An engineered high affinity Fbs1 carbohydrate binding protein for selective capture of N-glycans and N-glycopeptides

Abstract

A method for selective and comprehensive enrichment of N-linked glycopeptides was developed to facilitate detection of micro-heterogeneity of N-glycosylation. The method takes advantage of the inherent properties of Fbs1, which functions within the ubiquitin-mediated degradation system to recognize the common core pentasaccharide motif (Man3GlcNAc2) of N-linked glycoproteins. We show that Fbs1 is able to bind diverse types of N-linked glycomolecules; however, wild-type Fbs1 preferentially binds high-mannose-containing glycans. We identified Fbs1 variants through mutagenesis and plasmid display selection, which possess higher affinity and improved recovery of complex N-glycomolecules. In particular, we demonstrate that the Fbs1 GYR variant may be employed for substantially unbiased enrichment of N-linked glycopeptides from human serum. Most importantly, this highly efficient N-glycopeptide enrichment method enables the simultaneous determination of N-glycan composition and N-glycosites with a deeper coverage (compared to lectin enrichment) and improves large-scale N-glycoproteomics studies due to greatly reduced sample complexity.

Metabolic precision labeling enables selective probing of O-linkedN-acetylgalactosamine glycosylation

“…4A, Data S1) and were extended by the downstream glycosylation machinery. For instance, biosynthetic considerations allowed the assignment of the disaccharide β-Gal-(1-3)-α-GalNAzMe-(Thr*) on the glycopeptide TTPPT*TATPIR of human fibronectin, along with other glycoforms and even a di-glycosylated peptide TTPPT*T*ATPIR (Data S1). DMSO feeding did not lead to discernible signal.…”

“…Application of these techniques to glycobiology relies on the suitability of detection reagents. Antibodies, lectins, and engineered glycosidases have been instrumental, but are somewhat restricted to sterically accessible epitopes(3–6). Monosaccharides with bioorthogonal, chemically-editable functionalities have allowed a complementary view into glycobiology by entering early glycosylation events (7–11).…”

Cytosolic N‐Glycans: Triggers for Ubiquitination Directing Proteasomal and Autophagic Degradation

“…Because FBXO2, FBXO6, and FBXO27 recognize the common core pentasaccharide motif (Man 3 GlcNAc 2 ) of N‐glycans, they can bind a wide range of N‐glycans . However, they preferentially bind high‐mannose glycans . Like FBXO2 and FBXO6, FBXO27 binds glycoproteins modified with high‐mannose N‐glycans, but can also interact with glycoproteins modified with complex‐type N‐glycans .…”

Increasing the Affinity of an O‐Antigen Polysaccharide Binding Site in Shigella flexneri Bacteriophage Sf6 Tailspike Protein

“…This strategy has been further extended by taking into account evolutionary profiles or “re‐epitoping” of antibodies and validation by crystal structure analysis . For engineering of carbohydrate binding proteins, phage or plasmid display techniques have been employed . Computational approaches also used ROSETTA, but flexible ligands and water network distributions impede predictions via docking or Monte Carlo simulations with rotamer libraries.…”