Distribution of sialic acids on mucins and gels: a defense mechanism

Moist mucosal epithelial interfaces that are exposed to external environments are dominated by sugar epitopes, some of which (e.g., sialic acids) are involved in host defense. In this study, we determined the abundance and distribution of two sialic acids to assess differences in their availability to an exogenous probe in isolated mucins and mucous gels. We used atomic force microscopy to obtain force maps of human preocular mucous and purified ocular mucins by probing and locating the interactions between tip-tethered lectins Maackia amurensis and Sambucus nigra and their respective receptors, α-2,3 and α-2,6 N-acetylneuraminic (sialic) acids. The rupture force distributions were not affected by neighboring sugar-bearing molecules. Energy contours for both lectin-sugar bonds were fitted to a two-barrier model, suggesting a conformational change before dissociation. In contrast to data from purified mucin molecules, the preocular gels presented numerous large clusters (19,000 ± 4000 nm2) of α-2,6 sialic acids, but very few small clusters (2000 ± 500 nm2) of α-2,3 epitopes. This indicates that mucins, which are rich in α-2,3 sialic acids, are only partially exposed at the surface of the mucous gel. Microorganisms that recognize α-2,3 sialic acids will encounter only isolated ligands, and the adhesion of other microorganisms will be enhanced by large islands of neighboring α-2,6 sialic acids. We have unveiled an additional level of mucosal surface heterogeneity, specifically in the distribution of pro- and antiadhesive sialic acids that protect underlying epithelia from viruses and bacteria

Heterogeneity and persistence length in human ocular mucins

Atomic force microscopy (AFM) has been used to investigate the heterogeneity and flexibility of human ocular mucins and their subunits. We have paid particular attention, in terms of theory and experiment, to the problem of inducing the polymers to assume equilibrium conformations at a surface. Mucins deposited from a buffer containing Ni2+ ions adopt extended conformations on mica akin to those observed for DNA under similar conditions. The heterogeneity of the intracellular native mucins is evident from a histogram of contour lengths, reflecting, in part, the diversity of mucin gene products expressed. Reduction of the native mucin with dithiothreitol, thereby breaking the S=S bonds between cysteine residues, causes a marked reduction in polymer length. These results reflect the modes of transport and assembly of newly synthesized mucins in vivo. By modifying the worm-like chain model for applicability to two dimensions, we have confirmed that under the conditions employed mucin adsorbs to mica in an equilibrated conformation. The determined persistence length of the native mucin, 36 nm, is consistent with that of an extended, flexible polymer; such characteristics will influence the properties of the gels formed in vivo

Atomic force microscopy of the submolecular architecture of hydrated ocular mucins.

High-resolution atomic force microscopy has been applied to the imaging of intact human ocular mucins in a near-physiological buffer. The mucins displayed a range of lengths from several hundred nanometers to several microns. By varying the ionic composition of the imaging environment, it was possible to image molecules rigidly fixed to the substrate and the motion of single molecules across the substrate. From static molecular images, high-resolution line profiles show a variation of up to +/-0.75 nm in thickness along the molecule. This variation is localized in regions of several tens of nanometers. It is interpreted in terms of the varying glycosylation along the mucin and is consistent with the known size of oligosaccharides in ocular mucins. The dynamic images indicate the possibility of following mucin interactions in situ