Mucin-type O-glycosylation is controlled by short- and long-range glycopeptide substrate recognition that varies among members of the polypeptide GalNAc transferase family

Abstract

A large family of UDP-GalNAc:polypeptide GalNAc transferases (ppGalNAc-Ts) initiates and defines sites of mucin-type Ser/Thr-O-GalNAc glycosylation. Family members have been classified into peptide- and glycopeptide-preferring subfamilies, although both families possess variable activities against glycopeptide substrates. All but one isoform contains a C-terminal carbohydrate-binding lectin domain whose roles in modulating glycopeptide specificity is just being understood. We have previously shown for several peptide-preferring isoforms that the presence of a remote Thr-O-GalNAc, 6-17 residues from a Ser/Thr acceptor site, may enhance overall catalytic activity in an N- or C-terminal direction. This enhancement varies with isoform and is attributed to Thr-O-GalNAc interactions at the lectin domain. We now report on the glycopeptide substrate utilization of a series of glycopeptide (human-ppGalNAc-T4, T7, T10, T12 and fly PGANT7) and peptide-preferring transferases (T2, T3 and T5) by exploiting a series of random glycopeptide substrates designed to probe the functions of their catalytic and lectin domains. Glycosylation was observed at the -3, -1 and +1 residues relative to a neighboring Thr-O-GalNAc, depending on isoform, which we attribute to specific Thr-O-GalNAc binding at the catalytic domain. Additionally, these glycopeptide-preferring isoforms show remote lectin domain-assisted Thr-O-GalNAc enhancements that vary from modest to none. We conclude that the glycopeptide specificity of the glycopeptide-preferring isoforms predominantly resides in their catalytic domain but may be further modulated by remote lectin domain interactions. These studies further demonstrate that both domains of the ppGalNAc-Ts have specialized and unique functions that work in concert to control and order mucin-type O-glycosylation.

Expression system for structural and functional studies of human glycosylation enzymes

“…In addition, kinetic analysis was performed on several of these purified recombinant products before and after tag removal with TEV protease. The results, exemplified by ST6GALNAC2 (Supplementary Table 4), indicated that the enzymes had comparable activity as GFP fusion proteins or released catalytic domains17,28. As the construct library was being completed, we further characterized several of these expression products and the results effectively identified GT substrate specificities28–30, corrected the glycan linkage elaborated by a GT31, resulted in chemoenzymatic synthesis of novel glycan structures32, and supported selective exoenzymatic labeling (SEEL)33 of cell surface glycans, demonstrating the utility of our expression vector library as a tool for various glycoenzyme studies and applications in glycobiology.…”

O-Glycome Beam Search Arrays for Carbohydrate Ligand Discovery

“…The O-glycans are known to express species-and tissue-specific antigens that change during cell differentiation and oncogenesis (2,6), and mediate some of the above-mentioned mucin functions (7). However, the involvements of specific O-glycan structures in many processes are yet to be determined (2), assignments being hampered by the diversity of extensions at the core-monosaccharide, GalNAc, and multiplicity of chain lengths and branching patterns (5,8,9), and often the limited amounts of glycans available. Thus high-throughput and microscale methodologies are needed for deconvolution and functional assignments of individual O-glycans within O-glycomes as cues to large scale syntheses of desired glycan sequences for exploitation (10,11).…”

Inhibitor of ppGalNAc-T3-mediated O-glycosylation blocks cancer cell invasiveness and lowers FGF23 levels

“…Inhibition of ppGalNAc-T3 occurred with an IC 50 of 7 µM and was undetectable against ppGalNAc-T2. T3Inh-1 also lacked activity against ppGalNAc-T6 (Figure 2B), which is the isozyme considered most closely related to ppGalNAc-T3 (Bennett et al, 2012 ; Revoredo et al, 2016).

10.7554/eLife.24051.011Figure 2.T3Inh-1 is a direct mixed-mode inhibitor of ppGalNAc-T3.( A ) Comparison of T2 and T3 sensor activation at the indicated concentrations of T3Inh-1 (n = 3 ± SEM).

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