Tryptophan C-mannosylation is an unusual type of protein glycosylation whereby an α-D-mannose is covalently attached to a tryptophan side chain with the formation of a carbon-carbon bond. This protein modification is predominantly found on type I cytokine receptors and thrombospondin repeat domains, though it is found on many other proteins, including RNase 2, hyaluronidases and siglecs. The first gene to encode a Tryptophan C-mannosyltransferase was discovered in Caenorhabditis elegans, which has catalysed the discovery and characterisation of homologues from humans and apicomplexan parasites.
The study of tryptophan C-mannosylation has been hampered by a dearth of tools for producing proteins with the modification, detecting the modification, and for inhibiting protein modification. To address this issue we have engineered a simple microbial expression system to produce a wide variety of human proteins with and without tryptophan mannosylation. These proteins enabled us to assess the impact of C-mannosylation on protein fold and function, which provided insights into the significance of tryptophan mannosylation. Using these proteins we have generated and validated monoclonal antibodies (mAbs) specific for tryptophan mannosylation and obtained structural insights into how these mAbs recognise C-mannosyl tryptophan. With these antibodies and our microbial expression system, we have also been able to generate the first inhibitors of tryptophan C-mannosyltransferases by applying a rational inhibitor design approach to the creation of substrate-mimics. These inhibitors work in cell culture and represent an excellent starting points for the development of chemical probes with more drug-like properties.