.

Differential expression of Helios, Neuropilin-1 and FoxP3 in head and neck squamous cell carcinoma (HNSCC) patients.

In recent years, studies have begun to explore the immune involvement in head and neck tumors. Advanced stage head and neck squamous cell carcinoma (HNSCC) has a poor prognosis with low survival rates with high level of immune infiltrates. Tregs (regulatory T cells) play a crucial role in constructing an immunosuppressive tumor microenvironment. In the present study, we highlighted specific Treg markers and its factors in HNSCC solid tumors and peripheral blood of cancer patients. By histopathology and immunofluorescence staining, we observed differential expression of CD4, CD25, Foxp3, Helios and Neuropilin-1. Further, we analyzed the expression of Foxp3, Helios, Neuropilin-1 and GARP by qPCR and flow cytometry in whole blood and found to be elevated in HNSCC patients in comparison with healthy donors. Additionally, IFN-γ, TGF-ß, IL-6, IL-2, IL-10 and TNF-α expressions were also found to be relatively increased in the head and neck cancer patients when compared with healthy donors. Our findings emphasize that Tregs may be involved in promoting tumor progression. Helios and Neuropilin-1 could be potent markers in identifying subsets of Tregs. Association of soluble factors could sculpture the activity of Tregs. With further research, Treg markers and its associated soluble factors could be employed to block Tregs trafficking to the tumor, thus enlightening a potential strategy for targeting human cancers.

Sub-Micellar Concentration of Sodium Dodecyl Sulphate Prevents Thermal Denaturation Induced Aggregation of Plant Lectin, Jacalin.

The irreversible thermal unfolding of jacalin, the lectin purified from jackfruit seeds was accompanied by aggregation, where intermolecular interactions among the subunits are favoured over intramolecular interactions. The extent of aggregation increased as a function of temperature, time and protein concentration. The anionic surfactant, sodium dodecyl sulphate (SDS) significantly suppressed the formation of aggregates as observed by turbidity measurements and Rayleigh scattering assay. Moreover, far UV-CD spectra indicate that the protein ß sheet transforms into α helical structure, when denatured in the presence of 3 mM SDS. Further, jacalin when heated in the presence of SDS partially retained the hemagglutination activity when jacalin-SDS mixture was diluted to 1:8 factor since 3 mM SDS was found to lyse the red blood cells. Thus, SDS only altered the aggregation behaviour of jacalin by preventing intermolecular hydrogen bonding among the exposed residues but did not completely stabilize the native conformation.

Sustained mitogenic effect on K562 human chronic myelogenous leukemia cells by dietary lectin, jacalin.

Dietary lectins have been shown to affect the proliferation of human cancer cell lines. The anti-proliferative effects of lectins from varied sources have been extensively studied and in some cases, the underlying mechanism has been explored. Except for peanut agglutinin (PNA), the mitogenic effects of no other lectins have been studied in detail. In the present study, we have shown that jacalin, lectin purified from jackfruit (Artocarpus integrifolia) seeds act as a mitogen for K562, the Bcr-Abl expressing erythroleukemia cell line (K562) and the effect was found to be dose dependent. K562 cells remained in the proliferative state for a longer period even after the withdrawal of jacalin stimulation, thus jacalin was found to induce sustained mitogenic effect on K562 cells. Further, conditioned media from K562 cells treated with jacalin were observed to have the similar mitogenic effect even in the presence of galactose. Importantly, galactose which is a known ligand for jacalin will interact with functionally active jacalin present in the conditioned media and neutralise its effect. In addition, jacalin treatment also resulted in increased mRNA expression levels of pro-inflammatory cytokines including IL-1ß, IL-6 and IFN-γ. Our results indicate that jacalin induces secretion of soluble molecules, which maybe responsible for this observed increased proliferation of K562 cells.

The E3 ligase Itch and deubiquitinase Cyld act together to regulate Tak1 and inflammation.

Chronic inflammation has been strongly associated with tumor progression, but the underlying mechanisms remain elusive. Here we demonstrate that E3 ligase Itch and deubiquitinase Cyld formed a complex via interaction through 'WW-PPXY' motifs. The Itch-Cyld complex sequentially cleaved Lys63-linked ubiquitin chains and catalyzed Lys48-linked ubiquitination on the kinase Tak1 to terminate inflammatory signaling via tumor necrosis factor. Reconstitution of wild-type Cyld but not the mutant Cyld(Y485A), which cannot associate with Itch, blocked sustained Tak1 activation and proinflammatory cytokine production by Cyld(-/-) bone marrow-derived macrophages. Deficiency in Itch or Cyld led to chronic production of tumor-promoting cytokines by tumor-associated macrophages and aggressive growth of lung carcinoma. Thus, we have identified an Itch-Cyld-mediated regulatory mechanism in innate inflammatory cells.

Molecular characterization of tlyA gene product, Rv1694 of Mycobacterium tuberculosis: a non-conventional hemolysin and a ribosomal RNA methyl transferase.

BACKGROUND: Mycobacterium tuberculosis is a virulent bacillus causing tuberculosis, a disease responsible for million deaths each year worldwide. In order to understand its mechanism of pathogenesis in humans and to help control tuberculosis, functions of numerous Mycobacterium tuberculosis genes are being characterized. In this study we report the dual functionality of tlyA gene product of Mycobacterium tuberculosis annotated as Rv1694, a 268 amino acid long basic protein. RESULTS: The recombinant purified Rv1694 protein was found to exhibit hemolytic activity in vitro. It showed concentration and time-dependent hemolysis of rabbit and human erythrocytes. Multiple oligomeric forms (dimers to heptamers) of this protein were seen on the membranes of the lysed erythrocytes. Like the oligomers of conventional, well-known, pore-forming toxins, the oligomers of Rv1694 were found to be resistant to heat and SDS, but were susceptible to reducing agents like ß-mercaptoethanol as it had abolished the hemolytic activity of Rv1694 indicating the role of disulfide bond(s). The Rv1694 generated de novo by in vitro transcription and translation also exhibited unambiguous hemolysis confirming the self assembly and oligomerization properties of this protein. Limited proteolytic digestion of this protein has revealed that the amino terminus is susceptible while in solution but is protected in presence of membrane. Striking feature of Rv1694 is its presence on the cell wall of E. coli as visualized by confocal microscopy. The surface expression is consistent with the contact dependent haemolytic ability of E. coli expressing this protein. Also, immune serum specific to this protein inhibits the contact dependent hemolysis. Moreover, Rv1694 protein binds to and forms stable oligomers on the macrophage phagosomal membranes. In addition to all these properties, E. coli expressing Rv1694 was found to be susceptible to the antibiotic capreomycin as its growth was significantly slower than mock vector transformed E. coli. The S30 extract of E. coli expressing the Rv1694 had poor translational activity in presence of capreomycin, further confirming its methylation activity. Finally, incorporation of methyl group of [3H]-S-adenosylmethionine in isolated ribosomes also confirmed its methylation activity. CONCLUSIONS: The Rv1694 has an unusual dual activity. It appears to contain two diverse functions such as haemolytic activity and ribosomal RNA methylation activity. It is possible that the haemolytic activity might be relevant to intra-cellular compartments such as phagosomes rather than cell lysis of erythrocytes and the self-assembly trait may have a potential role after successful entry into macrophages by Mycobacterium tuberculosis.

Modulation of PP2A activity by Jacalin: is it through caveolae and ER chaperones?

Plant lectins have been reported to affect the proliferation of different human cancer cell line probably by binding to the specific carbohydrate moieties. In the present study, Badan labeled single cysteine mutant (present in the caveolin-1 binding motif) of jacalin (rJacalin) was found to penetrate the target membrane, indicating a protein-protein or protein-membrane interaction apart from its primary mode of binding i.e. protein-carbohydrate interaction. Further, Jacalin treatment has resulted in the movement of the GFP-Caveolin-1 predominantly at the cell-cell contact region with much restricted dynamics. Jacalin treatment has resulted in the perinuclear accumulation of PP2A and dissociation of the PHAP1/PP2A complex. PP2A was found to act as a negative regulator of ERK signaling and a significant decrease in the phosphorylation level of MEK and AKT (T308) in A431. In addition, we have also identified several ER resident proteins including molecular chaperones like ORP150, Hsp70, Grp78, BiP of A431 cells, which were bound to the Jacalin-sepharose column. Among various ER chaperones that were identified, ORP150 was found to present on the cell surface of A431 cells.

Membrane bound monomer of Staphylococcal alpha-hemolysin induces caspase activation and apoptotic cell death despite initiation of membrane repair pathway.

BACKGROUND: Wild type Staphylococcal alpha-hemolysin (alpha-HL) assembly on target mammalian cells usually results in necrotic form of cell death; however, caspase activation also occurs. The pathways of caspase activation due to binding/partial assembly by alpha-HL are unknown till date. RESULTS: Cells treated with H35N (a mutant of alpha-HL that remains as membrane bound monomer), have been shown to accumulate hypodiploid nuclei, activate caspases and induce intrinsic mitochondrial apoptotic pathway. We have earlier shown that the binding and assembly of alpha-HL requires functional form of Caveolin-1 which is an integral part of caveolae. In this report, we show that the caveolae of mammalian cells, which undergo a continuous cycle of 'kiss and run' dynamics with the plasma membrane, have become immobile upon the binding of the monomer. The cells treated with H35N were unable to recover despite activation of membrane repair mechanism involving caspase-1 dependent activation of sterol regulatory element binding protein-1. CONCLUSIONS: This is for the first time we show the range of cellular changes and responses that take place immediately after the binding of the monomeric form of staphylococcal alpha-hemolysin.

Taxol and 10-deacetylbaccatinIII induce distinct changes in the dynamics of caveolae.

Taxol treatment of HeLa cells resulted in a transient recruitment of Caveolin-1 to the cell surface followed by internalization. Interestingly, 20min after 10-deacetylbaccatinIII (10-DAB) treatment, the caveolae displayed faster 'kiss and run' dynamics while BaccatinIII (BacIII) did not induce any change. Sustained phosphorylation of Caveolin-1 is observed upon treatment and between Taxol and 10-DAB, the former shows phosphorylated Raf-1, ERK1/2 and hyperphosphorylated Bcl-2 while the later showed much less magnitude of the same. BacIII treatment did not induce phosphorylation of Raf-1 or Bcl-2. It is possible that Taxol might act on multiple targets and the side chain may be crucial.

Stress-induced phosphorylation of caveolin-1 and p38, and down-regulation of EGFr and ERK by the dietary lectin jacalin in two human carcinoma cell lines.

We have examined the A431 (human epidermoid carcinoma) and HT29 (human colorectal carcinoma) cellular responses evoked by lectins of dietary origin, Jacalin of Artocarpus integrifolia (native jacalin; nJacalin), peanut agglutinin (PNA) of Arachis hypogea, and recombinant single-chain jacalin (rJacalin), which has the same protein backbone but approximately 100-fold less affinity for carbohydrates than nJacalin. All three lectins (nJacalin, rJacalin, and PNA) are cycotoxic inhibitors of proliferation of A431 cells. However, cells recover once jacalin but not PNA have been removed from the growth medium. Treatment of nJacalin results in morphologically visible cell rounding while retaining the membrane integrity when treated at 40 microg ml(-1), but treatment with PNA did not induce such changes. The observed cell rounding was found to be due to stress as the phosphorylation of caveolin-1 (at tyr14), p38 but not c-Jun N-terminal kinase were up-regulated, while PNA did not up-regulate the phosphorylation of the same. Jacalin also down-regulated the phosphorylation of the epidermal growth factor receptor and extracellular signal regulated kinase in contrast to PNA, which failed to down-regulate the same. Confocal microscopic studies reveal that jacalin is not internalized, unlike the lectin of Agaricus bisporous. Analysis of the proteins that bind to an nJacalin-sepharose column revealed the binding of six to eight proteins, and significant among them is a protein at approximately 110 kDa, which appears to be oxygen-regulated protein 150 (ORP150) (endoplasmic reticulum chaperone) as identified by its isoelectric point, two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and mass spectrometric analysis. This 110-kDa band is detectable with anti-Hsp70 antibody because ORP150 has homology with Hsp70. Confocal microscopic studies reveal the presence of Hsp70-like proteins on the surface of A431 cells as revealed by immunostaining with anti-Hsp70 antibody. Moreover, overexpression of ORP150 in A431 cells has resulted in a dramatic protection of A431 cells against jacalin-induced toxicity, confirming that the jacalin-induced cytotoxicity is mediated through ORP150, and impairment of ORP150 functions with the help of jacalin makes the cells more susceptible to death due to stress. Our studies suggest that the cellular responses, as a consequence of lectin binding, may not be exclusively mediated by carbohydrate binding property alone, but other factors such as protein-protein interactions may also contribute to the observed cellular responses.