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M.S. Berry, T.J. McMaster, A.N. Round, D.J. Brayshaw; Mucin Interactions in Real Time . Invest. Ophthalmol. Vis. Sci. 2003;44(13):2501.
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Purpose: Atomic force microscopy (AFM) offers quantitative measurements of biophysical characteristics of individual molecules and molecular assemblies. To enhance the characterisation of human ocular mucins we have used purified intracellular mucins to demonstrate the capabilities of AFM and derive characteristics unavailable through "bulk" biochemistry: equilibrium conformations of mucins with different glycosylation densities and antibody binding to these glycoforms. Methods: Imaging after initial deposition of mucins, and imaging in situ in real time were used to study purified human conjunctival mucins on hydrophobic, hydrophyllic, negatively- and positively-charged substrates. Reduction of the largest mucins resulted in a length distribution with a mode of 100nm. This mode, consistent with the predominance of one mucin species, led us to analyse the distribution of MUC5AC C-termini by reaction with the antibody 45M1. Native and reduced mucins were chemically bound to an AFM tip and lowered onto an agarose gel, in which they were allowed to diffuse. Incursion in the gel and adhesions were assessed during tip withdrawal. Results: In situ real time imaging confirmed that mucins are entangled in solution. Extended molecules can be trapped by positive charges on the substrate. The equilibrium conformations of the largest mucin polymers varied with the density of glycosylation. A short incubation resulted in 45M1 antibodies bound to short polymers with a dumb bell appearance. Stiffer, longer polymers required longer reaction times. The flexibility of polymers controls behaviour in solution and penetration through gels. The influence of length and charge distribution on diffusion through a gel has been investigated by comparing the forces needed to extract DNA and mucin polymers from agarose gels. Native mucins penetrated a 1% agarose gel further than DNA of comparable lengths, but established more and stronger interactions with the gel. Reduced mucins penetrated further than their native parent sample and were easier to remove from the gel. Conclusions: The flexibility of human ocular mucins and their propensity to form interaction are affected by their glycosylation density. Enhanced movement through a gel might explain the physiological role of mucin cleavage at or after secretion.
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