The role of oligosaccharide decorations in mucin interaction capacities : assessment by high resolution force probes

Abstract

Mucins are large proteins glycosylated with oligosaccharide groups. They are secreted from epithelial cells or embedded in the apical side of the epithelial cell and promote cell survival in different ways. Mucins also provide the structural framework of the mucus barrier, act as a lubricant and trap microorganisms. The mucosal barriers in different organs in the body have different functions. In the airways the mucus traps foreign particles and protect against microorganisms whereas in the stomach the mucins form an adherent unstirred layer to protect against damage from the gastric acid. Mucins are structurally diverse, and large variations are seen e.g. in the oligosaccharide groups. These variations are seen between different tissues in the body and even within the same tissue. It is likely that the structure of the oligosaccharides plays an important part in the diversity of functions mucins have in different tissues. Tandem repeat sequences that are rich in threonine, serine and proline are found in all mucins. The number of such sequences and their structure varies among different mucins and function as a scaffold for glycosylation which means that the structure of glycosylation in these domains are highly ordered and have a locally high density of specific sequences. The structure and function of mucins can change due to the development of diseases. One example is airway diseases, where structural changes in the mucins lead to changes in e.g. the rheological properties of mucus. Rheological properties of mucus are also seen to be different in different healthy tissues. The mechanisms explaining how mucins are organized to produce the observed properties are not very well understood. Some cancer tumors also express mucins which are significantly different from the mucins expressed in the normal cells the tumor arose from. A group of these mucins express oligosaccharide groups called tumor associated antigens. Tumor associated antigens can evoke an immune response, but are not necessarily unique to the tumor cells. Antibodies against tumor associated antigens are widely used as diagnostic tools in cancer diagnosis. By understanding the binding properties of mucins better, they could possibly be more extensively used in both cancer diagnosis and treatment. In this work the adhesion properties of mucins containing well defined truncated carbohydrate side chains have been studied. It provides information of the interaction properties of cancer associated mucins as well as on the interaction properties of the specific oligosaccharides in the side chains. The homotypic interaction study showed that all the mucin samples investigated had the ability for self-adhesion but the fraction of interaction was largest for Tri-PSM presenting a mixture of T and Tn cancer antigens and Tn-PSM containing only the Tn cancer antigen. The observed most likely rupture forces were in the range 27-50 pN at a force loading rate of 2 nN/s for all samples investigated. The force-loading rate spectra show that the forces obtained are in the same range for all the mucin samples but there are some variations in the parameters of the energy landscape for the different samples. The heterotypic interaction study showed that Tn-PSM and Tri-PSM have the capacity of interacting with all samples investigated. The most likely rupture forces were determined to range from 18-31 pN at a force loading rate of approximately 0.5 nN/s. The observed rupture forces were in the same range as observed for the homotypic interactions. It is noteworthy that the fraction of interaction is dependent on the immobilization configuration of the measurement setup. This suggests that there is a mucin molecules interacting on the sample surface. This interaction might occupy the binding sites on the surface and thereby block for interactions with the mucin anchored to the AFM tip. In both studies two different analysis models were used, the Bell-Evans formalism and the Dudko-Hummer-Szabo model. The two models gave similar values of the energy landscape, and the differences between the models were seen to be systematic, suggesting that when comparing results it is important that the same model is used for determining the parameters. Homotypic interactions were also investigated by optical tweezers. This method has the advantages of a lower noise level and access to lower loading rates. At low loading rates a plateau in the force loading rate plots were revealed at low loading rates, indicating that rebinding is possible after an unbinding event at low loading rate regimes. Studies of beads functionalized with GalNAc groups anchored by a short PEG linker or with synthetic polymers with either GalNAc or NeuNGl groups, revealed that the force range measured for Tn-PSM is independent of the protein backbone. This conclusion is based on the observed identical force-loading rate plots for Tn- PSM and the synthetic polymer and the GalNAc functionalized beads. Moreover both GalNAc and NeuNGl have the ability for self-interactions and the measured forces are in the same range for the two groups. This means that both of these groups can be responsible for the self-interaction of the mucin samples. The optical tweezers measurements revealed that the force loading rate plots depend on the retraction speed of the measurements. This indicates the possibility of multivalent interactions. As the mucin molecules have multiple repeating units of the carbohydrate side chains, multivalency is an expected property for mucin interaction. The determined rupture forces and the parameters of the energy landscape for the homotypic interactions of Tn-PSM and Tri-PSM was in the same range for the AFM and OT measurements. This shows that the determined parameters by the Bell-Evans formalism can be compared for different systems also when rebinding and multivalency is a possibility. Mucoadhesive molecules such as alginate and chitosan have been used for pharmaceutical applications. These polymers have also been proposed for use in drug delivery systems. A characterization of the energy landscape and the binding properties of these polymers can therefore provide valuable information. The mucoadhesive properties of chitosan were investigated at different solvent conditions and the results were compared with the mucoadhesive properties determined for alginate. The chitosans shows a higher fraction of interactions when compared to the alginates, as expected from an electrostatic argument. Chitosan is positively charged at the used conditions whereas alginate is negatively charged. Nevertheless, the binding events in both systems were found to be similar which suggest the presence of other interaction forces, in addition to electrostatic, such as hydrophobic interactions.