Tryptophan C-mannosylation is an unusual metazoan co-translational modification found on many cell-surface receptors and extracellular proteins. It is the only type of protein glycosylation that involves the formation of a carbon-carbon bond between sugar and polypeptide. This unique chemistry is performed by the tryptophan C-mannosyltransferases, which are integral ER membrane proteins that utilise dolichol-phosphate mannose (Dol-P-Man) to glycosylate the C-2 position of tryptophan side chains within the WXXW consensus sequence.^[1,2]  Little else is known about tryptophan C-mannosylation or the enzymes that install this modification, largely due to limitations in the existing approaches to installing, detecting and blocking the modification.

We have developed alternative approaches to these problems to provide fresh insights into the biology of tryptophan C-mannosylation. Engineering C-mannosylation pathways into Pichia pastoris has provided a convenient microbial expression platform for the production of near-homogenous glycoforms on a multi-milligram scale. Efficient chemical syntheses of C-mannosyl tryptophan building blocks has enabled the use of solid-phase peptide synthesis for the production of pure glycoforms. Together, these methods for the production of C-mannosylated glycoforms have facilitated an examination of how C-mannosylation impacts protein stability, function and enzymatic activity. They have also led to the first monoclonal antibodies capable of detecting the modification.

In addition, we have established an in vitro assay of tryptophan C-mannosyltransferase activity, which have been used to explore the enzyme’s substrate preference and perform mutagenesis studies. These experiments revealed that the tryptophan C-mannosyltransferases are more promiscuous than previously appreciated. This assay, our newfound knowledge of enzyme substrate preference, and new tools for detecting tryptophan mannosylation, have enabled us to develop the first C-mannosyltransferase inhibitor, which also happened to be active in cells. Collectively, this suite of tools provides a strong foundation for further exploration of tryptophan C-mannosylation biology.