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Mucin Research
A three-dimensional AFM image of large ocular mucin From McMaster et al. 1999
What are mucins, and what do they do?
Mucins are glycoconjugates with most oligosaccharides O-linked to a Serine or Threonine in the peptide core. Epithelial mucins are found on all moist, mucosal, epithelia and are thought to combine mechanical protection functions with chemical and immune mucosal defense. Mucins are "chatty" molecules: some mucins span the apical membrane of epithelial cells and are linked to signalling intracellular pathways; these molecules bear potential ligands for cells of the immune system and are candidates for modulating the traffic of these molecules through mucosal surfaces. The Jekyl and Hyde nature of mucins is manifest in their interactions with bacteria: generally mucins are antiadhesive, but some of their oligosaccharides can serve as bacterial ligands. Furthermore mucin secretion can specifically respond to colonisation by a bacterial species.
Why ocular mucins?
The surface of the eye is kept moist and lubricated by the tear film: mucins anchor this fluid to the epithelium and protect the surface from bacterial, chemical and physical invasion. The mucin-rich preocular fluid is the transparent matrix through which oxygen and nutrients reach the avascular cornea, and the vehicle that eliminates contaminants from the ocular surface. Moreover mucins bridge between mucosal and systemic immunity. Mucins are synthesized by all the cells of the external ocular epithelia, by the lacrimal gland and the nasolacrimal sac and ducts. In addition the conjunctiva has specialized goblet cells that store secreted mucins.
Impression cytology of normal conjunctiva: goblet cells with MUC5AC mucin
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| Mica | TFF2-G on mica | TFF2-U on mica |
Trefoil factor peptides are intimately associated with mucins. The spatial arrangement of mucin-trefoil aggregates appears to be modulated by TFF glycosylation.
We are grateful to Dr Lars Thim for the gift of Trefoil Factor Peptides
Mucins from normal human conjunctival tissue and ocular surface
We have focused on biochemical attributes that are thought to be linked to mucin molecular function. Groups around the world have paid more attention to other aspects of mucin biology: more detailed descriptions of features described below can be found in the linked papers and in the references cited.
In the cells of the conjunctival epithelium MUC1, MUC2, MUC4 and MUC5AC are present in the buoyant density range 1.3-1.5g/ml, that is characteristic of mature glycosylation mucins [Berry 1996]. MUC 16 has also been identified on the ocular surface epithelia (Argueso et al. Invest. Ophthalmol. Vis. Sci. (2003) 44: 2487-95). MUC7, a small secreted mucin with antibacterial properties in the saliva, is secreted by both conjunctiva and the lacrimal gland (Jumblatt et al. Cornea (2003) 22: 41-45; Corrales et al. Curr. Eye Res. (2003) 27: 323-8; Corrales et al. Cornea (2003) 22: 665-71).
In the lacrimal gland, sac and ducts, another antimicrobial mucin is added to the ocular repertoire, MUC5B [Paulsen 2003, Paulsen 2004, Paulsen 2004].
Lacrimal Gland Mucins
MUC1 and MUC4 are membrane spanning [Paulsen 2003], as is MUC16. The list of transmembranal mucin species is likely to grow as identification techniques improve. The extracellular mucin domains are shed or cleaved, a process likely to contribute to preocular fluid turnover [Corfield 1997].
All mucin gene products identified in the tissue were recovered from washings of the ocular surface [Berry 2004], from tears [Paulsen 2004, Paulsen 2004] and from contact lenses [Corfield 1997]. Mucins harvested from conjunctival cells range from very large to relatively small hydrodynamic volumes [Corfield 1997]. On the ocular surface there is a relatively larger proportion of "medium" size mucins, while on contact lenses low glycosylation small hydrodynamic volume mucins are the dominant forms [Berry 2003].
In each subpopulation of mucins, fractionated according to density of glycosylation and hydrodynamic volume, there are mucins with diverse subunit charge, including glycoforms of MUC5AC. In subunits with medium negative charge sialylated epitopes are prevalent, while in high charge subunits we detected additional sulphated groups [Ellingham 1999]. Interestingly, and despite the multidipersity of these molecules, purified intracellular mucins probed with a general glycosylation reagent display electrophoretic patterns that are conserved among individuals irrespective of age and sex [Berry 1996]. Ocular surface mucins and mucins that adhere to contact lenses have subunits that are less and more charged than their intracellular homologues [Berry 2004, Berry 2002, Berry 2002].
MUC5AC in impression cytology
In an almost physiological buffer, mucins look like irregular pearl strings ranging from a few microns to a few hundred nanometers in length [McMaster 1999], with diameters of only few nanometers. The low height of the molecule indicates that in ocular mucins oligosaccharide chains are short. Tn and sialylTn epitopes, considered cancer-linked in other mucosae [Berry 1996, McMaster 1999], are common. Other epitopes of note are fucosylated and sialylated ologiosaccharides, e.g. Lewis epitopes that might serve as ligands to patrolling immune cells. Though not yet demonstrated for the ocular surface, it is known that neutrophil elastase and Cathepsin G cause mucin release in vivo and in vitro. Neutrophils dominate the ocular surface under closed lids.
The Biophysical Characterisation of Mucins
Hydrated ocular mucins are linear polymers with a propensity to entangle: their conformation and flexibility depends on the density of glycosylation [Round 2002, Round 2004]. Rapid deposition from solution onto a positively charged surface reveals mucin tangles, suggesting that entanglement is the preferred conformation in solution [Brayshaw 2004]. Adherence to surfaces are not restricted to specialised molecular domains. Some are mediated through positive ions in solution that bridge between negative charges on mucin and surface [Berry 2001]. There are no adhesions between mucin-coated surfaces, which might explain why blinking does not activate the sensory nerves on the ocular surface how mucins wrap and eliminate particles from the ocular surface. Last, but not least, hydrated preocular fluid imaged with an Atomic Force Microscope has the appearance of a gel: a gel with irregularities much smaller than the wavelengths of visible light. Disulphide bond disruption results in the rapid dissolution of the gel (Berry, Brayshaw and McMaster, submitted). More of atomic force microscopy on the SPM Web site (spm.phy.bris.ac.uk).
Equilibrium conformation of human ocular mucins with different glycosylation densities (reflected in polymer buoyancy). From Round et al. 2004