High-throughput carbohydrate microarray analysis of 24 lectins
High-throughput carbohydrate microarray analysis of 24 lectins
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DOI：
10.1002/anie.200600591
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发表时间：
2006-01-01
影响因子：
16.6
通讯作者：
Gildersleeve, Jeffrey C.
中科院分区：
文献类型：
--
作者：
Manimala, Joseph C.;Roach, Timothy A.;Gildersleeve, Jeffrey C.
Lectins, non-immunoglobulin proteins that bind carbohydrates, play a central role in a wide range of biological processes such as cell–cell recognition, viral and bacterial pathogenesis, and inflammation.[1, 2] Moreover, they are used extensively as research tools, diagnostics, and therapeutics. For example, mistletoe lectin is in clinical trials as an anticancer agent.[3] Therefore, a fundamental understanding of carbohydrate–protein interactions and comprehensive information on lectin specificity is critical. Unfortunately, evaluation of lectin specificity is not trivial. One common method involves measuring the binding of lectins to cells, tissues, and glycoproteins. This approach frequently uncovers interesting and useful binding properties, however, cells, tissues, and glycoproteins display complex mixtures of carbohydrate epitopes. Therefore, it is exceedingly difficult to determine the specific carbohydrate structures being recognized by the lectin. An alternative approach involves measuring binding to structurally defined carbohydrate epitopes through techniques such as isothermal calorimetry (ITC), mono-and oligosaccharide inhibition studies, enzymelinked lectin assays (ELLA), and surface plasmon resonance assays (SPR). Unfortunately, these methods can be labor intensive, require large amounts of carbohydrates, and/or be difficult to perform in a high-throughput fashion. Moreover, these studies have typically been limited to the small number of carbohydrate epitopes that were readily accessible. Although lectin specificity has been studied often, much more comprehensive information is still needed. Carbohydrate microarrays are an emerging technology for the high-throughput evaluation of carbohydrate-macromolecule interactions.[4–27] Analogous to DNA and protein arrays, carbohydrate microarrays contain numerous carbohydrate epitopes immobilized on a solid support in a miniaturized fashion. The microarray format allows one to rapidly evaluate many potential interactions with a minimal amount of sample. Our group has recently developed a carbohydrate microarray and a highly sensitive assay to detect binding.[21] To maximize throughput, each slide contained 16 wells with an entire array printed in each well (see Figure1a). To illustrate the capabilities of our microarray and study lectin sepcificity, 24 lectins were evaluated at eight different concentrations by using the microarray. As one of the largest and most-comprehensive lectin studies ever reported, the results should be a useful resource for scientists conducting basic and applied research with lectins. Interestingly, microarray analysis revealed unexpected ligands for many of the lectins.Our approach for fabricating arrays involves printing carbohydrate–bovine serum albumin (BSA)/human serum albumin (HSA) conjugates and glycoproteins on epoxidefunctionalized glass microscope slides. This strategy permits immobilization of both structurally discrete synthetic carbohydrates as well as natural carbohydrates presented on glycoproteins. Our first array contained 29 components. To increase the diversity, 23 additional glycans were chemically synthesized and 21 were purchased (see the Supporting Information). The array then contained 73 different components: 4 controls, 54 BSA/HSA conjugates, and 15 glycoproteins (see Figure 1c).
