Using lectins in biomarker research: addressing the limitations of sensitivity and availability

Abstract

Carbohydrates have fundamental roles throughout biology, yet they have not been as well studied as proteins and nucleic acids, in part due to limitations in the experimental tools. Improved methods for studying glycans could spur significant advances in the understanding and application of glycobiology. The use of affinity reagents, such as lectins and glycan-binding antibodies, is a valuable complement to methods involving mass spectrometry and chromatography. This article addresses two limitations that have prevented the broader experimental use of glycan-binding proteins: sensitivity and availability. The sensitivity limitation stems from the poor affinity that many glycan-binding proteins have as isolated analytical reagents. To address this problem, I propose making use of multivalent interactions between lectins and glycans, mimicking those frequently found in the biological setting. Recent experiments show that a practical technique for producing lectin multimers can significantly improve detection sensitivity. The second limitation, availability, is the difficulty of finding and obtaining glycan-binding proteins that recognize less common or arbitrarily defined glycan structures. To address this problem, I propose translating the wealth of existing glycan array data into a quantitative, searchable database of the specificities of glycan-binding proteins. Such a resource would allow us to more easily identify proteins with defined specificities and perform detailed comparisons between reagents. Solutions to these two limitations could lead to the more effective use of, and a broader range of, glycan-binding reagents.

Figure 1. Lectin-based detection of glycans in microarray formats

The types of microarrays depicted are lectin arrays, antibody-lectin sandwich arrays, and glycoprotein arrays. A detection strategy using a fluorescent dye is depicted, although other detection methods could be used, such as surface-plasmon resonance or chemiluminescence. Antibody-lectin sandwich arrays (middle row) enable the detection of glycans on specific proteins captured from biological solutions. Multiple antibodies are immobilized on a planar support, and the captured proteins are probed using biotinylated detection antibodies, followed by fluorescence detection using phycoerythrin-labeled streptavidin.

Figure 2. Detection using multimerized lectins

The left panel shows the conventional method of labeling and detecting individual lectins. The right panel shows a method of linking together lectins through the multiple binding sites of streptavidin and through an anti-streptavidin antibody.

Figure 3. Comparative analysis of the motif specificities of plant lectins

The data from 113 different glycan array incubations of 84 unique plant lectins were analyzed using the Motif Segregation method, and the logged p values (multiplied by the sign of the z score) are clustered by similarity among both the motifs (columns) and the rows (lectins). This analysis shows the potential for assembling quantitative information from multiple glycan-binding to enable searching and comparative studies. Reprinted from reference [13] with permission from Oxford Journals.