Australian researchers at the Walter and Eliza Hall Institute of Medical Research (WEHI) in Melbourne have developed new yeast- and antibody-based methods to determine how tryptophan C-mannosylation, an unusual protein glycosylation modification, impacts the stability and function of disease-relevant proteins.
Reported in the February 5, 2021, edition of Nature Chemical Biology, the study of how certain sugars modify proteins can not only be used to map the prevalence of this protein modification in healthy tissues, but may also lead to the development of drugs targeting aberrant glyosylation for diseases, including muscular dystrophy and cancer.
"There is an unmet need for treating diseases such as muscular dystrophy, although I should stress that all we've shown is that a protein involved in some muscular dystrophies is modified with tryptophan C-mannosylation and it remains unknown if this is required for protein function, or even if it contributes to the onset of some muscular dystrophies," said study leader Ethan Goddard-Borger.
"Although several other studies have shown the importance of tryptophan C-mannosyaltion for protein stability, ours provides a more comprehensive examination of this phenomenon and is the first report of the modification impacting protein function," said the associate professor and laboratory head of WEHI's Chemical Biology Division.
Approximately 90% of proteins on the surface of human cells and half of the cells' total proteins are glycosylated, with modifications ranging from the addition of a single sugar, to long complex polymer chains. However, their distribution, variability and biological functions remain poorly understood.
The objectives of the new WEHI study were therefore to derive an improved understanding of the roles of aberrant glycosylation in both health and diseases including cancers.
"A range of diseases feature aberrant cellular glycosylation, including congenital disorders of glycosylation and lysosomal storage disorders, among others, including cancers," said Goddard-Borger.
"Many, but not all, cancer cells possess aberrant glycosylation. However, as every cancer is different and evolves over time, it's difficult to make broad statements about which tumor subsets do and don't have aberrant glycosylation," he told BioWorld.
Nevertheless, "this knowledge may yield new therapeutic strategies, although first a better understanding of what constitutes 'normal' glycosylation is required, before we can further develop drugs targeting protein glycosylation."
Glycosylation usually occurs on a protein's nitrogen or oxygen atoms, but can also occur on carbon atoms via tryptophan C-mannosylation, which is particularly poorly understood, prompting the WEHI team to develop tools and methods to investigate the phenomenon.
New tools & methods
"The methods we have developed combine state-of-the-art mass spectrometry techniques with recombinant antibody tools generated at WEHI," said Goddard-Borger.
"These methods will enable researchers to easily install this unusual modification on nearly any protein, allowing investigation of its effect on protein stability and function," he added.
Specifically, "we have shown that a common feature of tryptophan C-mannosylation is that it stabilizes proteins. Diverse, unrelated proteins all appear to be more stable once modified and some proteins' functions can be modulated by tryptophan C-mannosylation."
Goddard-Borger said the new the tools will also enable the abundance of this poorly understood protein modification to be determined in healthy and diseased tissues, while boosting global efforts to map and understand protein glycosylation in health and disease.
"We've reported some really unexpected results regarding the prevalence of this modification in healthy brain tissue," he noted.
"We found the modification on many proteins that were not previously known to be modified, and at sites on the protein that were not expected to be modified, which may help identify therapeutic targets."
Regarding drug development, "blocking tryptophan C-mannosyaltion destabilizes some proteins and alters their functions. If those proteins are involved in the onset of a disease, inhibiting tryptophan C-mannosyaltion may prevent that particular disease, which is the focus of our ongoing investigations," Goddard-Borger told BioWorld Science.
The WEHI researchers are now extending this work to map the modification across most tissues in the body, in order to better understand the biology of this form of protein glycosylation, as well as its role in cancer and muscular dystrophies.
"Determining what proteins are modified, and where they are modified, is the important first step towards a better understanding of the biological roles of this protein modification," said Goddard Borger.
Therefore, in the near future "we hope to provide a more complete picture of the mammalian C-glycome; a study that will be enabled by the tools we have reported here."