Marina A. Sablina

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.

Neoglycoconjugates Based on Dendrimer Poly(aminoamides)

Neoglycoconjugates containing 4, 8, 32, and 64 terminal residues of B-disaccharide (BDI) or N-acetylneuraminic acid (Neu5Ac) attached to poly(aminoamide)-type dendrimers (PAMAMs) were synthesized. The ability of BDI conjugates to bind natural xenoantibodies (anti-BDI antibodies) and the ability of Neu5Ac conjugates to inhibit the hemagglutinin-mediated adhesion of influenza virus were studied. The biological activity of PAMAM conjugates turned out to be higher than that of free carbohydrate ligands, but less than that of multivalent glycoconjugates based on other types of synthetic polymeric carriers. A conformational analysis of PAMAM matrices and resulting conjugates was performed to determine the statistical distances between carbohydrate ligands. The computations revealed the tendency of the PAMAM chains toward compaction and formation of dense globules. The process results in a decrease in the distances between the carbohydrate ligands in the conjugates and, hence, could affect the ability of glycoconjugates to efficiently bind the polyvalent carbohydrate-recognizing proteins.

P-selectin blocking potency of multimeric tyrosine sulfates in vitro and in vivo

P-selectin blocking potency was investigated using synthetic monomeric and polymeric anionic compounds containing sulfate groups such as O-sulfotyrosine (sTyr) and/or sulfated Lewis structures. A non-carbohydrate-containing polyacrylamide conjugate sTyr-PAA (80% mol of sTyr) was a remarkably potent inhibitor of P-selectin binding in vitro, having an IC(50) value of 6 ng/mL (equivalent to 10 nM calculated on the basis of sTyr residues or 0.1 nM calculated by the mass of the macromolecule). The inhibitory effect of sTyr-PAA (80%) towards P-selectin is significantly greater than that of fucoidan (IC(50), 100 ng/mL). However, sTyr-PAA (80%) was less effective than fucoidan at reducing neutrophil extravasation in an in vivo rat model of peritonitis.

Immobilization of Polyacrylamide-Based Glycoconjugates on Solid Phase in Immunosorbent Assays

Our experience in coating of solid surfaces with glycans, mainly for obtaining routine glycoarrays based on immunological plates, is summarized. Three polystyrene coating techniques are described: direct physical adsorption, covalent binding, and immobilization using the biotin tag. Protocols for studies on anticarbohydrate antibodies are considered, with special emphasis on the application niches of different immobilization techniques as related to the specificity of each method of glycan-binding protein assay, as well as the problems of background binding and quantitative estimation of the results.

Synthesis of mono- and di-O-sulfates of spacer-armed lactose

A series of spacer-armed lactose derivatives with one or two O-sulfo groups in various positions, namely, 6-O-Su-Lac, 3´,6-O-Su2-Lac, 6´-O-Su-Lac, 6,6´-O-Su2-Lac, and 3´-O-Su-Lac (Su = SO3H), was synthesized starting from the same precursor. All sulfated disaccharides were obtained in the form of 2-aminoethyl glycosides.

Synthesis of modified ether phospholipids

A number of modified ether phospholipids with additional substituents in the 2 position of the C(1)-alkyl chain, 1-O-[2′-(R,S)-hydroxyhexadecyl]-2-O-methyl-rac-glycero-3-phosphocholine, 1-O-[2′-(R,S)-acetoxyhexadecyl]-2-O-methyl-rac-glycero-3-phosphocholine, 1-O-[2′-(R,S)-hydroxyhexadecyl]-2-chloro-2-deoxy-rac-glycero-3-phosphocholine, and 1-O-[2′-(R,S)-acetoxyhexadecyl]-2-chloro-2-deoxy-rac-glycero-3-phosphocholine, have been synthesized.

Synthetic glyco-O-sulfatome for profiling of human natural antibodies

Our understanding of biological role of glycans O-sulfation remains at the level of beginners due to microheterogeneity, lability and other difficulties of exact structural assignment. Partially, problem of functional investigations, especially determination of glycoepitope specificity of carbohydrate-binding proteins could be solved with the help of synthetic glycans of certain structure. Here we summing up our synthetic efforts in creation of synthetic O-sulfatome, and bring together all the synthesized in our group sulfated glycans, both existing in nature, yet undiscovered but biochemically licit, and completely unnatural. All glycans have aminoalkyl spacer group allowing immobilization on a chip. We exemplify the capabilities of O-sulfoglycan microarray (containing >70 ligands) for profiling human natural antibodies; for a number of glycans O-sulfation dramatically changes interaction with human antibodies.