Abstract
Secreted and transmembrane mucins are important components of innate defence at the body’s mucosal surfaces. The secreted mucins are large, polymeric glycoproteins, which are largely responsible for the gel-like properties of mucus secretions. The cell-tethered mucins, however, are monomeric but are typically composed of two subunits, a larger extracellular subunit which is heavily glycosylated while the smaller more sparsely glycosylated subunit has a short extracellular region, a single-pass transmembrane domain, and a cytoplasmic tail. These two families of mucins represent high-molecular-weight glycoproteins containing serine and threonine-rich domains that are the attachment sites for large numbers of O-glycans. The high-M r and high sugar content have been exploited for the separation of mucins from the majority of components in mucus secretions. In this chapter, we describe current and well-established methods (caesium chloride density-gradient centrifugation, gel-filtration and anion-exchange chromatography, and agarose gel electrophoresis) for the extraction and purification of gel-forming and cell-surface mucins which can subsequently be used for a variety of structural and functional studies.
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References
Lindén SK, Sutton P, Karlsson NG, Korolik V, McGuckin MA. (2008) Mucins in the mucosal barrier to infection. Mucosal. Immunol. 1, 183–197.
Thornton DJ, Rousseau K, McGuckin MA. (2008) Structure and function of the polymeric mucins in airways mucus. Annu. Rev. Physiol. 70, 459–486.
Hattrup CL, Gendler SJ. (2008) Structure and function of the cell surface (tethered) mucins. Annu. Rev. Physiol. 70, 431–457.
Holmén JM, Karlsson NG, Abdullah LH, Randall SH, Sheehan JK, Hansson GC, Davis, CW. (2004) Mucins and their O-Glycans from human bronchial epithelial cell cultures. Am J Physiol Lung Cell Mol Physiol. 287, 824–834.
Kirkham S, Sheehan JK, Knight D, Richardson PS, Thornton DJ. (2002) Heterogeneity of airways mucus: variations in the amounts and glycoforms of the major oligomeric mucins MUC5AC and MUC5B. Biochem. J. 361, 537–546.
Thornton DJ, Khan N, Mehrotra R, Howard M, Veerman E, Packer NH, Sheehan JK. (1999) Salivary mucin MG1 is comprised almost entirely of different glycosylated forms of the MUC5B gene product. Glycobiology 9, 293–302.
Sherblom A, Carraway KL. (1980) A complex of two cell surface glycoproteins from ascites mammary adenocarcinoma cells. J. Biol. Chem. 255, 12051–12059.
Hull SR, Sheng Z, Vanderpuye O, David C, Carraway KL. (1990) Isolation and partial characterization of ascites sialoglycoprotein-2 of the cell surface sialomucin complex of 13762 rat mammary adenocarcinoma cells. Biochem. J. 265, 121–129.
Davies JR, Carlstedt I. (1999) Isolation of large gel-forming mucins. In ‘Methods in Molecular Biology. Glycoprotein methods and protocols.’ pp 3–13. Humana Press NJ, USA.
Herrmann A, Davies JR, Lindell G, Mårtensson S, Packer N, Swallow D, Carlstedt I. (1999) Studies on the “insoluble” glycoprotein complex from human colon. Identification of reduction-insensitive MUC2 oligomers and C-terminal cleavage. J. Biol. Chem. 274, 15828–15836.
Hovenberg HW, Davies JR, Herrmann A, Lindén CJ, Carlstedt I. (1996) MUC5AC, but not MUC2, is a prominent mucin in respiratory secretions. Glycoconj. J. 13, 839–847.
Hovenberg HW, Davies JR, Carlstedt I. (1996) Different mucins are produced by the surface epithelium and the submucosa in human trachea: identification of MUC5AC as a major mucin from the goblet cells. Biochem. J. 318, 319–324.
Thornton DJ, Carlstedt I, Howard M, Devine PL, Price M, Sheehan JK (1996) Respiratory mucins: identification of core proteins and glycoforms. Biochem. J. 316, 967–975.
Wickström C, Davies JR, Ericsen G, Veermann E, Carlstedt I. (1998) MUC5B is a major gel-forming, oligomeric mucin from human salivary gland, respiratory tract and endocervix: identification of glycoforms and C-terminal cleavage. Biochem. J. 334, 685–693.
Thornton DJ, Howard M, Khan N, Sheehan JK. (1997) Identification of two glycoforms of the MUC5B mucin in human respiratory mucus. Evidence for a cysteine-rich sequence repeated within the molecule. J. Biol. Chem. 272, 9561–9566.
Thornton DJ, Gray T, Nettesheim P, Howard M, Koo JS, Sheehan JK. (2000) Characterisation of the MUC5AC and MUC5B mucins synthesised by cultured normal human tracheobronchial epithelial cells. Am. J. Physiol. Lung Cell. Mol. Physiol. 278, L1118–1128.
Rousseau K, Wickström C, Whitehouse DB, Carlstedt I, Swallow DS (2003) New monoclonal antibodies to non-glycosylated domains of the secreted mucins MUC5B and MUC7. Hybrid. Hybridom. 22, 293–299.
Sylvester PA, Myerscough N, Warren BF, Carlstedt I, Corfield AP, Durdey P, Thomas MG. (2001) Differential expression of the chromosome 11 mucin genes in colorectal cancer. J. Pathol. 195, 327–335.
Wickström C, Christersson C, Davies JR, Carlstedt I. (2000) Macromolecular organization of saliva: identification of ‘insoluble’ MUC5B assemblies and non-mucin proteins in the gel phase. Biochem. J. 351, 421–428.
Davies JR, Kirkham S, Svitacheva N, Thornton DJ, Carlstedt I. (2007) MUC16 is produced in tracheal surface epithelium and submucosal glands and is present in secretions from normal human airway and cultured bronchial epithelial cells. Int. J. Biochem. Cell Biol. 39, 1943–1954.
Acknowledgements
The authors acknowledge support from The Crafoord Foundation, Sweden; The Knowledge Foundation (Biofilms—Research Centre for Biointerfaces), Malmö University, Sweden; The Swedish Patent Revenue Fund for Research in Preventive Odontology; The Swedish Dental Association; BBSRC; The Wellcome Trust; MRC; and the Horserace Betting Levy Board, UK.
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Davies, J.R., Wickström, C., Thornton, D.J. (2012). Gel-Forming and Cell-Associated Mucins: Preparation for Structural and Functional Studies. In: McGuckin, M., Thornton, D. (eds) Mucins. Methods in Molecular Biology, vol 842. Humana Press. https://doi.org/10.1007/978-1-61779-513-8_2
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DOI: https://doi.org/10.1007/978-1-61779-513-8_2
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