N. V. Bovin

Further refinement of the description of the ligand‐binding characteristics for the galactoside‐binding mistletoe lectin, a plant agglutinin with immunomodulatory potency

The galactoside‐binding lectin from mistletoe (Viscum album L.) is a biological response modifier, eliciting e.g. enhanced secretion of cytokines. This immunological activity warrants the further analysis of its ligand‐binding properties with special attention paid to blood group epitopes. To avoid the microheterogeneity and complexity of naturally occurring glycoproteins, chemically strictly defined neoglycoconjugates and a panel of synthetic oligosaccharides were employed in solid‐phase assays for direct binding and assessment of the relative inhibitory capacity. Since label incorporation into the lectin, although performed under protective conditions, or surface immobilization by adsorption to plastic may affect its affinity characteristics, the extent of neoglycoconjugate binding in the absence of any interfering substance and in the presence of oligosaccharides was determined comparatively with labeled and with immobilized lectin. In principle, these two factors could be excluded to markedly alter binding features. In addition to lactose, the blood group determinants H and B were strongly reactive. A fucose residue can thus especially be accommodated to the binding site when linked to the non‐reducing unit. N‐Acetyllactosamine was nearly as potent as an inhibitor as lactose. Lec and the A determinant were notably inferior to the other ABH blood group epitopes. Lea and Lex and their sialylated derivatives displayed only very weak binding capacity. Among the two natural isomers of sialyllactose, the α2,6‐form displayed a higher level of inhibitory capacity than the α2,3‐derivative. Isomeric variants of the Thomsen‐Friedenreich antigen, too, reduced lectin binding to the lactose‐carrying polymer. Their capacities were surpassed by those of the H and the B determinants and a related form of the latter, the P1 epitope. An overlap of specificity with the immunomodulatory human galectin‐3 is thus measurable for H/B‐like structures. The documented differential reactivity of the mistletoe lectin to blood group oligosaccharides may have a bearing on the responsiveness of blood group‐positive cell populations.

Antitumour activity of cytotoxic liposomes equipped with selectin ligand SiaLeX, in a mouse mammary adenocarcinoma model

The overexpression of lectins by malignant cells compared with normal ones can be used for the targeting of drug-loaded liposomes to tumours with the help of specific carbohydrate ligands (vectors). Recently we have shown that liposomes bearing specific lipid-anchored glycoconjugates on a polymeric matrix bind in vitro to human malignant cells more effectively and, being loaded with a lipophilic prodrug of merphalan, reveal higher cytotoxic activity compared with unvectored liposomes. In this study, carbohydrate-equipped cytotoxic liposomes were tested in vivo in a mouse breast cancer model, BLRB-Rb (8.17)1Iem strain with a high incidence of spontaneous mammary adenocarcinoma (SMA). Firstly, a cell line of the SMA was established which was then used to determine the specificity of the tumour cell lectins. After screening of the lectin specificity of a number of fluorescent carbohydrate probes, SiaLe(X) was shown to be the ligand with the most affinity, and a lipophilic vector bearing this saccharide was synthesised. Then different liposomal formulations of the synthetic merphalan lipid derivative and SiaLe(X) vector were prepared and applied in the treatment of mice with grafted adenocarcinomas. The results of the tumorigenesis data show that the therapeutic efficacy of merphalan increases sharply after its insertion as a lipophilic prodrug into the membrane of SiaLe(X)-vectored liposomes.

Mammalian galectins: structure, carbohydrate specificity, and functions.

Galectins are a family of β-galactoside binding lectins, homological by a sequence of the carbohydrate-binding site. In this review literature data about structure and carbohydrate specificity of galectins are discussed. The role of galectins in the regulation of cell adhesion in immune response, inflammation, and cancer progression is considered.

Fucoidan inhibits leukocyte recruitment in a model peritonial inflammation in rat and blocks interaction of P‐selectin with its carbohydrate ligand

Neutrophil recruitment into systemic inflammatory sites in vivo is thought to be initiated by selectin‐mediated endothelial adherence. The effect of fucoidan (natural sulfated polymer of L‐fucose) on the selectin dependent PMN migration into rat peritoneum following the induction of inflammation by peptone injection was studied. Peritonitis was characterized by an increase in the total cell number (from 45.3×106 to 91.6×106rat), and by highly elevated PMN content (from 0.2% to 58%) in the rat peritoneal cavity 3 h after peptone injection. Intravenous administration of fucoidan was found to reduce, in a dose‐dependent manner, neutrophil migration into peritoneum. Fucoidan in a dose as low as 0.8 mg per rat caused 96.8% reduction of neutrophil extravasation. The inhibitory effect of fucoidan was also dependent on the time intervals between the peptone and fucoidan injections. The maximal inhibitory effect of fucoidan was observed within the first 15 rain after the induction of peritonitis and it was maintained at a level of 80% during 1.5 h. Administration of fucoidan 2.5 h after peptone injection had practically no effect on PMN extravasation. Since P‐selectin is known to play a key role at the earlier stages of PMN extravasation, it was suggested that the inhibitory effect of fucoidan was mostly due to its interaction with P‐selectin. The in vitro experiments demonstrated the high affinity of fucoidan for both isolated P‐selectin and P‐selectin in plasma membranes of activated platelets.

Probing sialic acid binding Ig-like lectins (siglecs) with sulfated oligosaccharides

Soluble siglecs-1,-4,-5,-6,-7,-8,-9, and-10 were probed with polyacrylamide glycoconjugates in which: 1) the Neu5Ac residue was substituted by a sulfate group (Su); 2) glycoconjugates contained both Su and Neu5Ac; 3) sialoglycoconjugates contained a tyrosine-O-sulfate residue. It was shown that sulfate derivatives of LacNAc did not bind siglecs-1,-4,-5,-6,-7,-8,-9, and-10; binding of 6′-O-Su-LacNAc to siglec-8 was stronger than binding of 3′SiaLacNAc. The relative affinity of 3′-O-Su-TF binding to siglecs-1,-4, and-8 was similar to that of 3′SiaTF. 3′-O-Su-Lec displayed two-fold weaker binding to siglec-1 and siglec-4 than 3′SiaLec. The interaction of soluble siglecs with sulfated oligosaccharides containing sialic acid was also studied. It was shown that siglecs-1,-4,-5,-6,-7,-9, and-10 did not interact with these compounds; binding of 6-O-Su-3′SiaLacNAc and 6-O-Su-3′SiaTF to siglec-8 was weaker than that of the corresponding sulfate-free sialoside probes. Siglec-8 displayed affinity to 6′-O-Su-LacNAc and 6′-O-Su-SiaLex, and defucosylation of the latter compound led to an increase in the binding. Sialoside probes containing tyrosine-O-sulfate residue did not display increased affinity to siglecs-1 and-5 compared with glycoconjugates containing only sialoside. Cell-bound siglecs-1,-5,-7, and-9 did not interact with 6-O-Su-3′SiaLacNAc, whereas the sulfate-free probe 3′SiaLacNAc demonstrated binding. In contrast, the presence of sulfate in 6-O-Su-6′SiaLacNAc did not affect binding of the sialoside probe to siglecs. 6′-O-Su-SiaLex displayed affinity to cell-bound siglecs-1 and-5; its isomer 6-O-Su-SiaLex bound more strongly to siglecs-1,-5, and-9 than SiaLex.

Sialoside-binding macrophage lectins in phagocytosis of apoptotic bodies

Elimination of apoptotic bodies is one of the important functions of macrophages. The aim of this work was to study the role of macrophage lectins in this process. Macrophage lectins were probed with neoglycoconjugates Glyc-PAA-fluo where carbohydrate is linked to fluorescein-labeled polyacrylamide (MW 30 kD). It was shown that neoglycoconjugates containing a Neu5Acα2-3Gal fragment bound to macrophages isolated from blood of healthy donors. Besides, carbohydrate chains containing the same fragment were revealed on apoptotic bodies. Phagocytosis of apoptotic bodies by macrophages was inhibited with sialooligosaccharide ligands of siglec-5 and MAbs to siglec-5. Thus, siglec-5 expressed on macrophages could participate in phagocytosis of apoptotic bodies. In addition, the role of siglecs in engulfment of apoptotic bodies by tumor-associated macrophages was studied. The phagocytic potency of macrophages isolated from blood of breast cancer patients was lower than engulfment ability of macrophages obtained from healthy donors and depended on tumor degree. Staining of macrophages obtained from blood of tumor patients with sialylated Glyc-PAA-fluo probes was more intense than that of macrophages from healthy donors; phagocytosis of apoptotic bodies by tumor associated macrophages was inhibited by carbohydrates that are known to be ligands for siglecs.

Structure, functions, and biosynthesis of glycoconjugates of Leishmania spp. cell surface

Cell surface of leishmaniasis causal agent, a parasitic member of Protozoa of Leishmania genus, is covered by thick glycocalix consisting of various phosphatidylinositol-anchored molecules. This review deals with the structure and biosynthesis of the main phosphoglycans and glycoproteins of Leishmania cell surface, many of which incorporate the rare natural D-arabinopyranose, and the problem concerning the involvement of these molecules in support of Leishmania survival during their intricate life cycle is discussed.

Carbohydrate specificity of chicken and human tandem-repeat-type galectins-8 in composition of cells

The network of adhesion/growth-regulatory galectins in chicken (chicken galectin, CG) has only one tandemrepeat-type protein, CG8. Using a cell-based assay and probing galectin reactivity with a panel of fluorescent neoglycoconjugates (glycoprobes), its glycan-binding profile was determined. For internal validation, human galectin-8 (HG8) was tested. In comparison to HG8, CG8 showed a rather similar specificity: both galectins displayed high affinity to blood group ABH antigens as well as to 3′-sialylated and 3′-sulfated lactosamine chains. The most remarkable difference was found to be an ability of HG8 (but not CG8) to bind the disaccharide Galβ1-3GlcNAc (Lec) as well as branched and linear oligolactosamines. The glycan-binding profile was shown to be influenced by glycocalix of the cell, where the galectin is anchored. Particularly, glycosidase treatment of galectin-loaded cells led to the change of the profile. Thus, we suppose the involvement of cis-glycans in the interaction of cell-anchored galectins with external glycoconjugates.

Monomeric and Multimeric Blockers of Selectins: Comparison of in vitro and in vivo Activity

The potency of the oligosaccharides SiaLex, SiaLea, HSO3Lex, and HSO3Lea, their conjugates with polyacrylamide (PAA, 40 kD), and other monomeric and polymeric selectin inhibitors has been compared with that of the polysaccharide fucoidan. The following assay systems were used: 1) a 96-well assay based either on the use of recombinant E-, P-, and L- selectins or an analogous assay with natural P-selectin isolated from human platelets; 2) a platelet-based P-selectin cell assay; and 3) a rat model of peritoneal inflammation. IC50 values for the neoglycoconjugate SiaLea-PAA were 6, 40, and 85 µM for recombinant E-, P-, and L-selectins, respectively; all monomeric inhibitors were about two orders of magnitude weaker. PAA-conjugates, containing as a ligand tyrosine-O-sulfate (sTyr) in addition to one of the sialylated oligosaccharides, were the most potent synthetic blockers in vitro. Compared with fucoidan, the most potent known P- and L-selectin blocker, the bi-ligand glycoconjugate HSO3Lea-PAA-sTyr displayed similar inhibitory activity in vitro towards L-selectin and about ten times lower activity towards P-selectin. All of the tested synthetic polymers displayed a similar ability to inhibit neutrophil extravasation in the peritonitis model (in vivo) at 10 mg/kg. The data provide evidence that monomeric SiaLex is considerably more effective as a selectin blocker in vivo than in vitro, whereas the opposite is true for fucoidan and the bi-ligand neoglycoconjugate HSO3Lea-PAA-sTyr.

Saccharide-assisted delivery of cytotoxic liposomes to human malignant cells

The overexpression of lectins by malignant cells was applied for in vitro targeting of liposomes equipped with a saccharide vector and loaded in the lipid phase with a lipid derivative of anticancer agent sarcotysine. The lectin specificity of human leukemia HL‐60 and human lung adenocarcinoma ACL cells was revealed by tests with fluorescein‐labeled sugar probes. With the help of fluorescent lipid dye it was shown that active saccharide ligands increased the level of the vectored liposome binding to malignant cells by 50‐80% as compared to liposomes without vector or with inactive one. The degree of liposome/cell membrane fusion was monitored fluorometrically and was shown to be complete and independent of the vectors. The targeted drug‐loaded liposomes had the cytotoxic activity 2‐4 times higher as compared to the vector‐free ones.