Evanescent-field fluorescence-assisted lectin microarray: a new strategy for glycan profiling

Abstract

Glycans have important roles in living organisms with their structural diversity. Thus, glycomics, especially aspects involving the assignment of functional glycans in a high-throughput manner, has been an emerging field in the postproteomics era. To date, however, there has been no versatile method for glycan profiling. Here we describe a new microarray procedure based on an evanescent-field fluorescence-detection principle, which allows sensitive, real-time observation of multiple lectin-carbohydrate interactions under equilibrium conditions. The method allows quantitative detection of even weak lectin-carbohydrate interactions (dissociation constant, K(d) > 10(-6) M) as fluorescent signals for 39 immobilized lectins. We derived fully specific signal patterns for various Cy3-labeled glycoproteins, glycopeptides and tetramethylrhodamine (TMR)-labeled oligosaccharides. The obtained results were consistent with the previous reports of glycoprotein and lectin specificities. We investigated the latter aspects in detail by frontal affinity chromatography, another profiling method. Thus, the developed lectin microarray should contribute to creation of a new paradigm for glycomics.

Lectin Microarray Reveals Binding Profiles of Lactobacillus casei Strains in a Comprehensive Analysis of Bacterial Cell Wall Polysaccharides

“…3, each strain had a unique binding profile. Similar to previously reported glycoproteins (20) and mammalian cells (37), the bacterial cells had their own molecular patterns of glycosylation on their cell surfaces. It can be also speculated that YIT 0005, YIT 0006, and YIT 0180 T have similar outer surface PS structures.…”

“…A novel technique called lectin microarray was developed to profile the complex features of glycans expressed in various forms (2,14,20,37,38). We adopted a unique evanescentfield-activated fluorescence detection principle to detect highly sensitive and reproducible lectin-glycoconjugate interactions on a glass side (20,38).…”

“…We adopted a unique evanescentfield-activated fluorescence detection principle to detect highly sensitive and reproducible lectin-glycoconjugate interactions on a glass side (20,38). Using this detection principle, Tateno et al (2007) developed an application method enabling detection of direct interaction between lectins and whole mammalian cells (37).…”

A lectin array-based methodology for the analysis of protein glycosylation

“…Recently, several reports on lectin arrays have appeared in the literature [41][42][43][44]. In all of the approaches, lectins are arrayed onto glass slides, and the binding of either intact glycoproteins [41][42][43] or whole cells [44] is measured.…”

Uncovering the Binding Specificities of Lectins with Cells for Precision Colorectal Cancer Diagnosis Based on Multimodal Imaging

“…These characteristics make lectins very useful for glycoanalysis and medicine development 16, 17, 18, 19, 20, 21. Due to their minimization of sample consumption and high throughput format for analyzing multiple targets simultaneously, lectin microarrays have been employed in the glycomics study 22, 23, 24, 25, 26. However, the interactions of lectins with glycans (dissociation constant, K d  = 10 −4 –10 −7 m ) are much weaker than the interactions of antigens with antibodies ( K d  = 10 −8 –10 −12 m ).…”