Surface Plasmon Resonance Study of Protein−Carbohydrate Interactions Using Biotinylated Sialosides

Abstract

Lectins are carbohydrate binding proteins found in plants, animals, and microorganisms. They serve as important models for understanding protein-carbohydrate interactions at the molecular level. We report here the fabrication of a novel sensing interface of biotinylated sialosides to probe lectin-carbohydrate interactions using surface plasmon resonance spectroscopy (SPR). The attachment of carbohydrates to the surface using biotin-NeutrAvidin interactions and the implementation of an inert hydrophilic hexaethylene glycol spacer (HEG) between the biotin and the carbohydrate result in a well-defined interface, enabling desired orientational flexibility and enhanced access of binding partners. The specificity and sensitivity of lectin binding were characterized using Sambucus nigra agglutinin (SNA) and other lectins including Maackia amurensis lectin (MAL), concanavalin A (Con A), and wheat germ agglutinin (WGA). The results indicate that alpha2,6-linked sialosides exhibit high binding affinity to SNA, while alteration in sialyl linkage and terminal sialic acid structure compromises the affinity by a varied degree. Quantitative analysis yields an equilibrium dissociation constant (KD) of 777 +/- 93 nM for SNA binding to Neu5Ac alpha2,6-LHEB. Transient SPR kinetics confirms the K D value from the equilibrium binding studies. A linear relationship was obtained in the 10-100 microg/mL range with limit of detection of approximately 50 nM. Weak interactions with MAL, Con A, and WGA were also quantified. The control experiment with bovine serum albumin indicates that nonspecific interaction on this surface is insignificant over the concentration range studied. Multiple experiments can be performed on the same substrate using a glycine stripping buffer, which selectively regenerates the surface without damaging the sialoside or the biotin-NeutrAvidin interface. This surface design retains a high degree of native affinity for the carbohydrate motifs, allowing distinction of sialyl linkages and investigation pertaining to the effect of functional group on binding efficiency. It could be easily modified to identify and quantify binding patterns of any low-affinity biologically relevant systems, opening new avenues for probing carbohydrate-protein interactions in real time.

Antibody blood-brain barrier efflux is modulated by glycan modification

“…The first hypothesis is that all endogenous 4G8 exhibit reduced efflux because they all present sialic acid in a variety of linkage states and locations that are not detectable by surface SNA binding methods. In one version of this hypothesis, α2,3 or α2,8 sialyalted glycans dominate 4G8 and that α2,6 Sia-Gal linkages are a minor glycan component [54–57]. In a second version of this hypothesis, Fc sialylation or other buried glycan groups with sialic acid are ubiquitous on all 4G8.…”

“…1, the coefficient β = 5.5 × 10 −10 (mol*s)/(L*RU) incorporates all known variables of IgG properties and dimensions of the Biacore T200 flow cell [52, 53]. The SNA flow cell provides the concentration of IgG with surface accessible sialic acid-α2,6-galactose linkage, denoted here as [IgG SNA ] [54]. Since both 6E10 and 4G8 bind OAβ, the OAβ flow cell provides the total IgG concentration, denoted here as [IgG OAβ ].…”

“…Lectins used to probe these specific configurations are SNA (Sia-α2,6-Gal), ECL (Gal-β1,4-GlcNAc), MAL I (Sia-α2,3-Gal-β1,4-GlcNAc), MAL II (Sia-α2,3-Gal-β1,3-GalNAc), and Siglec-11 (Sia-α2,8-Sia) [54–57]. Using these lectin probes, the On-Chip method was used to determine the presence of surface glycans on 4G8 and 6E10 with their particular sialic acid target permutations.…”

Microarray-Based Technology for Glycomics Analysis

“…Recently, several microarray platforms with nonfluorescence detection techniques including mass spectrometry (MS) [25,39,78,79], surface plasmon resonance (SPR) [52,53,[80][81][82][83], scanning ellipsometry (imaging) [84], and resonant light scattering (RLS) of nanoparticle [85,86], have developed for glycomic analysis. For instance, Cheng and coworkers reported the fabrication of a carbohydrate microarray with surface plasmon resonance imaging (SPRi) detection system for studying interactions of carbohydrates with lectins (see Fig.…”

Electrochemical synthesis of nanostructured gold film for the study of carbohydrate–lectin interactions using localized surface plasmon resonance spectroscopy

“…The interaction between carbohydrate and lectin is chosen because this type of interaction is predominant in many fundamental biological processes such as cellular recognition, inflammation, signal transduction, cell adhesion, and cancer cell metastasis. 56,57 Concanavalin A (Con A), 104 kDa, originally obtained from Jack bean, Canavalia ensiformis , is used as a model lectin. 58 It is a mannose specific lectin 59 and exists as tetramer above pH 7.0 and dimer below 6.0.…”