Yeast- and antibody-based tools for studying tryptophan C-mannosylation

Area of Science:

• Biochemistry
• Molecular Biology
• Proteomics

Background:

• Tryptophan C-mannosylation is an uncommon co-translational protein modification found in metazoans and apicomplexan protists.
• The biological roles and prevalence of this modification remain largely unknown due to a lack of effective tools for its study.

Purpose of the Study:

• To engineer a yeast system for producing proteins with and without tryptophan C-mannosylation.
• To investigate the impact of tryptophan C-mannosylation on protein stability and function.
• To develop tools for detecting and analyzing tryptophan C-mannosylation.

Main Methods:

• Engineered a yeast expression system to produce diverse proteins with and without tryptophan C-mannosylation.
• Conducted mutagenesis studies to identify key residues in the glycosyltransferase and substrates involved in catalysis.
• Generated and characterized monoclonal antibodies against tryptophan C-mannosylation.
• Utilized these antibodies for proteomic analysis of the brain C-glycome.

Main Results:

• Successfully produced a library of C-mannosylated and non-C-mannosylated proteins in yeast.
• Identified critical residues for the catalytic activity of the glycosyltransferase and its substrates.
• Developed specific monoclonal antibodies for detecting tryptophan C-mannosylation.
• Discovered numerous novel tryptophan C-mannosylation sites in brain proteins, including those with non-canonical consensus sequences.

Conclusions:

• The engineered yeast system is a valuable tool for studying tryptophan C-mannosylation.
• The developed antibodies facilitate proteomic analysis, expanding our understanding of the brain C-glycome.
• This study significantly advances the field by providing tools and revealing new insights into tryptophan C-mannosylation.