Recent Advancements in Arrayed Technologies and Emerging Themes in the Identification of Glycan-Protein Interactions

Joeh, Eugene; Vilen, Zak; O’Leary, Timothy R.; Huang, Mia L.

doi:10.1021/bk-2020-1346.ch001

Cited by 2 publications

(2 citation statements)

References 85 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In 2002, the high-throughput platform through printed glycan microarrays was introduced to study glycan-GBP interactions ( Fukui, Feizi, Galustian, Lawson, & Chai, 2002 ; Park & Shin, 2002 ; Wang, Liu, Trummer, Deng, & Wang, 2002 ; Willats, Rasmussen, Kristensen, Mikkelsen, & Knox, 2002 ). Additional microarray-based techniques such as shotgun glycomics and beam search arrays, surface plasmon resonance (SPR), the use of glycopolymers and glycodendrimers in microarrays, DNA encoded glycan arrays utilizing next generation sequencing, and lectin-affinity columns to capture interactions have all contributed to the growth of the array-based methods of studying glycan-GBP interactions ( Joeh, Vilen, O’Leary, & Huang, 2020 ). However, all of these methods drive focus onto the structure of the requisite binding glycans and require artificial, often static systems to study these interactions.…”

Section: Commentarymentioning

confidence: 99%

Mapping Interactions between Glycans and Glycan‐Binding Proteins by Live Cell Proximity Tagging

et al. 2021

Self Cite

View full text Add to dashboard Cite

Interactions between glycans and glycan‐binding proteins (GBPs) consist of weak, noncovalent, and transient binding events, making them difficult to study in live cells void of a static, isolated system. Furthermore, the glycans are often presented as protein glycoconjugates, but there are limited efforts to identify these proteins. Proximity labeling permits covalent tagging of the glycoprotein interactors to query GBP in live cells. Coupled with high‐resolution mass spectrometry, it facilitates determination of the proteins bearing the interacting glycans. In this method, fusion protein constructs of a GBP of interest with a peroxidase enzyme allows for in situ spatiotemporal radical‐mediated tagging of interacting glycoproteins in living cells that can be enriched for identification. Using this method, the capture and study of glycan‐GBP interactions no longer relies on weak, transient interactions, and results in robust capture and identification of the interactome of a GBP while preserving the native cellular environment. This protocol focuses on (1) expression and characterization of a recombinant fusion protein consisting of a peroxidase and the GBP galectin‐3, (2) corresponding in situ labeling and visualization of interactors, (3) and proteomic workflow and analysis of captured proteins for robust identification using mass spectrometry. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Expression, purification, and characterization of recombinant fusion protein Alternate Protocol 1: Manual Ni‐NTA purification of recombinant fusion protein Basic Protocol 2: In situ proximity labeling and evaluation by fluorescence microscopy Alternate Protocol 2: Western blot analysis of in situ proximity labeling Basic Protocol 3: Proximity labeling of cells for quantitative MS‐based proteomics with tandem mass tags

show abstract

Section: Commentarymentioning

confidence: 99%

Mapping Interactions between Glycans and Glycan‐Binding Proteins by Live Cell Proximity Tagging

et al. 2021

Self Cite

View full text Add to dashboard Cite

show abstract

“…The surface of every cell is coated with complex glycans, installed on lipids or as posttranslational modifications on proteins, forming a glycocalyx at which cellular interactions occur [1][2][3][4][5][6] . Cell-surface glycans mediate biological processes as diverse as cell-cell adhesion, bacterial and viral infection, and immune regulation [7][8][9] .…”

Section: Introductionmentioning

confidence: 99%

Chemoenzymatic Synthesis of Genetically-Encoded Multivalent Liquid N-glycan Arrays

Lin

Sojitra

Carpenter

et al. 2022

Preprint

View full text Add to dashboard Cite

A hallmark of cellular glycosylation is its chemical complexity and heterogeneity, which can be challenging to capture synthetically. Using chemoenzymatic synthesis on M13 phage, we produce a genetically-encoded liquid glycan array (LiGA) of biantennary complex type N-glycans. Ligation of azido-functionalized sialylglycosyl-asparagine derived from egg yolk to phage functionalized with 50-1000 copies of dibenzocyclooctyne produced divergent intermediate that can be trimmed by glycosidases and extended by glycosyltransferases to yield a library of phages with different N-glycans. Post-reaction analysis by MALDI-TOF MS provided a rigorous approach to confirm N-glycan structure and density, both of which were encoded in the bacteriophage DNA. The binding of this N-glycan LiGA by ten lectins, including CD22 or DC-SIGN expressed on live cells, uncovered an optimal structure/density combination for recognition. Injection of the LiGA into mice identified glycoconjugates with structures and avidity necessary for enrichment in specific organs. This work provides an unprecedented quantitative evaluation of the interaction of complex N-glycans with GBPs in vitro and in vivo.

show abstract

Recent Advancements in Arrayed Technologies and Emerging Themes in the Identification of Glycan-Protein Interactions

Cited by 2 publications

References 85 publications

Mapping Interactions between Glycans and Glycan‐Binding Proteins by Live Cell Proximity Tagging

Mapping Interactions between Glycans and Glycan‐Binding Proteins by Live Cell Proximity Tagging

Chemoenzymatic Synthesis of Genetically-Encoded Multivalent Liquid N-glycan Arrays

Contact Info

Product

Resources

About