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Publisher: Columbus, Ohio : American Chemical Society × Subject: Proteins ×

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Now showing 1 - 3 of 3
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    Secondary Structure and Glycosylation of Mucus Glycoproteins by Raman Spectroscopies
    (Columbus, Ohio : American Chemical Society, 2016) Davies, Heather S.; Singh, Prabha; Deckert-Gaudig, Tanja; Deckert, Volker; Rousseau, Karine; Ridley, Caroline E.; Dowd, Sarah E.; Doig, Andrew J.; Pudney, Paul D. A.; Thornton, David J.; Blanch, Ewan W.
    The major structural components of protective mucus hydrogels on mucosal surfaces are the secreted polymeric gel-forming mucins. The very high molecular weight and extensive O-glycosylation of gel-forming mucins, which are key to their viscoelastic properties, create problems when studying mucins using conventional biochemical/structural techniques. Thus, key structural information, such as the secondary structure of the various mucin subdomains, and glycosylation patterns along individual molecules, remains to be elucidated. Here, we utilized Raman spectroscopy, Raman optical activity (ROA), circular dichroism (CD), and tip-enhanced Raman spectroscopy (TERS) to study the structure of the secreted polymeric gel-forming mucin MUC5B. ROA indicated that the protein backbone of MUC5B is dominated by unordered conformation, which was found to originate from the heavily glycosylated central mucin domain by isolation of MUC5B O-glycan-rich regions. In sharp contrast, recombinant proteins of the N-terminal region of MUC5B (D1-D2-D′-D3 domains, NT5B), C-terminal region of MUC5B (D4-B-C-CK domains, CT5B) and the Cys-domain (within the central mucin domain of MUC5B) were found to be dominated by the β-sheet. Using these findings, we employed TERS, which combines the chemical specificity of Raman spectroscopy with the spatial resolution of atomic force microscopy to study the secondary structure along 90 nm of an individual MUC5B molecule. Interestingly, the molecule was found to contain a large amount of α-helix/unordered structures and many signatures of glycosylation, pointing to a highly O-glycosylated region on the mucin.
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    Detection of Protein Glycosylation Using Tip-Enhanced Raman Scattering
    (Columbus, Ohio : American Chemical Society, 2016) Cowcher, David P.; Deckert-Gaudig, Tanja; Brewster, Victoria L.; Ashton, Lorna; Deckert, Volker; Goodacre, Royston
    The correct glycosylation of biopharmaceutical glycoproteins and their formulations is essential for them to have the desired therapeutic effect on the patient. It has recently been shown that Raman spectroscopy can be used to quantify the proportion of glycosylated protein from mixtures of native and glycosylated forms of bovine pancreatic ribonuclease (RNase). Here we show the first steps toward not only the detection of glycosylation status but the characterization of glycans themselves from just a few protein molecules at a time using tip-enhanced Raman scattering (TERS). While this technique generates complex data that are very dependent on the protein orientation, with the careful development of combined data preprocessing, univariate and multivariate analysis techniques, we have shown that we can distinguish between the native and glycosylated forms of RNase. Many glycoproteins contain populations of subtly different glycoforms; therefore, with stricter orientation control, we believe this has the potential to lead to further glycan characterization using TERS, which would have use in biopharmaceutical synthesis and formulation research.
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    Polymer Brush-Functionalized Chitosan Hydrogels as Antifouling Implant Coatings
    (Columbus, Ohio : American Chemical Society, 2017) Buzzacchera, Irene; Vorobii, Mariia; Kostina, Nina Yu; de Los Santos Pereira, Andres; Riedel, Tomáš; Bruns, Michael; Ogieglo, Wojciech; Möller, Martin; Wilson, Christopher J.; Rodriguez-Emmenegger, Cesar
    Implantable sensor devices require coatings that efficiently interface with the tissue environment to mediate biochemical analysis. In this regard, bioinspired polymer hydrogels offer an attractive and abundant source of coating materials. However, upon implantation these materials generally elicit inflammation and the foreign body reaction as a consequence of protein fouling on their surface and concomitant poor hemocompatibility. In this report we investigate a strategy to endow chitosan hydrogel coatings with antifouling properties by the grafting of polymer brushes in a "grafting-from" approach. Chitosan coatings were functionalized with polymer brushes of oligo(ethylene glycol) methyl ether methacrylate and 2-hydroxyethyl methacrylate using photoinduced single electron transfer living radical polymerization and the surfaces were thoroughly characterized by XPS, AFM, water contact angle goniometry, and in situ ellipsometry. The antifouling properties of these new bioinspired hydrogel-brush coatings were investigated by surface plasmon resonance. The influence of the modifications to the chitosan on hemocompatibility was assessed by contacting the surfaces with platelets and leukocytes. The coatings were hydrophilic and reached a thickness of up to 180 nm within 30 min of polymerization. The functionalization of the surface with polymer brushes significantly reduced the protein fouling and eliminated platelet activation and leukocyte adhesion. This methodology offers a facile route to functionalizing implantable sensor systems with antifouling coatings that improve hemocompatibility and pave the way for enhanced device integration in tissue.