Bishnu P. Chatterjee

Jacalin Bound Plasma O-Glycoproteome and Reduced Sialylation of Alpha 2-HS Glycoprotein (A2HSG) in Rheumatoid Arthritis Patients

Glycosylation studies of plasma proteins can reveal information about the onset and progression of diseases, where in the glycan biosynthetic pathways are disturbed as in rheumatoid arthritis (RA). The present study was focused on analysis of O-linked glycoproteins of plasma in RA patients. Two dimensional gel electrophoresis of jacalin bound plasma of RA patients revealed a number of differentially expressed protein spots as compared to healthy controls. Eighteen protein spots were found to have statistically significant (p<0.05) difference in their expression level from four sets of gels and were identified by MALDI-TOF MS. Most of the identified proteins were predicted to be O-glycosylated proteins by Net–O-Gly 3.1 algorithm. Among these the alpha 2HS glycoprotein (A2HSG) was found to be down regulated whereas inter alpha trypsin inhibitor H4 (ITIH4) was up regulated and this was validated by Western blotting. The glycosylation studies showed the reduced N-linked sialylation of A2HSG in RA patients. Altered glycoprotein expression and functional as well as structural studies of glycans might help in the diagnosis of RA and understanding the disease pathogenesis.

Alterations of glycan branching and differential expression of sialic acid on alpha fetoprotein among hepatitis patients.

The level of serum glycoproteins and their glycosylation pattern change in liver diseases including hepatocellular carcinoma (HCC). Some of them, especially alpha fetoprotein (AFP), serve as useful biomarkers for HCC. The present investigation showed high level of AFP in hepatitis B cirrhosis (HBV-LC) and hepatitis C cirrhosis (HCV-LC) patients. However, increase of AFP level was not significantly high in chronic hepatitis B (HBV-CH) as determined by ELISA using monoclonal anti-human AFP (mAb-AFP). The differential expression of sialic acid linkage was observed in HBV-CH and HCV-LC by ELISA; the former bound strongly with Sambucus nigra agglutinin (SNA), which has exclusive binding specificity for NeuAcα2-6-, whereas HCV-LC reacted preferably with Maackia amurensis agglutinin (MAA) which recognizes NeuAcα2-3-. There was significantly high glycan branching in HBV-LC and HCV-LC in comparison to controls as illustrated by concanavalin A. This was further confirmed by Phaseolus vulgaris erythroagglutinin (E-PHA) and Datura stramonium agglutinin (DSA). Enhanced fucosylation of AFP was observed in HBV-LC, HCV-LC and HCC patients by ELISA using fucose binding Aleuria aurantia lectin; however, maximum binding was found in HCC. Fucosylation with α1-6 linkage was further confirmed by Lens culinaris agglutinin (LCA). From the above results it is concluded that the changes in concentration of AFP, differential expression of sialic acid, increase of glycan branching and fucosylation have a prognostic value of hepatitis and it could be possible that lectin-based assay with AFP can aid in diagnosis of hepatitis diseases besides clinical examination and routine laboratory investigation.

A study on antifungal activity of water-soluble chitosan against Macrophomina phaseolina

The objective of this study was to evaluate antifungal effect of water-soluble chitosan (s-chitosan) on Macrophomina phaseolina (M. phaseolina) causing jute seedling infection and monitor the change in activity of released enzymes during infection. The minimum inhibitory concentration (MIC) of s-chitosan for M. phaseolina was found at 12.5g/l and s-chitosan exhibited fungistatic mode of action against this pathogen. The application of s-chitosan (12.5g/l) during infection of jute seedlings by M. phaseolina inhibited fungal infection and length of the seedlings was found almost similar to seedlings without infection. M. phaseolina infected jute seedlings showed length of 22mm over 10 days of incubation and it increased to 58mm in presence of s-chitosan (12.5g/l) during incubation for 10 days. TEM study indicated presence of hyphae in the cortical and epidermal cells of fungus infected jute seedlings indicating colonization by the fungus and it disappeared after treatment with s-chitosan. The changes in enzyme profiles of jute seedling during prevention of fungal infection using s-chitosan helped in proper understanding of mode of action of s-chitosan as antifungal agent. The activity of defense related enzymes like chitosanase and peroxidase in infected seedlings was observed to be enhanced after treatment with s-chitosan.

Enhanced expression of α1-acid glycoprotein and fucosylation in hepatitis B patients provides an insight into pathogenesis

Altered glycosylation and concentration of α1-acid glycoprotein has been known to be related to the pathogenesis of the hepatic diseases. The present study investigated enhanced fucosylation of AGP in the sera of chronic hepatitis B (HBV-CH) and hepatitis B cirrhosis (HBV-LC) patients by high performance anion exchange chromatography and by ELISA using fucose binding Aleuria aurantia lectin. The concentration of AGP determined by ELISA using monoclonal anti-human AGP (mAb-AGP) showed high level of AGP in HBV-CH and HBV-LC patients. This was further judged by association constant (KA) measured by surface plasmon resonance analysis. There was no apparent linkage variation of sialic acid among different patient groups when tested with two sialic acid binding lectins viz., Maackia amurensis agglutinin (MAA, NeuAc α2-3-) and Sambucus nigra agglutinin (SNA, NeuAc α2-6-) respectively. There was no change of oligosaccharide branching in HBV-CH in comparison to controls whereas a slight change was observed in HBV-LC using ConA. The above results suggest that the changes in concentration of AGP and fucosylation have a prognostic value of hepatitis diseases and it could be possible to use AGP as diagnostic marker besides clinical examination and routine laboratory investigation.

Altered glycosylation, expression of serum haptoglobin and alpha-1-antitrypsin in chronic hepatitis C, hepatitis C induced liver cirrhosis and hepatocellular carcinoma patients

Liver cirrhosis with hepatitis C viral infection (HCV-LC) causes high risk to develop hepatocellular carcinoma (HCC). Besides diagnosis of liver cirrhosis by biochemical test, imaging techniques, assessment of structural liver damage by biopsy shows several disadvantages. Our aim was to monitor the changes in the expression level of serum proteins and their glycosylation pattern among chronic hepatitis C (HCV-CH), HCV-LC and HCC patients with respect to controls. 2D gel electrophoresis of HCV-CH, HCV-LC and HCC patients’ sera showed several protein spots, which were identified by LC-MS. The change in the expression of two prominent protein spots, haptoglobin (Hp) and alpha 1-antitrypsin (AAT) was evaluated by western blot and ELISA. The changes in glycosylation pattern of these serum proteins were assayed using different lectins. Increased level of Hp and AAT was observed in HCV-LC and HCC patients’ group whereas those were found to be present less in HCV-CH patient groups with respect to control as determined by ELISA using monoclonal antibodies. Decreased level of sialylation in both Hp and AAT was observed in HCV-LC and HCV-CH patients’ group whereas increased level of sialylation was observed in HCC patient groups by ELISA using Sambucus nigra agglutinin. On the other hand increased level of fucosylation in two serum glycoproteins was observed in HCV-LC and HCC patients’ group using Lens culinarris agglutinin. High glycan branching was found in HCV-LC and HCC patient groups in Hp but not in HCV-CH as determined by Datura stramonium agglutinin. However, there was no such change observed in glycan branching in AAT of HCV-CH and HCV-LC patients’ groups, to the contrary high glycan branching was observed in HCC patients’ group. Increased level of exposed galactose in both serum proteins was observed in both HCC patients’ group as determined by Ricinus communis agglutinin. The present glycoproteomics study could predict the progression of HCV-CH, HCV-LC and HCC without the need of liver biopsy.

Gold nanoparticle-based novel visual diagnostic method for the detection of specific IgE to test for food allergies

Food allergies are recognized as a major public health issue, especially in early childhood, with no preventive treatment. The diagnosis of food allergies currently relies on careful study of the patient’s history and diagnostic tests such as the skin prick test (SPT), serum specific IgE (immunoglobulin E) testing and, if indicated, oral food challenges, which are often risky. In this study, a sandwich-type immunocomplex was formed between different food allergens, specific IgE from sera and secondary anti-IgE conjugated with the enzyme horseradish peroxidase. Gold(III) chloride was subsequently added as a visual reader reagent. The color change from red to blue and the associated change in intensity at 550 nm was proportional to the concentration of remaining hydrogen peroxide after decomposition by horseradish peroxidase, which in turn triggers the aggregation of gold nanoparticles generating a blue color with a decrease in intensity at 550 nm. The generation of the visible blue color reports the presence of allergy issues compared to the red color in healthy subjects, and the consequent drop in intensity at 550 nm measures the amount of serum specific IgE, which corroborated the SPT results from the corresponding patients. This multiplexed visual diagnostic system for specific IgE against different food allergens using plasmonic phenomena of the aggregated gold nanoparticles has been a rapid, cheap, sensitive and high through put assay for the detection and determination of the severity of food allergens with a very low sample requirement (5 μl).

Macoma birmanica agglutinin recognizes glycoside clusters of β-GlcNAc/Glc and α-Man

Macoma birmanica agglutinin (MBA) that seems to play crucial roles in the innate immunity of marine bivalve, M. birmanica has been earlier defined as GlcNAc/Man specific. However, most complementary carbohydrate structures to its binding domain and ligand clustering in its recognition profile have not been established. In this study, the complete recognition profile of MBA was examined by enzyme-linked lectin-sorbent assay and inhibition assay. Among the monosaccharides tested, GlcNAc was more reactive followed by Man and Glc, others were non-reactive; revealing the importance of equatorial -NAc group at C-2, -OH group at C-4 and C-6, and pyranose conformation of hexose. Moreover, beta-glycosides of GlcNAc and Glc were more potent whereas for Man it was alpha-glycoside. MBA recognized both exposed and internal alpha-Man and beta-GlcNAc/Glc residues well with most linkages except (beta1-4). This binding pattern was further extended and confirmed by polyvalent glycoside clusters of GlcNAc(beta1-2)Man(alpha1-, which was a better inhibitor than Man(alpha1-2/3/6)Man(alpha1- or Man(alpha1-3/6)Man(beta1- present in well-defined naturally occurring glycoproteins. This broad range specificity explains the importance of MBA as an important pattern recognition molecule that provides more realistic picture of carbohydrate-based immune response triggering.

Structural Heterogeneity of Glycoform of Alpha-1 Acid Glycoprotein in Alcoholic Cirrhosis Patients

Altered glycosylation of serum proteins has been reported in different pathologic conditions. Changes in glycosylation of serum proteins in disease state have been extensively used for the development of noninvasive sensitive clinical tests for diagnostic purposes. The aim of the present research was to monitor the changes of glycoform of serum alpha1-acid glycoprotein (AGP) in alcoholic liver cirrhosis (ALC) patients which could be predicted as serological marker for diagnosis. AGP was isolated from the albumin depleted pooled sera of ALC patients as well as controls by monoclonal anti-AGP affinity column. The altered glycoforms of AGP was determined by HPLC mapping followed by mass spectrometry and GALAXY database search. N-glycans released from AGP by hydrazinolysis were labeled with 2-aminopyridine and separated by three successive HPLC columns, viz., DEAE, ODS and amide silica. Significant decrease of sialylation level was observed by HPLC in ALC patients group with respect to controls. The binding of SNA with AGP was found to be less in patients group than control by ELISA and lectin blotting using Sambucus nigra agglutinin (SNA). This variation of N-linked glycoforms and decreased level of sialic acid in AGP could be valuable for the diagnosis of ALC besides clinical examination and routine laboratory investigation that could be helpful for treatment strategy.

Glycosylation of Acute Phase Proteins: A Promising Disease Biomarker

All diseases including autoimmune diseases, chronic liver diseases and cancer are a serious health problem worldwide. The current gold standard to assess malignant changes in cells and structural liver damage is through a biopsy, which has several disadvantages. A non-invasive simple test to diagnose malignancy and liver pathology would be highly desirable. Protein glycosylation has drawn the attention of many researchers with an aim to achieve this goal. Glycosylation is the post-translational modification of many secreted proteins and it has been known for decades that structural changes in the glycan structures of serum proteins are an indication of carcinogenesis and liver damage. The aim of this paper is to give an overview of the altered protein glycosylation in different etiologies of liver diseases, autoimmune diseases, and cancer. Although individual carcinoma and liver diseases have their own specific markers, the same alteration seems to reappear continuously in all malignant transformation and liver diseases like hyperfucosylation, sialylation, increased branching and bisecting N-acetylglucosamine.

A glucose/mannose binding lectin from litchi (Litchi chinensis) seeds: Biochemical and biophysical characterizations

Background Lectins are highly important biomolecules to study several biological processes. A novel α-D-glucose/mannose specific lectin was isolated from the seeds of litchi fruits (Litchi chinensis) and its various biophysical and biochemical properties were studied. Methods Purification was done by successive Sephadex G 100 and Con A-Sepharose 4B affinity chromatography. SDS-PAGE, Surface Plasmon Resonance (SPR), steady state absorbance, fluorescence, time-correlated single-photon counting, circular dichroism and antibiofilm activity by measuring total protein estimation and azocasein degradation assay have been performed. Results The purified lectin is a homodimer of molecular mass ~ 54 kDa. The amount of lectin required for hemagglutination of normal human O erythrocytes was 6.72 µg/ml. Among the saccharides tested, Man-α-(1,6)-Man was found to be the most potent inhibitor (0.01 mM) determined by hemagglutination inhibition assay. Steady state and time resolved fluorescence measurements revealed that litchi lectin formed ground state complex with maltose (Ka=4.9 (±0.2)×104 M−1), which indicated static quenching (Stern-Volmer (SV) constant Ksv=4.6 (±0.2)×104 M−1). CD measurements demonstrated that litchi lectin showed no overall conformational change during the binding process with maltose. The lectin showed antibiofilm activity against Pseudomonus aeruginosa. Conclusions A novel homodimeric lectin has been purified from the seeds of litchi fruits (Litchi chinensis) having specificity for α-d-glucose/mannose. The thermodynamics and conformational aspects of its interaction with maltose have been studied in detail. The antibiofilm activity of this lectin towards Pseudomonus aeruginosa has been explored. General significance The newly identified litchi lectin is highly specific for α-d-glucose/mannose with an important antibiofilm activity towards Pseudomonus aeruginosa.