The mucosal surfaces of the body are protected by membrane-bound and secreted mucins.
The very large, oligomeric species which form mucus constitute a distinct subfamily sometimes referred to
as mucus glycoproteins.
Typical mucus glycoproteins are oligomers of subunits joined by disulphide bonds and contain hundreds
of oligosaccharide side chains enriched into 'clusters' on the protein core. Reduction of disulphide bonds
fragments mucins into their constituent subunits and subsequent proteolysis yields high-molecular weight
glycopeptides corresponding to the oligosaccharide clusters.
Depending on the object of the study, fractionation can be performed either on purified whole mucin
molecules or on mucin fragments.
SOLUBLE MUCINS AND INSOLUBLE MUCINS
Gastrointestinal mucins can be separated into 'soluble' and 'insoluble' species
We use gentle agitation in 6M-guanidinium chloride to break the interactions
between the mucus glycoproteins and thus to solubilize the mucus gel. This mild
extraction procedure helps to avoid the mechanical degradation often seen in
preparations of DNA and RNA, and we include proteinase inhibitors to protect
the mucin apoprotein from cleavage. Most gastric mucins are solubilized by this
procedure, but the major part of the intestinal ones is 'insoluble' even after
prolonged extraction (Carlstedt et al., 1993).
Approach for intestinal mucins is to subject the tissue to several rounds of extraction and then purify the glycoproteins as subunits after reduction of the extraction residue. Using this approach, shown that the 'insoluble' glycoprotein complex contains virtually all the MUC2 in human intestine (Carlstedt et al., 1995). Other isolation procedures may lead to selective loss of the insoluble species or, where for example vigorous mechanical treatment is used, the isolation of undefined fragments.
BUOYANT DENSITY, SIZE AND CHARGE CAN
BE EXPLOITED FOR MUCIN PURIFICATION AND FRACTIONATION
Mucins are rich in carbohydrate, and thus have a higher buoyant density than conventional proteins while the buoyant density is usually lower than that of proteoglycans and nucleic acids. Density-gradient centrifugation in CsCl can thus be used to separate mucins from proteins and DNA. The large size and (usually) negative charge of mucins are also properties that can be used distinguish them from other molecules.
HOW TO DO IT ?
To isolate mucins from tissue, the mucosa is preferably removed from the underlying connective tissue. At this stage, enrichment of mucins from the surface epithelium and from the glands (stomach) or crypts (colon) can be achieved by first loosely scraping the mucosal surface and then subjecting the remaining tissue to a more vigorous mechanical treatment. The strategy used for extracting and purifying mucins from proteins, lipids and nucleic acids follows that established in the past (summarised by Carlstedt and Sheehan, 1984). After extraction, high-speed centrifugation is used to recover the solubilized material and the residue re-extracted until no more material is brought into solution. Mucins in the extraction residue (the major part of the intestinal mucins) are solubilized by reduction followed by alkylation. The extracts are then dialysed against e.g. 10 vols. of extractant to remove low-molecular weight contaminants before density-gradient centrifugation.
A two-stage procedure is usually used, i.e. separation of mucins from proteins is first undertaken in CsCl/4M-guanidinium chloride and DNA is then removed in CsCl/0.5M-guanidinium chloride. However, intestinal mucin subunits often separate well from both DNA and protein in CsCl/4M-guanidinium chloride. Fractions are analysed for density, protein (e.g. absorbance at 280 nm that will also detect the presence of nucleic acids), antibody or lectin reactivity, carbohydrate, etc. The laboratory with little experience in carbohydrate analysis may benefit from using the slot-blot technique developed by Thornton et al. (1989).
Mucins chromatograph in the void volume of most commercially available gels. The chromatographic process is perturbed by the presence of large amounts of DNA, which usually also elutes in the void volume and mucins and DNA are thus not separated. We therefore use density-gradient centrifugation as the first purification step(s) followed, where necessary, by gel chromatography.
Mucins sometimes separate into 'high-density' and 'low-density' populations in the density gradient used
to purify them (see e.g. Carlstedt et al., 1995). Mucins can usually also be subfractionated into distinct
populations using ion-exchange chromatography and agarose gel electrophoresis of the cognate subunits.
PAGE is not recommended with oligomeric mucins or even their subunits since the large molecules do not
penetrate the gel well. Ion-exchange chromatography has a higher capacity than electrophoresis and is thus
the first choice in preparative work. The eluent may be supplemented with urea and a detergent (e.g. CHAPS)
to minimise interactions with the chromatographic matrix. Eperience in ion-exchange chromatography works
better with mucin subunits than with whole mucins. Good examples of how combinations of density-gradient
centrifugation, ion-exchange chromatography and agarose gel electrophoresis can be used to fractionate
mucins are shown by Thornton et al. (1996).