[Conformational polymorphysm of G-rich fragments of DNA ALU-repeats. I. Potential noncanonical structures]. / Konformatsionnyi polimorfizm G-bogatykh fragmentov ALU-povtorov DNK. I. Nekanonicheskie struktury.

In this paper, we report results of systematic studies of conformational polymorphism of G-rich DNA fragments from Alu repeats. Alu retrotransposones are primate-specific short interspersed elements. Using the Alu sequence from the prooncogen bcl2 intron and the consensus AluSx sequence as representative examples, we determined characteristic Alu sites that are capable of adopting G-quadruplex (GQ) conformations (i.e., potential quadruplex sites - PQSAlu), and demonstrated by bioinformatics methods that those sites are Alu-specific in the human genome. Genomic frequencies of PQSAlu were assessed (~1/10000 b.p.). The sites were found to be characteristic of young (active) Alu families (Alu-Y). A recombinant DNA sequence bearing the Alu element from the human bcl2 gene (304 b.p.) and its PQS-mutant (Alu-PQS) were constructed. The formation of noncanonical structures in Alubcl2 dsDNA and the absence of such structures in the case of Alu-PQS were shown using DMS-footprinting and AFM microscopy. Expression vectors bearing wild-type and mutant Alu insertions in the promoter regions were obtained, and the effects of these insertions on the expression of the reporter gene in НЕК293 and HeLa cell lines were compared. Our findings on the spatial organization of Alu repeats may provide insight into the mechanisms of genomic rearrangements which underlie many oncological and neurodegenerative diseases.

[Synthesis of Oligocaccharides Containing Internal and Terminal Fragment Galß1-3GlcNAc].

Synthesis of oligocaccharides Galß1-3GlcNAcß-sp, GlcNAcß1-3Galß1-3GlcNAcß-sp, Galß1-3GlcNAcß1-3Galß1-3GlcNAcß-sp, Galß1-3GlcNAcß1-3Galß1-4GlcNAcß-sp, Galß1-3GlcNAcß1-6Galß1-4GlcNAcß-sp (sp = O(CH2)3NH2 or O(CH2)2NH2) was carried out using glycosylation by N-Troc-protected derivatives of glycosamine or disaccharide Galß1-3GlcN.

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 tandem-repeat-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 Galß1-3GlcNAc (Le(c)) 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.

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.

[The synthesis of linear trilactosamine].

Trilactosamine Galbeta1-4GlcNAcbeta1-3Galbeta1-4GlcNAcbeta1-3Galbeta1-4GlcNAcbeta-sp, where sp = O(CH2)3NH2 is a spacer, was synthesized. The tetrasaccharide fragment Galbeta1-4GlcNAcbeta1-3Galbeta1-4GlcNAcbeta-sp was obtained by successive glycosylation using elongation by one monosaccharide residue at a time, and the tetrasaccharide was then transformed into a hexasaccharide with a disaccharide glycosyl donor. A 2,2,2-trichloroethoxycarbonyl group was used for the protection of the glucosamine amino group.

[Synthesis of N-acetyllactosamine-containing oligosaccharides, galectin ligands].

The following spacered oligosaccharides were synthesized: GlcNAcbetal-3Galbetal-4GlcNAcbeta-sp, GlcNAcbetal-6Galbeta1-4GlcNAcbeta -sp, GlcNAcbeta -3(GlcNAcbeta1-6)Galbeta-4GllcNAcbeta-sp, Galbeta1-4GlcNAcbeta1-3Galbeta1-4GlcNAcbeta-sp, Galbeta1-4GlcNAcbetal-6Galbetal-4GlcNAcbeta-sp, Galbeta1-4GlcNAcbeta -3(Galbeta1-4GlcNAcbeta 1-6)Galbeta1-4GlcNAcbeta-sp, GlcNAcbeta1-3(Galbeta1-4GlcNAcbetal-6)Galbeta 1-4GlcNAcbeta-sp, and Galbeta1-4GlcNAcbetal-3(GlcNAcbetal-6)Galbetal-4GlcNAcbeta-sp (sp = O(CH2)2NH2). They represent N-acetyllactosamines substituted with N-acetylgly-cosamine or N-acetyllalctosamine residue at 03, O6, or at both positions of galactose. Glycosylation was achieved by coupling with N-trichloroethoxycarbonyl-protected glucosamine bromide in the presence of silver triflate.