printed-glycan-array-sensitive-technique-for-analysis-repertoire

Printed Glycan Array: A Sensitive Technique for the Analysis of the Repertoire of Circulating Anti-carbohydrate Antibodies in Small Animals

The repertoire of circulating anti-carbohydrate antibodies of a given individual is often associated with its immunological status. Not only the ...

Russian Academy of Sciences

Auckland University of Technology

Tissue lectins are emerging (patho)physiological effectors with broad significance. The capacity of adhesion/growth-regulatory galectins to form functional complexes with distinct cellular glycoconjugates is based on molecular selection of matching partners. Engineering of variants by changing the topological display of carbohydrate recognition domains (CRDs) provides tools to understand the inherent specificity of the functional pairing. We here illustrate its practical implementation in the case of human tandem-repeat-type galectin-8 (Gal-8). It is termed Gal-8 (NC) due to presence of two different CRDs at the N- and C-terminal positions. Gal-8N exhibits exceptionally high affinity for 3'-sialylated/sulfated β-galactosides. This protein is turned into a new homodimer, i.e., Gal-8 (NN), by engineering. The product maintained activity for lactose-inhibitable binding of glycans and glycoproteins. Preferential association with 3'-sialylated/sulfated (and 6-sulfated) β-galactosides was seen by glycan-array analysis when compared to the wild-type protein, which also strongly bound to ABH-type epitopes. Agglutination of erythrocytes documented functional bivalency. This result substantiates the potential for comparative functional studies between the variant and natural Gal-8 (NC)/Gal-8N.

Studying the Structural Significance of Galectin Design by Playing a Modular Puzzle: Homodimer Generation from Human Tandem-Repeat-Type (Heterodimeric) Galectin-8 by Domain Shuffling.

Dendritic cells (DCs) play crucial roles in innate and adaptive immune response, for which reason targeting antigen to these cells is an important strategy for improvement of vaccine development. To this end, we explored recognition of DCs lectins by glycans. For selection of the glycan "vector", a library of 229 fluorescent glycoprobes was employed to assess interaction with the CD14CD16CD83 blood mononuclear cell population containing the DCs known for their importance in antigen presentation to T-lymphocytes. It was found that: 1) the glycan-binding profiles of this CD14CD16CD83 subpopulation were similar but not identical to DCs of monocyte origin (moDCs); 2) the highest percentage of probe-positive cells in this CD14 CD16CD83 subpopulation was observed for GalNAcα1-2Galβ (A), (Neu5Acα) and three mannose-reach glycans; 3) subpopulation of CD14CD16 cells preferentially bound 4'-O-Su-LacdiNAc. Considering the published data on specificity of DCs binding, the glycans showing particular selectivity for the CD14 CD16CD83 cells are likely interacting with macrophage galactose binding lectin (MGL), siglec-7 and dectin-2. In contrast, DC-SIGN is not apparently involved, even in case of mannose-rich glycans. Taking into consideration potential in vivo competition between glycan "vectors" and glycans within glycocalyx, attempting to target vaccine to DCs glycan-binding receptors should focus on A and (Neu5Acα) as the most promising vectors.

Glycan recognition by human blood mononuclear cells with an emphasis on dendritic cells.

Galectins are involved in various biological processes, e.g. cell-cell and cell-matrix adhesion and the transmission of cellular signals. Despite the diversity of functions, little is known about the nature of their physiological cognate ligands on the cell surface and the localization of galectins in the glycocalyx, although this information is important for understanding the functional activity of galectins. In this work, localization of endogenous and exogenously loaded galectins in the glycocalyx was studied. The following main conclusions are drawn: 1) galectins are not evenly distributed within the glycocalyx, they are accumulated in patches. Patching is not the result of a cross-linking of cellular glycans by galectins. Instead, patch-wise localization is the consequence of irregular distribution of glycans forming the glycocalyx; 2) galectins are accumulated in the inner zone of the glycocalyx rather than at its outer face or directly in vicinity of the cell membrane; 3) patches are not associated with cell rafts.

Localization of Galectins within Glycocalyx.

Sepharose matrix without immobilized ligands binds antibodies from human blood serum or immunoglobulin preparations. The eluted antibodies bind bacterial polysaccharides having no structural similarity to agarose (Sepharose is a cross-linked polysaccharide agarose) with a high affinity. It is concluded that the identified antibodies are capable of recognizing spatial rather than linear epitopes of bacterial polysaccharides. This side activity of Sepharose matrix should be taken into account in isolating target antibodies and other proteins from human blood.

Article	Year
Studying the Structural Significance of Galectin Design by Playing a Modular Puzzle: Homodimer Generation from Human Tandem-Repeat-Type (Heterodimeric) Galectin-8 by Domain Shuffling. Molecules (Basel, Switzerland)\| PubMed ID: 28925965	2017
Glycan recognition by human blood mononuclear cells with an emphasis on dendritic cells. Glycoconjugate journal\| PubMed ID: 29388006	2018
Localization of Galectins within Glycocalyx. Biochemistry. Biokhimiia\| PubMed ID: 30195329	2018
Human antibodies eluted from ligand-free Sepharose capable of binding bacterial polysaccharides and sulfated glycans. Molecular immunology\| PubMed ID: 30583222	2019

Nicolai Bovin

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