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Non-canonical interactions between plant proteins and lectins cause false positives in lectin blots

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Author(s): Marshall Louis Reaves | Linda C. Lopez | Sasha M. Daskalova

Journal: Research in Plant Biology
ISSN 2231-5101

Volume: 1;
Issue: 4;
Start page: 49;
Date: 2011;
Original page

Keywords: plant proteins | lectin blot | MAA | SNA | VVA

ABSTRACT
Lectins are proteins that specifically recognize and non-covalently bind to solublecarbohydrates or to the carbohydrate moieties of glycoproteins or glycolipids. Historically,lectin-blot analysis has been widely used as a tool for structural characterization of manymammalian glycoconjugates. In the present study, we demonstrate that the application ofthis technique to screen sugar moieties of plant proteins results in numerous false positives.Plants lack the enzyme machinery necessary to perform sialylation, however many bandsappear upon probing of N. benthamiana L. leaf proteins with Maacia amurensis agglutinin(MAA) that recognizes specifically N-linked or core 2 O-linked glycans containingNeu5Ac/Gc-α2,3Galβ-1,4GlcNAc/Glc and O-linked glycans containing the trisaccharideNeu5Ac-3Galβ1-3GalNAc. The non-canonical binding is a direct result of samplepreparation for SDS PAGE, because native proteins do not show an affinity to MAA-agaroseresin. Moreover, inhibition with known hapten fails to prevent binding of MAA to plantproteins in lectin blots. We also provide evidence that interactions of a hydrophobic naturecontribute, at least in part, to the non-specific binding, and that other lectins – Sambucusnigra agglutinin (SNA) and Vicia villosa agglutinin (VVA) – also bind non-specifically toplant proteins. In conclusion, lectin blot analysis of plant proteins should always be verifiedby probing the binding specificity with a known hapten inhibitor alongside appropriatemammalian glycoprotein controls. Alternatively, non-specific binding can be avoided iflectin affinity chromatography of the native plant proteins is performed prior to lectin blotsanalysis.
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