Abstract
The extreme size, extensive glycosylation, and gel-forming nature of mucins make them a challenge to work with, and methodologies for the detection of mucins must take into consideration these features to ensure that one obtains both accurate and meaningful results. In understanding and appreciating the nature of mucins, this affords the researcher a valuable toolkit which can be used to full advantage in detecting, quantifying, and visualising mucins. The employment of a combinatorial approach to mucin detection, using antibody, chemical, and lectin detection methods, allows important information to be gleaned regarding the size, extent of glycosylation, specific mucin species, and distribution of mucins within a given sample. In this chapter, the researcher is guided through considerations into the structure of mucins and how this both affects the detection of mucins and can be used to full advantage. Techniques including ELISA, dot/slot blotting, and Western blotting, use of lectins and antibodies in mucin detection on membranes as well as immunohistochemistry and immunofluorescence on both tissues and cells grown on Transwell™ inserts are described. Notes along with each section advice the researcher on best practice and describe any associated limitations of a particular technique from which the researcher can further develop a particular protocol.
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References
Hattrup, C. L., and Gendler, S. J. (2008) Structure and function of the cell surface (tethered) mucins. Annu Rev Physiol 70, 431–457.
Thornton, D. J., Rousseau, K., and McGuckin, M. A. (2008) Structure and function of the polymeric mucins in airways mucus. Annu Rev Physiol 70, 459–486.
Desseyn, J. L., Aubert, J. P., Van Seuningen, I., Porchet, N., and Laine, A. (1997) Genomic organization of the 3′ region of the human mucin gene MUC5B. J Biol Chem 272, 16873–16883.
Desseyn, J. L., Buisine, M. P., Porchet, N., Aubert, J. P., and Laine, A. (1998) Genomic organization of the human mucin gene MUC5B. cDNA and genomic sequences upstream of the large central exon. J Biol Chem 273, 30157–30164.
Buisine, M. P., Desseyn, J. L., Porchet, N., Degand, P., Laine, A., and Aubert, J. P. (1998) Genomic organization of the 3′-region of the human MUC5AC mucin gene: additional evidence for a common ancestral gene for the 11p15.5 mucin gene family. Biochem J 332 (Pt 3), 729–738.
Desseyn, J. L., Guyonnet-Duperat, V., Porchet, N., Aubert, J. P., and Laine, A. (1997) Human mucin gene MUC5B, the 10.7-kb large central exon encodes various alternate subdomains resulting in a super-repeat. Structural evidence for a 11p15.5 gene family. J Biol Chem 272, 3168–3178.
Carlstedt, I., Lindgren, H., Sheehan, J. K., Ulmsten, U., and Wingerup, L. (1983) Isolation and characterization of human cervical-mucus glycoproteins. Biochem J 211, 13–22.
Carlstedt, I., and Sheehan, J. K. (1984) Is the macromolecular architecture of cervical, respiratory and gastric mucins the same? Biochem Soc Trans 12, 615–617.
Mantle, M., and Allen, A. (1978) A colorimetric assay for glycoproteins based on the periodic acid/Schiff stain [proceedings]. Biochem Soc Trans 6, 607–609.
Thornton, D. J., Howard, M., Devine, P. L., and Sheehan, J. K. (1995) Methods for separation and deglycosylation of mucin subunits. Anal Biochem 227, 162–167.
Gerken, T. A., Gupta, R., and Jentoft, N. (1992) A novel approach for chemically deglycosylating O-linked glycoproteins. The deglycosylation of submaxillary and respiratory mucins. Biochemistry 31, 639–648.
Raju, T. S., and Davidson, E. A. (1994) New approach towards deglycosylation of sialoglycoproteins and mucins. Biochem Mol Biol Int 34, 943–954.
Hong, J. C., and Kim, Y. S. (2000) Alkali-catalyzed beta-elimination of periodate-oxidized glycans: a novel method of chemical deglycosylation of mucin gene products in paraffin embedded sections. Glycoconj J 17, 691–703.
Kim, Y. S., Gum, J., Jr., and Brockhausen, I. (1996) Mucin glycoproteins in neoplasia. Glycoconj J 13, 693–707.
Young, P. R. (1989) Enhancement of immunoblot staining using a mixed chromogenic substrate. J Immunol Methods 121, 295–296.
Randell, S. H., Walstad, L., Schwab, U. E., Grubb, B. R., and Yankaskas, J. R. (2001) Isolation and culture of airway epithelial cells from chronically infected human lungs. In Vitro Cell Dev Biol Anim 37, 480–489.
Holmen, J. M., Karlsson, N. G., Abdullah, L. H., Randell, S. H., Sheehan, J. K., Hansson, G. C., and Davis, C. W. (2004) Mucins and their O-Glycans from human bronchial epithelial cell cultures. Am J Physiol Lung Cell Mol Physiol 287, L824–L834.
Gray, T. E., Guzman, K., Davis, C. W., Abdullah, L. H., and Nettesheim, P. (1996) Mucociliary differentiation of serially passaged normal human tracheobronchial epithelial cells. Am J Respir Cell Mol Biol 14, 104–112.
Diamandis, E. P., Christopoulos, T.K. (1989) Immunoassay, Academic Press, San Diego.
McGuckin, M. A., Devine, P. L., Ramm, L. E., and Ward, B. G. (1994) Factors effecting the measurement of tumor-associated MUC1 mucins in serum. Tumour Biol 15, 33–44.
Thornton, D. J., Holmes, D. F., Sheehan, J. K., and Carlstedt, I. (1989) Quantitation of mucus glycoproteins blotted onto nitrocellulose membranes. Anal Biochem 182, 160–164.
Ayre, D., Hutton, D. A., and Pearson, J. P. (1994) The use of wheat germ agglutinin to improve binding of heterogeneous mucin species to nitrocellulose membranes. Anal Biochem 219, 373–375.
Jay, G. D., Culp, D. J., and Jahnke, M. R. (1990) Silver staining of extensively glycosylated proteins on sodium dodecyl sulfate-polyacrylamide gels: enhancement by carbohydrate-binding dyes. Anal Biochem 185, 324–330.
Acknowledgements
Michael McGuckin is supported by an NHMRC Senior Research Fellowship. Ceri Harrop is supported by a grant from the Dr. Hadwen Trust for Humane Research.
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Harrop, C.A., Thornton, D.J., McGuckin, M.A. (2012). Detecting, Visualising, and Quantifying Mucins. 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_3
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DOI: https://doi.org/10.1007/978-1-61779-513-8_3
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