Enhanced Self-Association of Mucins Possessing the T and Tn Carbohydrate Cancer Antigens at the Single-Molecule Level

Abstract

Mucins are linear O-glycosylated glycoproteins involved in inflammation, cell adhesion and tumorigenesis. Cancer associated mucins often possess increased expression of the T (Galβ1,3GalNAcαThr/Ser) and Tn (GalNAcαThr/Ser) cancer antigens, which are diagnotic markers for several cancers including colon cancer. We have used AFM based single-molecule forced unbinding under near physiological conditions to investigate the self-interactions between porcine submaxillary mucin (PSM) as well as between PSM analogs possessing various carbohydrates including the T- and Tn-antigen. Distributions of unbinding forces and corresponding force loading rates were determined for force loading rates from 0.18 nN/s to 39 nN/s, and processed to yield most probable unbinding forces f* and lifetimes of the interactions. Parameter f* varied in the range 27 to 50 pN at force loading rates of about 2 nN/s among the various mucins. All mucin samples investigated showed self-interaction, but the tendency was greatest for PSM displaying only the Tn-antigen (Tn-PSM) or a mixture of Tn-, T-antigen and the trisaccharide Fucα1,2Galβ1,3GalNAc (Tri-PSM). Weaker self-interactions were observed for native PSM (Fd-PSM), which consists of a nearly equal mixture of the longer core 1 blood group A tetrasaccharide (GalNAcα1,3(Fucα1,2)Galβ1,3GalNAcαSer/Thr) and Tn-antigen. The data are consistent with the truncated Tn and T glycans enhancing self-interaction of the mucins. These carbohydrate cancer antigens may thus play an active role in the disease by constitutively activating mucin and mucin-type receptors by self-association on cells.

Direct Determination of Chitosan–Mucin Interactions Using a Single-Molecule Strategy: Comparison to Alginate–Mucin Interactions

“…This emerges from the observation of no major decline in the magnitudes of the forces following repeated trials, the presence of the unbinding events being specific to the molecular pair [49,62] and the magnitude up to about 0.2 nN being much less than 1-3 nN being the force needed to break covalent bonds.…”

“…The control experiments probing (i) chitosan against the aminosilanized surfaces (Figure 2), alginate functionalized tips against (ii) freshly cleaved mica or (iii) aminosilanized mica [49], and (iv) immobilized mucin towards aminosilanized surfaces [62] show that the interactions observed are specific to the chitosan-mucin and alginate-mucin molecular pairs.…”

“…The forced dissociation of the molecular pairs, mucin and either alginate or chitosan, were conducted using covalently anchored biopolymers following previous reported protocols [49,61,62]. The pig gastric mucin (PGM) was covalently linked to mica surfaces, and chitosan and alginates were covalently linked to AFM tips (OTR4, Bruker AFM Probes, Camarillio, CA, USA, triangular cantilevers with nominal spring constant 0.02 nN/nm) following previously developed protocols.…”

“…The actual velocities were selected to limit data collection time for the required statistics while at the same time providing apparent loading rates over an extended range to the parameters determined for the energy landscape. We have previously reported on control experiments between alginate functionalized tips and freshly cleaved mica or aminosilanized mica [49] as well as between immobilized mucin and aminosilanized surfaces [62]. Control experiments relevant for the chitosan-mucin interaction consisted of probing the chitosan functionalized AFM tip with the aminsilanized surface, i.e., the surface used for conjugation of the mucin.…”

Atomic force microscopy measurements reveal multiple bonds between Helicobacter pylori blood group antigen binding adhesin and Lewis b ligand

“…It is worth considering alternative reasons for the apparent difference, beyond the models used. First, one of the two bonds observed in the AFM measurements could form more slowly than the short dwell time used in the optical tweezers experiments, and therefore may not have been as prevalent in the previously reported force histograms [43,47]. Alternatively, the recombinant, purified protein could be more conformationally heterogeneous than the membrane-bound form.…”