title: Secondary Structure and Glycosylation of Mucus Glycoproteins by Raman Spectroscopies.
abstract: 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.
authors: Wellcome Trust Centre for Cell-Matrix Research, Faculty of Biology, Medicine and Health, University of Manchester , Manchester M13 9PL, United Kingdom.
authors: H
authors: H
authors: Davies-Strickleton
authors: Institute of Physical Chemistry and Abbe Center of Photonics, Friedrich-Schiller University Jena , Helmholtzweg 4, 07743 Jena, Germany.
authors: Prabha
authors: P
authors: Singh
authors: Leibniz Institute of Photonic Technology , Albert-Einstein-Strasse 9, 07745 Jena, Germany.
authors: Tanja
authors: T
authors: Deckert-Gaudig
authors: Institute of Physical Chemistry and Abbe Center of Photonics, Friedrich-Schiller University Jena , Helmholtzweg 4, 07743 Jena, Germany.|Leibniz Institute of Photonic Technology , Albert-Einstein-Strasse 9, 07745 Jena, Germany.
authors: Volker
authors: V
authors: Deckert
authors: Wellcome Trust Centre for Cell-Matrix Research, Faculty of Biology, Medicine and Health, University of Manchester , Manchester M13 9PL, United Kingdom.
authors: Karine
authors: K
authors: Rousseau
authors: Wellcome Trust Centre for Cell-Matrix Research, Faculty of Biology, Medicine and Health, University of Manchester , Manchester M13 9PL, United Kingdom.
authors: Caroline E
authors: CE
authors: Ridley
authors: School of Chemistry, Manchester Institute of Biotechnology, University of Manchester , Manchester M1 7DN, United Kingdom.
authors: Sarah E
authors: SE
authors: Dowd
authors: School of Chemistry, Manchester Institute of Biotechnology, University of Manchester , Manchester M1 7DN, United Kingdom.
authors: Andrew J
authors: AJ
authors: Doig
authors: Unilever Discover , Sharnbrook, Bedfordshire MK44 1LQ, United Kingdom.
authors: Paul D A
authors: PD
authors: Pudney
authors: Wellcome Trust Centre for Cell-Matrix Research, Faculty of Biology, Medicine and Health, University of Manchester , Manchester M13 9PL, United Kingdom.
authors: David J
authors: DJ
authors: Thornton
authors: Wellcome Trust Centre for Cell-Matrix Research, Faculty of Biology, Medicine and Health, University of Manchester , Manchester M13 9PL, United Kingdom.|School of Science, RMIT University , Melbourne, Victoria 3001, Australia.
authors: Ewan W
authors: EW
authors: Blanch
mesh_terms: D006031:Glycosylation; D064368:Healthy Volunteers; D006801:Humans; D018625:Microscopy, Atomic Force; D055257:Mucin-5B / Q000737:chemistry* / Q000302:isolation & purification; D017433:Protein Structure, Secondary; D013059:Spectrum Analysis, Raman
chemical_list: C524578:MUC5B protein, human; D055257:Mucin-5B