posted on 2021-07-26, 15:07authored byJacob
F. Wardman, Peter Rahfeld, Feng Liu, Connor Morgan-Lang, Lyann Sim, Steven J. Hallam, Stephen G. Withers
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.