(Strept)avidin Binds Glycoconjugates

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Abstract

Fluorescently labeled and conjugated (strept)avidin is widely used for imaging of biotinylated molecules in immunological assays and histochemistry. The presented article shows that in addition to biotin, these proteins bind a number of glycans, including fragments of glycoprotein and glycolipid chains of mammals, in particular, antigens of the ABO blood system, oligolactosamines, 6-O-sulfated oligosaccharides. This interaction is inhibited in a dose-dependent manner by glycans in a form of polymeric (but not monomeric) conjugates at the micromolar concentration level, i.e. requires polyvalence. Taking into account the cluster organization of cell glycans (glycoproteins and glycolipids), when analyzing objects containing carbohydrates, this property is a potential source of error, that can be prevented by avoiding a large excess of (strept)avidin in the analytical system.

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About the authors

N. V. Shilova

Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences; Academician Kulakov National Medical Research Center for Obstetrics, Gynecology and Perinatology

Author for correspondence.
Email: pumatnv@gmail.com
Russian Federation, 117997, Moscow; 117997, Moscow

S. M. Polyakova

Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences

Email: pumatnv@gmail.com
Russian Federation, 117997, Moscow

A. Yu. Nokel

Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences; Academician Kulakov National Medical Research Center for Obstetrics, Gynecology and Perinatology

Email: pumatnv@gmail.com
Russian Federation, 117997, Moscow; 117997, Moscow

A. D. Lipatnikov

Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences

Email: pumatnv@gmail.com
Russian Federation, 117997, Moscow

E. A. Gordeeva

Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences

Email: pumatnv@gmail.com
Russian Federation, 117997, Moscow

M. V. Lavrentyeva

MIREA – Russian Technological University

Email: pumatnv@gmail.com
Russian Federation, 119571, Moscow

N. V. Bovin

Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences

Email: pumatnv@gmail.com
Russian Federation, 117997, Moscow

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4. Fig. 1. Glycans that showed the best interaction with Alexa 555-labelled streptavidin (at a concentration of 1 µg/ml). The glycan errey consisted of 174 glycans immobilised as polyacrylamide conjugates, Glyc-RAA [11]. The maximum possible fluorescence (OEF) value in this analytical system is about 65,000; signals from ligand-free spots, i.e. background, do not exceed 100 OEF. All monosaccharides except rhamnose are D-pyranoses. Trivial names of oligosaccharides are given in parentheses. See Appendix 1 for more details

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5. Fig. 2. Interaction of PC-labelled streptavidin (at a concentration of 0.1 µg/ml) with Glyc-PAA conjugates: a - concentration dependence of the interaction with glycoconjugates immobilised in the wells of a 96-well plate; b - dose-dependent inhibition of the interaction of labelled streptavidin with immobilised GlcNAcα-RAA by GlcNAcα-RAA and GlcNAcβ-RAA, and unconjugated GlcNAcα-sp and GlcNAcβ-sp (sp - spacer). OD - optical density

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6. Fig. 3. Decreasing the concentration of streptavidin when visualising the binding of RCA120 to its ligands did not affect the overall signal intensity or the interaction profile. The Pearson correlation coefficient is 0.92. See Appendix 2 for more details

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