Discovery and Development of Promiscuous O-Glycan Hydrolases for Removal of Intact Sialyl T-Antigen
| Autor: | Lyann Sim, Peter Rahfeld, Connor Morgan-Lang, Stephen G. Withers, Jacob F. Wardman, Feng Liu, Steven J. Hallam |
|---|---|
| Rok vydání: | 2021 |
| Předmět: |
Glycan
Erythrocytes Glycosylation CAZy Glycoside Hydrolases Swine Sequence analysis CHO Cells Biochemistry Substrate Specificity 03 medical and health sciences Cricetulus Bacterial Proteins Hydrolase Animals Humans 030304 developmental biology 0303 health sciences biology Chemistry 030302 biochemistry & molecular biology Mucins General Medicine Protein engineering carbohydrates (lipids) Streptococcus pneumoniae Mutation Mutagenesis Site-Directed biology.protein Molecular Medicine Phylogenetic profiling Sequence space (evolution) Function (biology) |
| Zdroj: | ACS Chemical Biology. 16:2004-2015 |
| ISSN: | 1554-8937 1554-8929 |
| DOI: | 10.1021/acschembio.1c00316 |
| Popis: | Mucin-type O-glycosylation (O-glycosylation) is a common post-translational modification that confers distinct biophysical properties to proteins and plays crucial roles in intercellular signaling. Yet, despite the importance of O-glycans, relatively few tools exist for their analysis and modification. In particular, there is a need for enzymes that can cleave the wide range of O-glycan structures found on protein surfaces, to facilitate glycan profiling and editing. Through functional metagenomic screening of the human gut microbiome, we discovered endo-O-glycan hydrolases from CAZy family GH101 that are capable of slowly cleaving the intact sialyl T-antigen trisaccharide (a ubiquitous O-glycan structure in humans) in addition to their primary activity against the T-antigen disaccharide. We then further explored this sequence space through phylogenetic profiling and analysis of representative enzymes, revealing large differences in the levels of this promiscuous activity between enzymes within the family. Through structural and sequence analysis, we identified active site residues that modulate specificity. Through subsequent rational protein engineering, we improved the activity of an enzyme identified by phylogenetic profiling sufficiently that substantial removal of the intact sialyl T-antigen from proteins could be readily achieved. Our best sialyl T-antigen hydrolase mutant, SpGH101 Q868G, is further shown to function on a number of proteins, tissues, and cells. Access to this enzyme opens up improved methodologies for unraveling the glycan code. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|