Abstract
Specialized secretory cells known as goblet cells in the intestine and respiratory epithelium are responsible for the secretion of mucins. Mucins are large heavily glycosylated proteins and typically have a molecular mass higher than 106 Da. These large proteins are densely substituted with short glycan chains, which have many important functional roles including determining the hydration and viscoelastic properties of the mucus gel that lines and protects the intestinal epithelium. In this protocol, we comprehensively describe the method for extraction of murine mucus and its analysis by agarose gel electrophoresis. Additionally we describe the use of High Iron Diamine-Alcian Blue, Periodic Acid Schiff’s-Alcian Blue and immune–staining methods to identify and differentiate between the different states of glycosylation on these mucin glycoproteins, in particular with a focus on sulphation and sialylation.
Keywords: Sialylation, Sulphation, Glycosylation, Mucin, Secreted mucin, Stored mucin, High Iron Diamine
Background
A layer of mucus protects the intestinal epithelium and primarily consists of mucins, water, proteins and inorganic salts. The viscous and gel-like properties of the mucus barrier, which enable it to physically protect and lubricate the mucous membranes, are conferred mainly by mucins. Mucins are large heavily glycosylated proteins and typically have a molecular mass higher than 106 Da. Mucins, however, are predominantly decorated with O-glycan sugars, which accounts for up to 80% of their molecular weight. The diverse site-specific and mucin-specific glycosylation patterns influence the properties of the mucin and therefore the mucus gel. It is well known that mucin glycosylation is altered in infection and disease (Arike et al., 2017; Hasnain et al., 2017). Here we describe methods to assess the amounts of intestinal mucins in murine models and assess the changes in glycosylation with a particular focus on sialylation and sulphation of mucins. Previous methods have not differentiated between mucins isolated from the secreted barrier or those stored within the goblet cells. Methods described here can be employed to assess the changes in secreted or goblet cell-stored mucins in each individual animal. Moreover, using high-iron diamine staining changes in amounts of mucins can also be correlated this with changes in mucin glycosylation.
Materials and Reagents
Equipment
Software
Procedure
An outline is provided for all procedures included in this protocol (Figure 3) Figure 3. Outlines of the procedures described
To determine the changes in the glycosylation of mucins, two separate methods can be utilized which are described in detail below. Periodic Acid Schiff’s-Alcian blue staining can differentiate between acidic and neutral mucins, whereas High Iron Diamine-Alcian Blue staining can be used to identify the changes in mucin sulphation and sialylation.
Data analysis
Recipes
Acknowledgments
A University of Queensland Postdoctoral Fellowship currently supports Sumaira Hasnain. The University Of Queensland Foundation Of Research Excellence Award to Sumaira Hasnain supports Ran Wang. The diamine method was originally described by Spicer (Spicer, 1965) and has been modified for our studies. David Thornton, John Sheehan and Ingemar Carlstedt (Thornton et al., 1994) originally developed the method of assessing mucins in human samples using western blotting on nitrocellulose membrane which was adapted for the animal studies.
References
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