Abstract B31: Development of fucose based pancreatic cancer biomarkers using modified lectins
Authors: Singh Sudhir , Pal Kuntal , Ensink Elliot , Yadav Jessica , Kletter Doron , Bern Marshall , Mehta Anand , Melcher Karsten , Haab Brian B. 
Published: October 2015

Abstract
Pancreatic cancer has poor prognosis because of ineffective and delayed diagnosis as well as treatment. Accurately identifying molecular changes could help with early detection and evaluation of treatment efficacy. Glycoprotein fucosylation is associated with various cancers. Interestingly, information at structural level such as diversity in fucose linkages can provide extensive details of disease state. But, lack of diagnostic reagents has impeded the research in identifying and interpreting these changes. This study is aimed to develop fucose based cancer biomarker reagents to identify important fucose linkages associated with cancer progression.
We hypothesized that a panel of lectins, each specific for a particular type of fucose presentation, could identify differences between glycoproteins that are not discernable using any individual lectin or measurements of total fucose. Using a database of glycan array data, we identified lectins with high specificity for diverse linkages of fucose. We selected three for further testing: CGL2 for detecting fucose in a 2’ linkage, CCL2 for detecting fucose in a 3’ linkage, and RSL for detecting fucose in all linkages. To improve quality, production consistency, and test various modes of detection the lectins were recombinantly expressed with avitag (biotin) and poly-histidine fusion tags.
The choice of terminus as well as the type of fusion tag could affect the molecular architecture and thereby lectin-glycan interaction. Therefore, two variants for each lectin (N terminus biotin and C terminus histidine and vice-versa) were expressed. The initial purification by size-exclusion chromatography showed similar results between the two tagging schemes for each protein. To test the accessibility of fusion tags and lectin multimerization with secondary detections (streptavidin or anti-poly-histindine), the two were pre-incubated prior to separation on an analytical scale size exclusion column resulting in protein elution that was entirely shifted to a higher molecular weight.
Specificity for recombinant lectins to their respective linkages was performed using thermostability assay that resulted in a stability shift of lectins with respective glycan target but not with off-target glycan. To examine if the use of particular tag or secondary reagent was optimal for detection we performed competitive assay. Lectin binding to spotted glycoproteins in microarray assay was reduced upon pre-incubation with a competing sugar but not upon pre-incubation with a non-specific sugar. This suggests that recombinant lectins with fusion tags were functional.
Lectins produced by recombinant expression showed specific glycan-binding using either a biotin or poly-histidine fusion tag at either terminus, but detection using the C-terminus tag was more reliable in each case. We probed the glycans on various glycoproteins with each lectin and predicted that the pattern of lectin binding would perform better than individual lectin measurements for identifying differences in fucose presentation. We tested this prediction using monoclonal antibodies against selected fucose-bearing glycans. Based on the known specificities of the antibodies, we could confirm differences in glycans predicted by the pattern of lectin binding but not by individual lectin measurements, thus confirming the ability to distinguish fucose presentations using a panel of lectins. This technique promises to help uncover details about the presentation of fucose on specific glycoproteins.
The ability of these lectins to distinguish subtle changes in glycan presentation with enough precision at early stage and enable comparisons between tumor grades could prove valuable diagnostic reagents for pancreatic cancer.
Citation Format: Sudhir Singh, Kuntal Pal, Elliot Ensink, Jessica Yadav, Doron Kletter, Marshall Bern, Anand Mehta, Karsten Melcher, Brian B. Haab. Development of fucose based pancreatic cancer biomarkers using modified lectins. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr B31.