Abstract
Protein glycosylation is one of the most abundant post‐translational modifications. However, detailed analysis of O‐linked glycosylation, a major type of protein glycosylation, has been severely impeded by the scarcity of suitable methodologies. Here, a chemoenzymatic method is introduced for the site‐specific extraction of O‐linked glycopeptides (EXoO), which enabled the mapping of over 3,000 O‐linked glycosylation sites and definition of their glycans on over 1,000 proteins in human kidney tissues, T cells, and serum. This large‐scale localization of O‐linked glycosylation sites demonstrated that EXoO is an effective method for defining the site‐specific O‐linked glycoproteome in different types of sample. Detailed structural analysis of the sites identified revealed conserved motifs and topological orientations facing extracellular space, the cell surface, the lumen of the Golgi, and the endoplasmic reticulum (ER). EXoO was also able to reveal significant differences in the O‐linked glycoproteome of tumor and normal kidney tissues pointing to its broader use in clinical diagnostics and therapeutics.
Highlights
Protein glycosylation is arguably the most diverse and sophisticated form of protein modification which drastically escalates protein heterogeneity to facilitate functional plasticity (Varki, 2011; Tran & Ten Hagen, 2013)
The O-linked glycopeptides are enzymatically released from the support using an endo-protease OpeRATOR that requires the presence of O-linked glycans to cleave on the N-terminal side of O-linked glycan-occupied Ser or Thr (Fig 1A)
To demonstrate proof of principle, bovine fetuin was analyzed and the six known O-linked glycosylation sites documented in the UniProt database were pinpointed at Ser-271, Thr-280, Ser-282, Ser-296, Thr-334, and Ser-341 (Dataset EV1)
Summary
Protein glycosylation is arguably the most diverse and sophisticated form of protein modification which drastically escalates protein heterogeneity to facilitate functional plasticity (Varki, 2011; Tran & Ten Hagen, 2013). Compared to N-linked glycosylation, the study of O-linked glycosylation has proved difficult due to the structural complexity of the glycans added and the technical challenges posed to definitively characterizing them (Jensen et al, 2010; Qin et al, 2017; Darula & Medzihradszky, 2018). Among the different types of O-linked glycosylation seen, O-linked N-acetyl-galactosamine (O-GalNAc) addition is a major type (Jensen et al, 2010; Chia et al, 2016; Darula & Medzihradszky, 2018). In contrast to N-linked glycosylation, where a consensus glycosylation motif has been identified, there is no consensus O-linked glycosylation motif for the amino acid residues surrounding the glycosylated Ser or Thr (Nishikawa et al, 2010; Darula & Medzihradszky, 2018). The presence of up to 20 GalNActransferases (GalNAc-Ts) for adding the initial sugar to amino acid residues in different sequence regions further complicates the dynamic regulation of O-linked glycosylation (Bennett et al, 2012). O-linked glycosylation can exhibit high heterogeneity in different cells, tissues, and diseases (Steentoft et al, 2013; Medzihradszky et al, 2015)
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