The recruitment of leukocytes from blood is one of the most important cellular processes in response to tissue damage and inflammation. This multi-step process includes rolling leukocytes and their adhesion to endothelial cells (EC), culminating in crossing the EC barrier to reach the inflamed tissue. Galectin-8 and galectin-9 expressed on the immune system cells are part of this process and can induce cell adhesion via binding to oligolactosamine glycans. Similarly, these galectins have an order of magnitude higher affinity towards glycans of the ABH blood group system, widely represented on ECs. However, the roles of gal-8 and gal-9 as mediators of adhesion to endothelial ABH antigens are practically unknown. In this work, we investigated whether H antigen–gal-9-mediated adhesion occurred between Jurkat cells (of lymphocytic origin and known to have gal-9) and EA.hy 926 cells (immortalized endothelial cells and known to have blood group H antigen). Baseline experiments showed that Jurkat cells adhered to EA.hy 926 cells; however when these EA.hy 926 cells were defucosylated (despite the unmasking of lactosamine chains), adherence was abolished. Restoration of fucosylation by insertion of synthetic glycolipids in the form of H (type 2) trisaccharide Fucα1-2Galβ1-4GlcNAc restored adhesion. The degree of lymphocyte adhesion to native and the “H-restored” (glycolipid-loaded) EA.hy 926 cells was comparable. If this gal-9/H (type 2) interaction is similar to processes that occur in vivo, this suggests that only the short (trisaccharide) H glycan on ECs is required.

Glycans of MVs are proposed to be candidates for mediating targeting specificity or at least promoting it. In contrast to exosomes, glycomic studies of MVs are largely absent. We studied the glycoprofile of endothelial cell-derived MVs using 21 plant lectins, and the results show the dominance of oligolactosamines and their α2-6-sialylated forms as N-glycans and low levels of α2-3-sialylated glycans. The low levels of α2-3-sialosides could not be explained by the action of extracellular glycosidases. Additionally, the level of some Man-containing glycans was also decreased in MVs. Spatial masking as the causative relationship between these low level glycans (as glycosphingolipids) by integral proteins or proteoglycans (thus, their lack of interaction with lectins) seems unlikely. The results suggest that integral proteins do not pass randomly into MVs, but instead only some types, differing in terms of their specific glycosylation, are integrated into MVs.

Many important aspects of intercellular glycosphingolipid (GSL) transport remain unknown, in particular how GSLs cross the glycocalyx of the cell-donor and cell-acceptor. The use of synthetic analogs instead of natural GSLs alleviates the study. However, in order to obtain adequate results, we must know how simplifications in the structure of a synthetic probe affect its properties of interest to us. In particular, how the replacement of a ceramide residue in GSL with a synthetic analog affects the release and insertion kinetics. To this end, we compared the properties of synthetic analogs, one of which contains natural ceramide moiety, while two others have lipid residues that are more convenient in terms of the probes chemical synthesis. FSL (Function-Spacer-Lipid) constructs containing DOPE (1,2-O-dioleoyl-sn-glycero-3-phospho ethanolamine), cholesterol (Chol), or C18-ceramide (Cer) lipid fragments (L) were compared. In all three probes, the lipid residues were linked to biotin with the hydrophilizing CMG2-spacer-arm. Using confocal microscopy, the similarity of the localization patterns of these three probes, both with each other and with the previously studied glycoprobes (F = glycan), was shown, namely: they were observed in the form of patches of an average size of 0.3 μm but were not detected at all in cell rafts. At the same time, the following differences were observed in the properties of the three studied FSLs: (1) the process of insertion of the ceramide construct reaches a plateau within three hours, while the other two within one hour; (2) the amount of inserted ceramide construct is less than the other two; and (3) release of the cholesterol construct from the cells where it is preinserted is slower than the other two. Thus, despite some quantitative differences, qualitatively all three constructs behave similarly, that is, they are interchangeable when studying the incorporation/release of the FSL into/from the cell.

Selective insertion of lipid probes from the external milieu into raft regions of the cell membrane would enable targeted studies of raft molecular organization and of raft-associated peptide and glycan components. In this work, we compared the insertion of several synthetic glycolipids into raft and non-raft membrane areas of Raji and EA.hy 926 endothelial cells. A glyco-cholesterol derivative in which the 3β oxygen atom is replaced by NH was selected as a candidate raft-selective probe. Although this glycolipid also inserted into other membrane areas, its raft-to-non-raft distribution ratio was higher than that of its O-analog (natural 3β-O-cholesterol).