Glycobiology Advance Access published online on February 6, 2008
Glycobiology, doi:10.1093/glycob/cwn009
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Galectin-loaded cells as platform for profiling of lectin specificity by fluorescent neoglycoconjugates: case study on galectins-1 and -3 and the impact of assay setting
1 Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry RAS, 117997, ul. Miklukho-Maklaya 16/10, Moscow, Russia
2 Institute of Physiological Chemistry, Faculty of Veterinary Medicine, Ludwig-Maximilians-University, Veterinärstr. 13, D-80539 Munich, Germany
* to whom correspondence should be addressed: N.V. Bovin, Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, 117997, ul. Miklukho-Maklaya 16/10, Moscow, Russia, fax: +7(495)3305592, e-mail: bovin{at}carbohydrate.ru
Received on October 2, 2007; accepted on February 1, 2008
The involvement of galectins as pleiotropic regulators of cell adhesion and growth in disease progression explains the interest to define their ligand-binding properties. Toward this end it is desirable to approach in vivo conditions to attain medical relevance. In order to simulate physiological conditions with cell surface glycans as by recognition sites and galectins as mediators of intercellular contacts we developed an assay using galectin-loaded Raji cells. Extent of surface binding of fluorescent neoglycoconjugates depended on the lectin presence and the type of lectin, nature of the probes’ carbohydrate headgroup and density of unsubstituted β–galactosides on the cell surface. Using the most frequently studied galectins-1 and -3, application of this assay led to rather equal binding levels for linear and branched oligomers of N-acetyllactosamine. A clear preference of galectin-3 for 
1–3-linked galactosylated lactosamine was noted. In parallel, panel of 24 neoglycoconjugates was tested as inhibitors of galectin binding from solution to N-glycans of surface-immobilized asialofetuin. These two assays differ in presentation of galectin and ligand, facilitating to spot assay-dependent properties. Under the condition of the cell assay, selectivity among oligosaccharides for the lectins was higher, and 3’-O-extraordinary affinity of galectin-1 to 3’-O-sulfated probes in solid-phase assay was lost in cell assay. Having herewith introduced and validated a cell assay, comprehensive profiling of ligand binding to cell-surface-presented galectins is made possible.
Key words: galectin / glycoprotein / lactosamine / lectin / neoglycoconjugate / oligosaccharide O-sulfate