Mapping the GALNT1 substrate landscape with versatile proteomics tools

Saei, A. A.; Lundstrom, S. L.; Lyu, H.; Gharibi, H.; Lu, W.; Fang, P.; Zhang, X.; Meng, Z.; Wang, J.; Gaetani, M.; Vegvari, A.; Gygi, S. P.; Zubarev, R. A.

doi:10.1101/2022.08.24.505189

bioRxiv · 10.1101/2022.08.24.505189

Mapping the GALNT1 substrate landscape with versatile proteomics tools

Saei, A. A.·Lundstrom, S. L.·Lyu, H.·Gharibi, H.·Lu, W.·Fang, P.·Zhang, X.·Meng, Z.·Wang, J.·Gaetani, M.·Vegvari, A.·Gygi, S. P.·Zubarev, R. A.

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Abstract

O-GalNAc type glycosylation is a common post-translational modification (PTM) of proteins catalyzed by polypeptide GalNAc transferases, but the substrate specificity of these transferases is poorly understood. Here we develop a strategy based on integral thermal proteome solubility profiling to identify and prioritize the protein substrates of polypeptide N-acetylgalactosaminyltransferase 1 (GALNT1). Combined with glycoprotein enrichment followed by HCD and soft EThcD gas-phase fragmentation technique, we uncover hundreds of novel GALNT1 substrates in two model human cell lines. GALNT1-mediated O-glycosylation is more common on Thr than Ser residues, with a strong preference for Pro at positions +3 and +4 in respect to O-glycosylation. These results implicate GALNT1 in potentially regulating proteins in several diverse pathways, including some unexpected processes, such as TCA cycle and DNA transcription. This study depicts a roadmap for identification of functional substrates for glycosyltransferases, facilitating fundamental insight into the role of glycosylation in homeostasis and disease.

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Saei, A. A., Lundstrom, S. L., Lyu, H., Gharibi, H., Lu, W., Fang, P., Zhang, X., Meng, Z., Wang, J., Gaetani, M., Vegvari, A., Gygi, S. P., Zubarev, R. A.. 2022-08-24. Mapping the GALNT1 substrate landscape with versatile proteomics tools. https://doi.org/10.1101/2022.08.24.505189

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