Abstract
A novel fucose-binding lectin (SL2-1) from the bacterium Streptomyces rapamycinicus was identified by analysis of metagenomic DNA sequences. SL2-1 belongs to a new group of bacterial fucose-specific lectins that have no similarity to known bacterial fucose-binding proteins, but are related to certain eukaryotic fucose-binding lectins. The 17 kDa protein was expressed recombinantly in E. coli and purified by affinity chromatography. Glycan microarray analysis with fluorescently labeled recombinant SL2-1 demonstrated its ability to bind to core α1-6 fucosylated N-glycans, but not to core α1-3 fucosylated N-glycans, or other α1-2, α1-3 and α1-4 fucosylated oligosaccharides. The minimal high affinity binding epitope of SL2-1 was α1-6 fucosylated di-n-acetylchitobiose. The recombinant lectin was efficient in detection of N-glycan core fucosylation using lectin blotting and lectin ELISA assays. Finally, a workflow using SL2-1 for selective and quantitative profiling of core fucosylated N-glycans using UPLC-HILIC-FLR analysis was established. The approach was validated for selective capture and analysis of core fucosylated N-glycans present in complex glycan mixtures derived from mammalian serum IgG.
Highlights
Fucosylation of N-glycans occurs throughout eukaryotes and plays an important role in a variety of cellular processes, including signaling, cell adhesion, fertilization and cell growth[1,2,3]
We show that this protein binds to α1-6 fucosylated chitobiose and defines a new group of fucose-specific lectins found in actinomycete bacteria
To investigate the glycan binding specificity of a member of this protein family, the hypothetical protein from Streptomyces rapamycinicus NRRL 5491 was selected for further study and was designated SL2-1
Summary
Fucosylation of N-glycans occurs throughout eukaryotes and plays an important role in a variety of cellular processes, including signaling, cell adhesion, fertilization and cell growth[1,2,3]. A molecular tool that discriminates between core and outer arm fucosylation, and that permits highly specific detection and quantification of core fucose is needed. Such a reagent would permit a better understanding of the biological role of core fucosylated N-glycans and would be useful in the identification of potential glycan based biomarkers of disease. We report identification and characterization of a protein from Streptomyces rapamycinicus with a previously unknown function We show that this protein binds to α1-6 fucosylated chitobiose and defines a new group of fucose-specific lectins found in actinomycete bacteria. Efficient use of SL2-1 for epitope-specific profiling of core fucosylated N-glycans present in complex mixtures of mammalian glycans was demonstrated
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