Chhabinath Mandal

Induction of glycosylation in human C-reactive protein under different pathological conditions.

As an acute-phase protein, human C-reactive protein (CRP) is clinically important. CRPs were purified from several samples in six different pathological conditions, where their levels ranged from 22 to 342 microg/ml. Small, but significant, variations in electrophoretic mobilities on native PAGE suggested differences in molecular mass, charge and/or shape. Following separation by SDS/PAGE, they showed single subunits with some differences in their molecular masses ranging between 27 and 30.5 kDa, but for a particular disease, the mobility was the same for CRPs purified from multiple individuals or pooled sera. Isoelectric focusing (IEF) also indicated that the purified CRPs differed from each other. Glycosylation was demonstrated in these purified CRPs by Digoxigenin kits, neuraminidase treatment and binding with lectins. The presence of N-linked sugar moiety was confirmed by N-glycosidase F digestion. The presence of sialic acid, glucose, galactose and mannose has been demonstrated by gas liquid chromatography, mass spectroscopic and fluorimetric analysis. Matrix-assisted laser-desorption ionization analysis of the tryptic digests of three CRPs showed systematic absence of two peptide fragments, one at the N-terminus and the other near the C-terminus. Model-building suggested that the loss of these fragments exposed two potential glycosylation sites on a cleft floor keeping the protein-protein interactions in pentraxins and calcium-dependent phosphorylcholine-binding qualitatively unaffected. Thus we have convincingly demonstrated that human CRP is glycosylated in some pathological conditions.

Oxidative inhibition of Hsp90 disrupts the super-chaperone complex and attenuates pancreatic adenocarcinoma in vitro and in vivo.

Pancreatic cancer is almost always fatal, in part because of its delayed diagnosis, poor prognosis, rapid progression and chemoresistance. Oncogenic proteins are stabilized by the Hsp90, making it a potential therapeutic target. We investigated the oxidative stress‐mediated dysfunction of Hsp90 and the hindrance of its chaperonic activity by a carbazole alkaloid, mahanine, as a strategic therapeutic in pancreatic cancer. Mahanine exhibited antiproliferative activity against several pancreatic cancer cell lines through apoptosis. It induced early accumulation of reactive oxygen species (ROS) leading to thiol oxidation, aggregation and dysfunction of Hsp90 in MIAPaCa‐2. N‐acetyl‐L‐cysteine prevented mahanine‐induced ROS accumulation, aggregation of Hsp90, degradation of client proteins and cell death. Mahanine disrupted Hsp90‐Cdc37 complex in MIAPaCa‐2 as a consequence of ROS generation. Client proteins were restored by MG132, suggesting a possible role of ubiquitinylated protein degradation pathway. Surface plasmon resonance study demonstrated that the rate of interaction of mahanine with recombinant Hsp90 is in the range of seconds. Molecular dynamics simulation showed its weak interactions with Hsp90. However, no disruption of the Hsp90‐Cdc37 complex was observed at an early time point, thus ruling out that mahanine directly disrupts the complex. It did not impede the ATP binding pocket of Hsp90. Mahanine also reduced in vitro migration and tube formation in cancer cells. Further, it inhibited orthotopic pancreatic tumor growth in nude mice. Taken together, these results provide evidence for mahanine‐induced ROS‐mediated destabilization of Hsp90 chaperone activity resulting in Hsp90‐Cdc37 disruption leading to apoptosis, suggesting its potential as a specific target in pancreatic cancer.

Effect of environmental pollutants on the c-reactive protein of a freshwater major carp, Catla catla

C-reactive proteins (CRP) have been affinity purified to electrophoretic homogeneity from the sera of major carp, Catla catla before and after exposure to environmental pollutants. Exposure to these pollutants elevate the levels of circulating CRPs to 2.8-3.5 times the normal values. Kinetic studies of metal intoxication indicate that a unique molecular variant of CRP is present in the serum at the peak level of acute phase induction, and this variant coexists with normal CRPs. Carbohydrate analysis and lectin binding reveals that these CRPs are glycoproteins differing significantly in total carbohydrate contents. Their electrophoretic mobilities in native gel are different but become identical on desialylation and deglycosylation implying that the molecular variants vary in the glycan parts. All these forms of CRP contain two nonidentical subunits of Mr 22 and 29 kDa. Examination of their immunological crossreactivity demonstrate their similarity in overall molecular topology but their differences in the quantitative extent of binding are reflected.

A colorimetric assay to evaluate the chemotherapeutic response of children with acute lymphoblastic leukemia (ALL) employing AchatininH: a 9-O-acetyl sialic acid binding lectin

Employing a 9-O-acetyl sialic acid binding lectin, Achatinin(H) (ATNH), we have reported a non-invasive, blood based lymphoproliferation assay which measures the maximal lymphoproliferative dose (MLD) of ATN(H) to assess the status of 9-O-acetylated sialoglycoconjugates (9-OAcSGs) in patients with Acute lymphoblastic leukemia (ALL) (Mandal C, Sinha D, Sharma V, Bhattacharya DK. O-acetyl sialic acid binding lectin, as a probe for detection of subtle changes on the cell surface induced during acute lymphoblastic leukemia [ALL] and its clinical application. Ind J Biochem Biophys 1997;34:82; Sinha D, Mandal C, Bhattacharya DK. Development of a simple blood based lymphoproliferation assay to assess the clinical status of patients with acute lymphoblastic leukemia. Leuk Res 1999;13:309-312; Sinha D, Mandal C, Bhattacharya DK. A novel method for prognostic evaluation of childhood acute lymphoblastic leukemia. Leukemia 1999;13[in press]). Although the expression of 9-OAcSGs clearly serves as an index of treatment outcome, the assay has limitations in that it requires radioisotopes, i.e. [3H]-TdR. Therefore a colorimetric assay was developed as an alternative approach. The pre-treatment MLD, as measured by the colorimetric assay, was 0.15 +/- 0.02 microg which progressively increased during consolidation therapy (1.40 +/- 0.39 microg), maintenance therapy (4.20 +/- 1.60 microg) and in followed-up cases (5.20 +/- 0.43 microg) but sharply declined following relapse (0.25 +/- 0.02 microg). The colorimetric assay also showed a good correlation with radiometric assay (r = + 0.93) and their mean coefficient of inter-assay precision were also comparable (15.53% versus 14.86%). We therefore propose that the colorimetric assay is a safe, non-radiometric, user-friendly alternative for assessing individual chemotherapeutic responses in childhood ALL.

O-acetylation of sialic acids is required for the survival of lymphoblasts in childhood acute lymphoblastic leukemia (ALL)

Exploiting the selective affinity of Achatinin-H towards 9-O-acetylneuraminic acid(α2-6)GalNAc, we have demonstrated the presence of 9-O-acetylated sialoglycoproteins (Neu5,9Ac2-GPs) on hematopoietic cells of children suffering from acute lymphoblastic leukemia (ALL), indicative of defective sialylation associated with this disease. The carbohydrate epitope of Neu5,9Ac2-GPsALL was confirmed by using several synthetic sialic acid analogues. They are functionally active signaling molecules as demonstrated by their role in mediating lymphoproliferative responses and consequential increased production of IFN-γ due to specific stimulation of Neu5,9Ac2-GPs on PBMCALL with Achatinin-H. Cells devoid of 9-O-acetylations (9-O-AcSA−) revealed decreased nitric oxide production as compared to 9-O-AcSA+ cells on exposure to IFN-γ. Under this condition, a decrease in viability of 9-O-AcSA− cells as compared to 9-O-AcSA+ cells was also observed which was reflected from increased caspase 3 activity and apoptosis suggesting the protective role of this glycotope. These Neu5,9Ac2-GPs are also capable of inducing disease-specific anti-Neu5,9Ac2-GPs antibodies in ALL children. Additionally, we have observed that disease-specific anti-Neu5,9Ac2-GPs have altered glycosylation profile, and they are incapable of exerting a few Fc-glycosylation-sensitive effector functions. These observations hint toward a disbalanced homeostasis, thereby enabling the cancer cells to escape host defense. Taken together, it may be hypothesized that Neu5,9Ac2-GPs and their antibodies play a prominent role in promoting the survival of lymphoblasts in ALL.

Variations in binding characteristics of glycosylated human C-reactive proteins in different pathological conditions

C-reactive protein (CRP) is a clinically important classic acute phase pentameric protein. It is thought to play an important role in immunomodulation. Earlier reports convincingly demonstrated that human CRP is differentially glycosylated in different pathological conditions. Although CRP is considered to be a clinically important molecule, changes in binding characteristics with appropriate ligands with respect to glycosylation remain unexplored. In an effort to demonstrate that these glycosylated molecular variants are capable of modulating their binding activity with different ligands, CRPs were affinity purified from six different clinical samples. Variable amounts of linkage-specific sialic acid derivatives were found in these CRPs with varying tryptophan contents. Differential binding patterns with antibodies against human CRP, human IgG, and other ligands like fibronectin, fetuin, and asialofetuin indicated that the purified CRPs differed significantly in their lectin-like interactions. Thus, we have convincingly demonstrated that differentially induced CRPs exhibited variable binding characteristics. These results may have far reaching practical applications for understanding acute phase responses. Published in 2004..

Mahanine, A DNA Minor Groove Binding Agent Exerts Cellular Cytotoxicity with Involvement of C-7-OH and −NH Functional Groups

Mahanine, a carbazole alkaloid is a potent anticancer molecule. To recognize the structure-activity correlation, mahanine was chemically modified. Antiproliferative activity of these derivatives was determined in 19 cancer cell lines from 7 different origins. Mahanine showed enhanced apoptosis compared to dehydroxy-mahanine-treated cells, indicating significant contribution of the C-7-OH group. O-Methylated-mahanine and N-methylated dehydroxy-mahanine-treated cells exhibited apoptosis only at higher concentrations, suggesting additional contribution of 9-NH group. Using biophysical techniques, we demonstrated that mahanine interacts with DNA through strong association with phosphate backbone compared to other derivatives but is unable to induce any conformational change in DNA, hence suggesting the possibility of being a minor groove binder. This was corroborated by molecular modeling and isothermal titration calorimetry studies. Taken together, the results of the current study represent the first evidence of involvement of C-7-OH and 9-NH group of mahanine for its cytotoxicity and its minor groove binding ability with DNA.

High level of sialate-O-acetyltransferase activity in lymphoblasts of childhood acute lymphoblastic leukaemia (ALL): enzyme characterization and correlation with disease status

Previous studies had established an over-expression of 9-O-acetylated sialoglycoproteins (Neu5,9Ac2-GPs) on lymphoblasts of childhood acute lymphoblastic leukaemia (ALL). Here, we report the discovery and characterization of sialate-O-acetyltransferase enzyme in ALL-cell lines and lymphoblasts from bone marrow of children diagnosed with B- and T-ALL. We observed a positive correlation between the enhanced sialate-O-acetyltransferase activity and the enhanced expression of Neu5,9Ac2-GPs in these lymphoblasts. Sialate-O-acetyltransferase activity in cell lysates or microsomal fractions of lymphoblasts of patients was always higher than that in healthy donors reaching up to 22-fold in microsomes. Additionally, the Vmax of this enzymatic reaction with AcCoA was over threefold higher in microsomal fractions of lymphoblasts. The enzyme bound to the microsomal fractions showed high activity with CMP-N-acetylneuraminic acid, ganglioside GD3 and endogenous sialic acid as substrates. N-acetyl-7-O-acetylneuraminic acid was the main reaction product, as detected by radio-thin-layer chromatography and fluorimetrically coupled radio-high-performance liquid chromatography. CMP and coenzyme A inhibited the microsomal enzyme. Sialate-O-acetyltransferase activity increased at the diagnosis of leukaemia, decreased with clinical remission and sharply increased again in relapsed patients as determined by radiometric-assay. A newly-developed non-radioactive ELISA can quickly detect sialate-O-acetyltransferase, and thus, may become a suitable tool for ALL-monitoring in larger scale. This is the first report on sialate-O-acetyltransferase in ALL being one of the few descriptions of an enzyme of this type in human.

Unraveling the C-reactive protein complement-cascade in destruction of red blood cells: potential pathological implications in Plasmodium falciparum malaria.

Background: Deficiencies of the complement-regulatory proteins on RBC (RBCMal) of patients with Plasmodium falciparum were reported. Here, we sought to determine the role of affinity-purified C-reactive protein from patients (CRPMal), in modulating the complement-regulatory proteins and downstream effect on complement-cascade. Methods: CRPMal was characterized by analytical ultracentrifuge and electrophoretic analysis. Surface plasmon resonance, Western blotting, co-immuno-precipitation, flow-cytometry and ELISA determined the binding of CRPMal with RBCMal. Modifications of membranes for RBCMal-CRPMal binding were explored by scanning electron microscopy, osmotic and turbulence fragility, hydrophobicity and oxyhemoglobin release. Flow-cytometry, ELISA, Western blotting and Scatchard analysis monitored the status of complement-regulatory proteins on RBCMal. Complement-activation via CRPMal was quantified by C3-deposition and hemolysis. Results: CRPMal binds specifically to RBCMal through distinct molecules. Such binding altered the normal discoid-shape of RBCMal with increased membrane fluidity and hydrophobicity. In the presence of CRP, RBCMal showed reduced complement-regulatory proteins (CR1 or CD35, CD55 and CD59) with decreased affinity. These changes caused enhanced C3-deposition and complement-mediated hemolysis. Conclusion: Taken together, we have established the contributory effect of CRPMal causing decreased complement-regulatory proteins, possibly providing a new mechanism of complement-fueled RBCMal destruction refractory to erythrophagocytosis and may account for pathogenesis of anemia.

O-acetyl sialic acid specific IgM in childhood acute lymphoblastic leukaemia

Initial studies have revealed an enhanced surface expression of O-acetylated sialoglycoconjugates (O-AcSGs) on lymphoblasts concomitant with high titres of IgG in childhood Acute Lymphoblastic Leukaemia (ALL) (Mandal C, Chatterjee M, Sinha D, Br J Haematol 110, 801–12, 2000). In our efforts to identify disease specific markers for ALL, we have affinity-purified IgM directed against O-AcSGs that reacts with three disease specific O-AcSGs present on membrane proteins derived from peripheral blood mononuclear cells (PBMC) of ALL patients. Antibody specificity towards O-AcSGs was confirmed by selective binding to erythrocytes bearing surface O-AcSGs, decreased binding with de-O-acetylated BSM and following pretreatment with O-acetyl esterase. Competitive inhibition ELISA demonstrated a higher avidity of IgM for O-AcSG than IgG. Flow cytometry demonstrated the diagnostic potential of purified O-AcSA IgM as binding was specific with ALL patients and minimal with other haematological disorders and normal individuals. It therefore may be adopted as a non-invasive approach for detection of childhood ALL. Taken together, the data indicates that carbohydrate epitopes having terminal O-AcSA α2 → 6 GalNAc determinants induce disease specific IgG and IgM, potentially useful molecular markers for childhood ALL.