Browsing by Author "Chacko, BK"

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    Dextran-binding human plasma antibody recognizes bacterial and yeast antigens and is inhibited by glucose concentrations reached in diabetic sera
    (MOLECULAR IMMUNOLOGY, 2003)
    Dextran-binding antibody was isolated in high yield from plasma of all 40 blood donors screened in a South Indian population. The antibody was purified by a single step affinity chromatography on Sephadex G100 using 1-O-methyl alpha-D-glucoside as eluant. Analysis of protein peaks obtained in size exclusion high pressure liquid chromatography (HPLC) revealed dominance of IgG and suggested the presence of polymeric IgA in this antibody. Methyl and para-nitrophenyl alpha-D-glucosides, in contrast to their beta-anomers, were very efficient inhibitors of binding of this antibody to dextran. Galactose and glucose were equally good inhibitors. Among disaccharide inhibitors sucrose was more efficient than maltose or melibiose. Hemoglobin artificially glycosylated to contain covalently-linked glucose or alpha-anomeric galactose was sugar-specifically recognized by this antibody. Galactose moieties in glycoproteins or polysaccharides were, however, not recognized. The dextran-binding antibody bound sugar-specifically to glycoconjugates from yeast (Saccharomyces cerevisiae) and to lipopolysaccharides from Klebsiella and group A Streptococci, but not to lipopolysaccharides from E. coli. Inhibition studies suggested glucose moiety with unsubstituted C2 and C4 and alpha-anomeric C1 as ideal for recognition by the dextran-binding antibody. Concentration of glucose required for 50% inhibition of binding of the purified antibody to polystyrene-coated dextran in phosphate buffered saline was above the glucose concentrations in normal sera, but well below those reached in diabetic sera. Binding of the antibody from dialysed plasma to immobilized dextran was lowered only marginally in presence of glucose at 4.5 mM (which nears normal serum glucose concentrations), but substantially in presence of the sugar at 20 mM and above which are reached in diabetic sera. If verified in vivo, inhibition of this antibody by high serum glucose may possibly be among reasons for the increased susceptibility of diabetics to infection. (C) 2003 Elsevier Science Ltd. All rights reserved.
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    Dual Specificity of Human Plasma Lactose-Binding Immunoglobulin to Anomers of Terminal Galactose Enables Recognition of Desialylated Lipoprotein(a) and Xenoantigens
    (SCANDINAVIAN JOURNAL OF IMMUNOLOGY, 2014) Sabarinath, PS; Chacko, BK; Appukuttan, PS
    Human plasma lactose-binding immunoglobulin (LIg) isolated by affinity chromatography on lactose-Sepharose was largely IgG with significant IgA and IgM contents. LIg-mediated agglutination of desialylated human RBC was inhibited equally by the alpha- and beta-anomers of methyl galactoside. Recognition of either the terminal alpha-galactose (TAG)-containing glycans of bovine thyroglobulin or the N-acetyl lactosamine (LacNAc)-terminating glycans of asialofetuin by LIg was inhibitable nearly as much by the alpha-galactoside melibiose as by the beta-galactoside lactose. Melibiose covalently conjugated to protein and coated on polystyrene wells captured several times more LIg molecules than its lactose analogue. LIg binding to bovine thyroglobulin or rabbit RBC membrane proteins, both bearing TAG was substantially reduced by prior treatment of the proteins with alpha-galactosidase to remove TAG though enzyme-treated glycans contained newly exposed LacNAc moieties. Desialylated O-linked oligosaccharides, however, were no ligand for LIg. Unlike LDL, plasma lipoprotein(a) [Lp(a)] coated on polystyrene well and desialylated by neuraminidase was recognized by LIg through terminal LacNAc moieties exposed by the enzyme on its apo(a) subunit. Further, same amount of added fluorescence-labelled LIg formed significantly more immune complex with Lp(a) in high Lp(a) plasma than in low Lp(a) plasma. Results suggest (1) possibility of a role for LIg in combating non-primate molecules and cells bearing TAG moiety and (2) a mechanism for Lp(a)-mediated vascular injury as diabetes, infections and inflammations induce greater release of neuraminidase into circulation.
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    Glutaraldehyde cross-linking of lectins to marker enzymes: Protection of binding site by specific sugars
    (INDIAN JOURNAL OF BIOCHEMISTRY & BIOPHYSICS, 2000)
    The role of bound specific sugars in protecting the sugar binding activity of several galactose binding proteins during their covalent conjugation to horse radish peroxidase by glutaraldehyde-mediated cross-linking was examined by: a) affinity matrix binding of the conjugate, b) enzyme linked lectin assay and c) hemagglutination assay. During conjugation using 1% glutaraldehyde, protection of jack fruit (Artocarpus integrifolia) lectin (jacalin) activity depended on concentration of specific sugar present during conjugation; optimum protection was offered by 50 mM galactose. This indicated the presence of one or more primary groups at the binding site of jacalin, which is (are) essential for sugar binding. On the other hand, such essential amino group(s) was not indicated at the sugar binding site of the peanut lectin, bovine heart galectin or of the human serum anti alpha-galactoside antibody, since exclusion of sugar during their conjugation to HRP did not diminish sugar binding activity. The differential behavior is discussed in the light of reported differences in sugar specificities. Results indicated that sugar mediated blocking of active site may be used in characterization of the latter in lectins.
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    Peanut (Arachis hypogaea) lectin recognizes alpha-linked galactose, but not N-acetyl lactosamine in N-linked oligosaccharide terminals
    (INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES, 2001)
    Peanut (Arachis hypogaea) agglutinin (PNA) is extensively used as tumour marker as it strongly recognises the cancer specific T antigen (Ga1 beta --> 3GalNAc-), but not its sialylated version. However, an additional specificity towards Gal beta1 --> 4GlcNAc (LacNAc), which is not tumour specific, had been attributed to PNA. For correct interpretation of lectin histochemical results we examined PNA sugar specificity using naturally occurring or semi-synthetic glycoproteins, matrix-immobilised galactosides and lectin-binding tissue glycoproteins, rather than mono- or disaccharides as ligands. Dot-blots, transfer blots or polystyrene plate coatings of the soluble glycoconjugates were probed with horse-radish peroxidase (HRP) conjugates of PNA and other lectins of known specificity. Modifications of PNA-binding glycoproteins, including selective removal of O-linked oligosaccharides and treatment with glycosidases revealed that Gal beta1 --> 4GlcNAc (LacNAc) was ineffective while terminal alpha -linked galactose (TAG) as well as exposed T antigen (Gal beta1 --> 3 GalNAc-) was excellent as sugar moiety in glycoproteins for their recognition by PNA. When immobilised, melibiose was superior to lactose in PNA binding. Results were confirmed using TAG-specific human serum anti-a-galactoside antibody. (C) 2001 Elsevier Science B.V. All rights reserved.
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    Peanut (Arachis hypogaea) lectin: Use in quantitation of desialylation of glycoproteins
    (INDIAN JOURNAL OF BIOCHEMISTRY & BIOPHYSICS, 2000) Chacko, BK; Appukuttan, PS
    A high affinity lectin from an easily available source, peanut (Arachis hypogaea) agglutinin (PNA) which specifically recognizes desialylated versions of sialylated oligosaccharides is a unique tool in glycoconjugate biotechnology. By a single step affinity chromatography on cross-linked guar galactomannan, PNA was purified to homogeneity with 19 times higher hemagglutinating activity than the sample prepared by existing methods involving defatting with organic solvents. Agglutinating activity of the new preparation remained unchanged for at least 6 months while PNA prepared from defatted seed lost activity within one week. Glycoproteins desialylated to varying degrees were prepared by treating bovine fetuin with 0.1 N H2SO4 at 80 degreesC for durations of 10 seconds and above. Enzyme-linked lectin assay of desialylation of differentially desialylated glycoproteins coated on microplates, using horse radish peroxidase (HRP) conjugate of PNA (PNA-HRP), along with sialic content assay revealed that PNA can be used as a quantitative probe for assay of desialylation in sialylated glycoproteins.