Localization of Galectins within Glycocalyx.

Galectins are involved in various biological processes, e.g. cell-cell and cell-matrix adhesion and the transmission of cellular signals. Despite the diversity of functions, little is known about the nature of their physiological cognate ligands on the cell surface and the localization of galectins in the glycocalyx, although this information is important for understanding the functional activity of galectins. In this work, localization of endogenous and exogenously loaded galectins in the glycocalyx was studied. The following main conclusions are drawn: 1) galectins are not evenly distributed within the glycocalyx, they are accumulated in patches. Patching is not the result of a cross-linking of cellular glycans by galectins. Instead, patch-wise localization is the consequence of irregular distribution of glycans forming the glycocalyx; 2) galectins are accumulated in the inner zone of the glycocalyx rather than at its outer face or directly in vicinity of the cell membrane; 3) patches are not associated with cell rafts.

Solid-phase assays for study of carbohydrate specificity of galectins.

We have recently shown that the carbohydrate-binding pattern of galectins in cells differs from that determined in artificial (non-cellular) test-systems. To understand the observed discrepancy, we compared several test-systems differing in the mode of galectin presentation on solid phase. The most representative system was an assay where the binding of galectin (human galectins-1 and -3 were studied) to asialofetuin immobilized on solid phase was inhibited by polyacrylamide glycoconjugates, Glyc-PAA. This approach permits us to range quantitatively glycans (Glyc) by their affinity to galectin, i.e. to study both high and low affinity ligands. Our attempts to imitate the cell system by solid-phase assay were not successful. In the cell system galectin binds glycoconjugates by one carbohydrate-recognizing domain (CRD), and after that the binding to the remaining non-bound CRD is studied by means of fluorescein-labeled Glyc-PAA. In an "imitation" variant when galectins are loaded on adsorbed asialofetuin or Glyc-PAA followed by revealing of binding by the second Glyc-PAA, the interaction was not observed or glycans were ordered poorly, unlike in the inhibitory assay. When galectins were adsorbed on corresponding antibodies (when all CRDs were free for recognition by carbohydrate), a good concentration dependence was observed and patterns of specificities were similar (though not identical) for the two methods; notably, this system does not reflect the situation in the cell. Besides the above-mentioned, other variants of solid-phase analysis of galectin specificity were tested. The results elucidate the mechanism and consequence of galectin CRD cis-masking on cell surface.

Determination of carbohydrate specificity of monoclonal antibodies against MUC1.

The carbohydrate specificity of 57 MAbs submitted to the ISOBM TD-4 Workshop on MUC1 were investigated by two versions of ELISA, direct binding and inhibition of binding. The following free saccharides and their polyacrylamide conjugates (Sug-PAA) were used: tetrasaccharides--SiaLe(x), Sia--Le(a); trisaccharides--Le(x), 3'HSO3Le(x), Le(a), 3'HSO3Le(a), 3'SiaLac, Atri, Btri; a number of disaccharides including TF, Hdi, SiaTn, LactNAc, and monosaccharides. It was shown that MAbs 143 and 167 interacted only with SiaLe(x), MAbs 127 and 128 only with Le(x). Antibodies 123 and 164 interacted preferably with Le(a) but also recognized Le(c). Antibody 151 recognized alpha GalNAc (Tn) and cross-reacted with beta GalNAc. Antibody 157 displayed high affinity to Atri and Atetr (type 1). Neither anti-TF nor anti-SiaTn antibodies were revealed.