Breakthrough in the sugar biology of multicellular organisms

In multicellular organisms, there are three types of protein glycosylation. N-glycosylation, O-mannosylation and C-mannosylation. All of these processes take place in the endoplasmic reticulum, and in all of them enzymes attach sugar residues to specific sites in newly forming protein.

While N- and O-glycosylation are well studied, the third form, C-mannosylation of tryptophan side chains, has long been a mystery to researchers. Although 20 percent of all secretory proteins, as well as membrane proteins, are affected by it, it was unclear until recently what the change was for, how the specific protein sequences are recognised and how the associated enzyme reaction is chemically possible at all.

In an international collaboration, researchers from ETH Zurich, the Walter and Eliza Hall Institute of Medical Research (WEHI) in Australia, the University of Chicago and the University of Bern have now elucidated the structure and function of the responsible enzyme, 'tryptophan C-mannosyltransferase' (CMT). The corresponding study was published in the latest issue of the journal Nature Chemical Biology.

CMT is a member of the category C (GT-C) glycosyltransferase enzymes, one of the three glycosyltransferase superfamilies. The most prominent member is oligosaccharyltransferase (OST), which is responsible for N-glycosylation.

Similar to the OST, the CMT also recognises highly specific sequences in proteins, with the difference, however, that in mammals four different CMTs occur simultaneously, which also recognise different protein sequences.

Sugars help immuno-receptors to the cell surface

Only in recent years, the necessary tools, such as special antibodies and mass spectrometry test methods, were developed in order to be able to investigate the extent of C-mannosylation. It was shown that this process occurs almost exclusively where cell-cell communication is essential, especially in cytokine receptors of the immune system and adhesion GPCRs. The latter serve as "sensory antennae" for growing neurons that make their way through the brain. "The topic is red-hot, especially for our understanding of the cell-cell communication of the immune system," explains Kaspar Locher, Professor of Structural Biology at ETH Zurich: "Signalling molecules such as cytokines direct the immune response during an infection. While these and their associated receptors have been intensively studied for decades, it has long been neglected that C-mannosylation determines whether a cytokine receptor reaches the cell surface to exert its function."

"With our insights into the structure of the enzymes involved, we now have a near-complete understanding of how C-mannosylation gets to these receptors," adds study first author Joël Bloch, former senior scientist in Locher's group.

Tailor-made molecular construction kit

The ETH researchers succeeded in producing the CMT enzyme in its pure form. With the help of chemists from WEHI (AUS) and the University of Bern, they built customised molecules that mimic CMT-specific protein sequences and sugar substrates. This allowed them to test the enzyme for its specific properties in the test tube for the first time.

The researchers quickly realised that the enzyme chemistry of CMT must be novel and completely different from that of OST. "In such a case, we can only find out the mechanism of an enzyme using high-resolution structural elucidation. The problem, however, was that CMT could not be crystallised until now and had too little mass for cryo-EM, because this technique is particularly difficult to apply to proteins below 100 kDa," Locher explains.