Oral Presentation HUPO 2019 - 18th Human Proteome Organization World Congress

Application of glycoproteomics to identify the plant Golgi localized UDP-GlcNAc transporter and mining the data for novel plant O-linked glycans (#104)

Joshua L Heazlewood 1
  1. The University of Melbourne, Melbourne, VIC, Australia

Glycosylation require activated glycosyl donors in the form of nucleotide sugars to drive processes such as post-translational modifications and polysaccharide biosynthesis. Many of these reactions occur in the endomembrane using cytosolic-derived nucleotide sugars. These are actively transported into the lumen by nucleotide sugar transporters (NSTs). We recently identified a plant UDP-GlcNAc transporter responsible for the delivery of substrate for the maturation of N-glycans and sphingolipids within the endomembrane. To determine the biochemical phenotype of the UDP-GlcNAc transporter loss-of-function mutants, we have applied glycoproteomic profiling. This necessitated the development of a HILIC enrichment and mass spectrometry-based workflow to detect, identify and quantify N-glycopeptides. Results indicated that that N-glycopeptides containing complex N-glycans (e.g. GlcNAc) were significantly reduced in plants lacking the UDP-GlcNAc transporter. In contrast, complex N-glycan structures from wild-type plants comprised a considerable proportion (35 %) of observed N-glycans i.e. those containing GlcNAc. Our findings indicate that the reference plant Arabidopsis contains a single UDP-GlcNAc transporter responsible for the maturation of complex N-glycans in Golgi lumen. The resultant N-glycoproteomic data was further mined to determine whether we could find any evidence for O-linked glycans in pant proteins. Since plants encode many proteins with epidermal growth factor (EGF) domains, we hypothesized that, similar to mammals, small O-linked glycans may also exist in plants. We have identified a number of high-confidence matches that would indicate that plant proteins may indeed harbour small O-linked glycan post translation modifications.