A lectin‐based gold nanoparticle assay for probing glycosylation of glycoproteins

Abstract

We report a glycoanalysis method in which lectins are used to probe the glycans of therapeutic glycoproteins that are adsorbed on gold nanoparticles. A model mannose-presenting glycoprotein, ribonuclease B (RNase B), and the therapeutic monoclonal antibody (mAb) rituximab, were found to adsorb spontaneously and non-specifically to bare gold nanoparticles such that glycans were accessible for lectin binding. Addition of a multivalent binding lectin, such as concanavalin A (Con A), to a solution of the modified gold nanoparticles resulted in cross-linking of the nanoparticles. This phenomenon was evidenced within 1 min by a change in the hydrodynamic diameter, D(H), measured by dynamic light scattering (DLS) and a shift and increase in absorbance of the plasmon resonance band of the gold nanoparticles. By combining the sugar-binding specificity and the cross-linking capabilities of lectins, the non-specific adsorption of glycoproteins to gold surfaces, and the unique optical reporting properties of gold nanoparticles, a glycosylation pattern of rituximab could be generated. This assay provides advantages over currently used glycoanalysis methods in terms of short analysis time, simplicity of the conjugation method, convenience of simple spectroscopic detection, and feasibility of providing glycan characterization of the protein drug product by using a variety of binding lectins.

Lectins as probes for assessing the accessibility of N‐linked glycans in relation to the conformational changes of fibronectin

“…The results suggest that lectins are useful and powerful probes for fine‐tuned biochemical studies of the conformation of ECM glycoproteins. In comparison with recent immobilized methods such as lectin arrays (Etxebarria et al ., ) or surface plasmon resonance (Sanchez‐Pomales et al ., ), the combined approach described herein could give more comprehensive insight into changes in glycan accessibility in response to slight structural modifications. Modification of cell surface protein glycosylation in vivo is associated with pathophysiological processes, for example, the ability of cancer cells to metastasize, leading to a poor prognosis (Brooks, ).…”

Luminescent Gold Nanodots for Detection of Heavy Metal Ions, Proteins and Bacteria

“…Con A is a member of the lectin family; it binds selectively to α-mannopyranosyl and α-glucopyranosyl residues (58). Although most lectin-carbohydrate interactions have low binding constants, usually over 10 3 -10 7 M -1 range (59), the extremely high local concentrations of carbohydrates found on the surfaces of carbohydrate-captured nanoparticles make them particularly useful for probing glycoproteins (60)(61)(62)(63)(64). Man-Au ND/Con A aggregates are formed through specific interactions between the mannose units and the multivalent Con A.…”

Multivalent Lactose–Ferrocene Conjugates Based on Poly (Amido Amine) Dendrimers and Gold Nanoparticles as Electrochemical Probes for Sensing Galectin-3

“…We tested the binding ability of AuNP@Fc-Lac toward Gal-3 by following their UV-vis spectra variations after the addition of the lectin. It is well-known that this technique is a very useful tool to demonstrate protein-induced aggregation of AuNPs due to the dependence of the surface plasmon resonance (SPR) band on interparticle distance [36,61,62]. Thus, upon AuNPs aggregation, coupling interactions between the surface plasmon fields of the nanoparticles take place, leading to a change of the local refractive index around the nanoparticles, a red shift of the SPR band, and (usually) a broadening that, on occasion, results in absorbance decay.…”