Development of recombinant Aleuria aurantia lectins with altered binding specificities to fucosylated glycans

Abstract

Changes in glycosylation have long been associated with disease. While there are many methods to detect changes in glycosylation, plant derived lectins are often used to determine changes on specific proteins or molecules of interest. One change in glycosylation that has been observed by us and by others is a disease or antigen associated increase in fucosylation on N-linked glycans. To measure this change, the fucose binding Aleuria aurantia lectin (AAL) is often utilized in plate and solution based assays. AAL is a mushroom derived lectin that contains five fucose binding sites that preferentially bind fucose linked (α-1,3, α-1,2, α-,4, and α-1,6) to N-acetyllactosamine related structures. Recently, several reports by us and by others have indicated that specific fucose linkages found on certain serum biomarker glycoprotein's are more associated with disease than others. Taking a site-directed mutagenesis approach, we have created a set of recombinant AAL proteins that display altered binding affinities to different analytes containing various fucose linkages.

Intrinsic hepatocyte dedifferentiation is accompanied by upregulation of mesenchymal markers, protein sialylation and core alpha 1,6 linked fucosylation

“…2C. Panel D shows the increased fucosylation over time using a recombinant Aleuria aurantia lectin with greater affinity for core fucose glycan, N224Q rAAL42 (see Methods). Figure 2C,D are consistent with the HPLC glycan analysis in that sialic acid composition changes and fucosylation increases in the PRH following time in culture.…”

“…Eleven of these contained grade I-II tumor tissue (defined as well-differentiated or moderately-differentiated tissue) and 5 contained grade II-III tumor tissue (defined as poorly differentiated). The level of fucosylation was analyzed by lectin histochemistry using the N224Q rAAL that has enhanced affinity for core fucosylated glycan as opposed to other fucose linkages42. In our analysis, only 2 out of the 11 grade I-II tumors stained positive with the N224Q rAAL.…”

“…In the case of lectin blotting, fucosylation was detected using a recombinant N224Q rAAL lectin. This is a recombinant AAL lectin that has been modified to increase binding to core fucosylated glycan42. For sialic acid detection, we used commercially purchased Sambucus nigra lectin (SNA, Vector Labs, Cat #B-1305), which binds preferentially to sialic acid attached to terminal galactose in α-2,6 and to a lesser degree, α-2,3 linkage.…”

Glycoprotein Disease Markers and Single Protein-omics

“…This is an important consideration for biomarker discovery and even more for the performance of clinical assays where the need for validation of the reagents cannot be overemphasized (34). Detailed mass spectrometric characterization of glycoprotein epitopes, development of engineered lectins, or optimization of carbohydrate-targeting immunoassays is expected to improve the quality of glycoprotein quantification (44,49). Alternative quantitative methods complementary to immunoassays will further facilitate use of glycoconjugates in disease monitoring (50 -52).…”

“…We are not aware of clinical assays using direct lectin recognition of carbohydrates. Nonetheless, lectin-based ELISAs have been developed, and engineered lectins with increased specificity and binding affinity should enhance biomarker discovery and reach clinical utility (44,45).…”