8 research outputs found

    CD44 Binding to Hyaluronic Acid Is Redox Regulated by a Labile Disulfide Bond in the Hyaluronic Acid Binding Site

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    <div><p>CD44 is the primary leukocyte cell surface receptor for hyaluronic acid (HA), a component of the extracellular matrix. Enzymatic post translational cleavage of labile disulfide bonds is a mechanism by which proteins are structurally regulated by imparting an allosteric change and altering activity. We have identified one such disulfide bond in CD44 formed by Cys77 and Cys97 that stabilises the HA binding groove. This bond is labile on the surface of leukocytes treated with chemical and enzymatic reducing agents. Analysis of CD44 crystal structures reveal the disulfide bond to be solvent accessible and in the–LH hook configuration characteristic of labile disulfide bonds. Kinetic trapping and binding experiments on CD44-Fc chimeric proteins show the bond is preferentially reduced over the other disulfide bonds in CD44 and reduction inhibits the CD44-HA interaction. Furthermore cells transfected with CD44 no longer adhere to HA coated surfaces after pre-treatment with reducing agents. The implications of CD44 redox regulation are discussed in the context of immune function, disease and therapeutic strategies.</p></div

    Aligned 3D total ion current chromatograms using Progenesis QI in the area of chromatographic space where MPB labelled GFETCR elutes for (A) control 2B4 cells and (B) TCEP-HCl reduced 2B4 cells.

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    <p>The peak corresponding to MPB labelled GFETCR is outlined in black and is visibly larger in the TCEP-HCl reduced cells. Other peptides are seen in both the control and TCEP-HCl reduced samples, ones with increased area after TCEP-HCl treatment suggests they are from proteins containing labile disulfide bonds.</p

    TCEP-HCl reduction of CD44 inhibits binding to HA.

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    <p>(A) hCD44-Fc and (B) mCD44-Fc binding to plates coated with 50 μg/ml HA. Columns represent non reduced control CD44-Fc (dark grey) and TCEP-HCl reduced CD44-Fc (light grey). Mean and SEM of peptide chromatographic areas from three experiments are shown, the differences between non reduced and TCEP-HCl reduced chimera: *, P < 0.05; **, P < 0.01. Titrations of Trx1 reduced (C) human CD44-Fc and (D) mouse CD44-Fc binding to plates coated with 50 μg/ml HA. For both mouse and human CD44-Fc titrations the binding curves represent non reduced control CD44-Fc (black circles) and Trx1 reduced CD44-Fc (black squares). Binding curves are constructed from data of three independent experiments and equilibrium dissociation constants were determined by fitting to a single binding site model.</p

    (A) Crystal structure of the HABD of murine CD44 in complex with an HA 8-mer (adapted from PDB ID:2JCQ).

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    <p>The protein backbone is represented as a blue cartoon. The HA 8-mer is shown as pink sticks. Disulfide bonds are shown as yellow sticks and the cysteine residues from which they are formed are numbered according to their position in the mouse CD44 sequence (human numbering is shown in parenthesis). (B) For each disulfide bond dark grey bars show solvent accessibility and light grey bars show bond strain energy calculated from the PDB coordinates (PDB ID:2JCQ for mouse and 1UUH for human, the coordinates for HA were removed from 2JCQ prior to analysis) <b>using</b> (<a href="http://powcs.med.unsw.edu.au/research/adult-cancer-program/services-resources/disulfide-bond-analysis-tool/disulfide-bond" target="_blank">http://powcs.med.unsw.edu.au/research/adult-cancer-program/services-resources/disulfide-bond-analysis-tool/disulfide-bond</a>).</p

    Levels of CD44 determined by flow cytometry analysis of (A) untransfected CHO K1 cells and (B) hCD44 transfected CHO cells.

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    <p>Light grey trace is isotype control, black trace is anti-CD44. (C) Non reduced SDS-PAGE gel showing reduced proteins on the surface of hCD44 transfected CHO cells after reduction (green bands) overlaid with a western blot of CD44 (red). (D) non reduced SDS-PAGE gel of eluted proteins which contain reduced disulfide bonds (green bands) from a CD44 immunoprecipitation of hCD44 transfected CHO cells that had been reduced with either TCEP-HCl or Trx1.</p

    Cell adhesion assay showing the effect of TCEP-HCl and Trx1 reduction of untransfected and hCD44 transfected CHO cells binding to HA coated plates.

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    <p>Binding data are compositions of three independent experiments all normalised assuming non reduced CHO-hCD44 binding to HA coated wells as 100% response. Mean and SEM of total fluorescence are shown. The differences from control: *, P < 0.05; **, P < 0.01; ***, P < 0.001; ***, P < 0.0001</p

    Cell types and treatments for which the allosteric disulfide has been observed.

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    <p><sup>a</sup> gamma-interferon-inducible lysosomal thiol reductase.</p><p><sup>b</sup> MLR supplemented with staphylococcal enterotoxin A super antigen.</p><p><sup>c</sup>–is not detected in the mass spectrometry run.</p><p>Cell types and treatments for which the allosteric disulfide has been observed.</p
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