Visualization of sialyl Lewis(X) glycosphingolipid microdomains in model membranes as selectin recognition motifs using a fluorescence label.

Selectin-induced leukocyte rolling along the endothelial surface is an essential step in the cellular immune response. For efficient recognition, the relevant carbohydrate epitope sialyl Lewis(X) (sLe(X); alpha-Neup5Ac-(2-->3)-beta-Galp-(1-->4)-[alpha-Fucp-(1-->3)]GlcpNAc) has to be arranged in clusters. We describe the synthesis of the sLe(X)-glycosphingolipid (sLe(X)-GSL) with a NBD fluorescence label in the tail region, which allows the direct visualization of sLe(X)-GSL microdomains to very low concentrations (0.01mol%) in various planar phosphocholine matrices by fluorescence microscopy. Cell rolling experiments of E-selectin expressing cells along these membranes confirmed that the fluorescence analog behaves similar to the naturally occuring sLe(X)-GSL. This is direct evidence for recent hypotheses on multivalent sLe(X) binding as molecular basis for selectin-mediated cell rolling.

The influence of various structural parameters of semisynthetic sulfated polysaccharides on the P-selectin inhibitory capacity.

Selectin-mediated leukocyte rolling along the endothelium is of key importance for maintaining the cellular immune response. The anti-inflammatory activities of heparin have partly been related to inhibition of P-selectin binding. Heparin, however, suffers from its heterogeneous variable structure, the animal origin and multiple in vivo effects. As P-selectin is a promising target for anti-inflammatory approaches, we focused on P-selectin inhibition by other sulfated polysaccharides and compared them with six heparins. We examined 15 structurally defined semisynthetic sulfated glucans, non-animal-derived from the linear glucans phycarin, curdlan or pullulan. The derivatives gradually differ in their degree of sulfation, molecular weight, and glycosidic linkage. The inhibitory capacity was analysed in a parallel plate flow chamber, detecting the rolling of U937 cells on P-selectin layers. Unfractionated heparins displayed variabilities between different preparations. Considering fractionated heparins, exceeding of a minimal mass is essential for activity. Comparing the glucan sulfates, charge density is the most important parameter for P-selectin binding. Highly sulfated derivatives are excellent inhibitors, the reduced cell binding up to 16.2+/-6.4% strongly exceeded the heparin activities. Molecular weight is of minor effects, while glycosidic backbone linkage holds certain importance. To check the P-selectin inhibition in vivo, heparin and one phycarin sulfate were tested using intravital microscopy of microvasculature in mice. Both compounds significantly reduced the rolling fractions of activated platelets on endothelium as effective as a blocking P-selectin antibody. Our study indicates that semisynthetic glucan sulfates with optimal structures block P-selectin excellently and might become promising candidates for anti-inflammatory drugs to replace heparin for certain applications.

Human milk oligosaccharides affect P-selectin binding capacities: in vitro investigation.

OBJECTIVE: In the initial phase of cellular immune response, selectins mediate the emigration of leukocytes from the blood stream into inflammatory regions. Human milk oligosaccharides (HMOs) possess binding epitopes of selectin ligands such as sialyl Lewis(x) and sialyl Lewis(a) and therefore might impair the interaction of selectins with cellular ligands. Neutral, acidic, sialylated, or fucosylated HMO fractions with polymerization degrees of 3 to 50 were investigated regarding this interaction in a dynamic flow chamber model that considers physiologic shear stress conditions. METHODS: Human milk oligosaccharides were compared with kappa-carrageenans and pectin oligosaccharides to deduce structure-activity relations. Fucoidan and sialyl Lewis(x) served as positive controls. RESULTS: All HMO fractions affected P-selectin ligand binding capacity but were not comparable to fucoidan. The activity of the acidic HMO fraction resembled sialyl Lewis(x) in decreasing the binding of the ligand to P-selectin. CONCLUSION: Human milk oligosaccharides modulate rather than block the function of P-selectin.

Lessons learned from clustering of fluorinated glycolipids on selectin ligand function in cell rolling.

Selectin-induced leukocyte rolling along the endothelium is an essential step in the cellular immune response. Since clustering of binding epitopes is thought to be crucial for selectin-ligand interaction, we focused on requirements of ligand clusters in a flow chamber study. Neoglycolipids bearing the binding epitope sialyl Lewis X (sLeX) were used as artificial ligands in model membranes. sLeX ligands or matrix lipids or both were applied with partially fluorinated alkyl chains to increase the ligand cluster separation tendency. Cluster size, their inner structure, and separation distance were evaluated with high resolution by scanning force microscopy (SFM) and correlated with binding or rolling of E-selectin-expressing cells. Fluorination of only one component, sLeX ligand or matrix lipid, leads to a very high separation tendency and impeded cell rolling, although firm cell adhesion could be observed down to 0.005 mol % ligand concentration. As a sign of total immiscibility, cluster size increased with ligand concentration, and resulting excessive ligand densities within the clusters prevent cell rolling. Fluorination of both sLeX ligands and matrix created small clusters which could serve as rolling patches. Our results confirmed that cluster size and separation distance controlled by a certain miscibility of ligand and matrix as well as a sufficiently diluted ligand concentration within the clusters are necessary for cell rolling. Within this work, selectin ligand clustering and its ability to mediate cell rolling are presented as a balance between multivalency of binding and sufficient flexibility of the single epitopes. This might be helpful for better understanding the function of the natural selectin ligands.

Galactinol synthase1. A novel heat shock factor target gene responsible for heat-induced synthesis of raffinose family oligosaccharides in Arabidopsis.

Heat shock factors (HSFs) are transcriptional regulators of the heat shock response. The major target of HSFs are the genes encoding heat shock proteins (HSPs), which are known to have a protective function that counteracts cytotoxic effects. To identify other HSF target genes, which may be important determinants for the generation of stress tolerance in Arabidopsis, we screened a library enriched for genes that are up-regulated in HSF3 (AtHsfA1b)-overexpressing transgenic plants (TPs). Galactinol synthase1 (GolS1) is one of the genes that is heat-inducible in wild type, but shows constitutive mRNA levels in HSF3 TPs. The generation and analysis of TPs containing GolS1-promoter::beta-glucuronidase-reporter gene constructs showed that, upon heat stress, the expression is transcriptionally controlled and occurs in all vegetative tissues. Functional consequences of GolS1 expression were investigated by the quantification of raffinose, stachyose, and galactinol contents in wild type, HSF3 TPs, and two different GolS1 knockout mutants (gols1-1 and gols1-2). This analysis demonstrates that (1) raffinose content in leaves increases upon heat stress in wild-type but not in the GolS1 mutant plants; and (2) the level of raffinose is enhanced and stachyose is present at normal temperature in HSF3 TPs. These data provide evidence that GolS1 is a novel HSF target gene, which is responsible for heat stress-dependent synthesis of raffinose, a member of the raffinose family oligosaccharides. The biological function of this osmoprotective substance and the role of HSF-dependent genes in this biochemical pathway are discussed.

Functional microdomains of glycolipids with partially fluorinated membrane anchors: impact on cell adhesion.

Functional microdomains of glycolipids were designed by mixing neoglycolipids with partially fluorinated alkyl (F-alkyl) chains and matrix lipids with alkyl chains. Fluorescence images of the mixed lipid monolayers at the air-water interface demonstrated that it is possible to control both size and distribution of the microdomains by means of the strong demixing of alkyl and F-alkyl membrane anchors, while the carbohydrate head groups seemed to play a rather minor role. These microdomains in monolayers could be transferred onto hydrophobized substrates and subjected to experiments in a dynamic flow chamber. The results obtained here clearly indicated that the dynamic adhesion of Chinese hamster ovarial cells expressing E-selectin (CHO-E cells) on a lipid monolayer containing microdomains of sialyl Lewisx (sLex) can be both enhanced and reduced by controlled demixing of ligands and matrices. Moreover, the same clusters of sLex could also be formed in giant lipid vesicles, which can be used as a model cell that locally expresses biospecific functions.

Cooperation between lateral ligand mobility and accessibility for receptor recognition in selectin-induced cell rolling.

Selectin-induced leukocyte rolling along the endothelial surface is an essential step in the immune response. Several in vitro studies showed that this cell rolling is a highly regulated adhesion phenomenon, controlled by the kinetics and forces of selectin-ligand interactions. In the flow chamber study presented here, we focused on the requirements on the ligand structure in this context. A series of neoglycolipids bearing the binding epitope Sialyl Lewis X was synthesized and used as artificial ligands. These lipids differed in their spacer structures between headgroup and membrane anchor, resulting in a gradual variation in accessibility and mobility of the binding epitope when immobilized in model membranes. Consequently, analysis of cell rolling along such membranes allowed correlation of ligand structures and functionality. All model membranes containing such ligands were further characterized by film balance measurements, epifluorescence, and atomic force microscopy. Generally, the glycolipids exhibited a high tendency for lateral aggregation, but the resulting clusters were of different morphology. This was also reflected by strong differences in the rolling experiments. Our results confirm that, in addition to a sufficient headgroup accessibility, the cell rolling process is governed by two further interdependent factors: (i) the headgroup flexibility caused by the intramolecular uncoupling between the headgroup and the hydrophobic moiety due to introduction of a spacer, and (ii) the stiffness of the molecules resulting from their supramolecular arrangement in clustered assemblies. Since both factors are influenced simultaneously by the spacer modification, we present for the first time a clear correlation between structural aspects of selectin ligands and their ability to mediate cell rolling. This might help to develop a better understanding for the function of the natural selectin ligands.