Emerging Paradigms for the Initiation of Mucin-type Protein O-Glycosylation by the Polypeptide GalNAc Transferase Family of Glycosyltransferases

Abstract

Mammalian mucin-type O-glycosylation is initiated by a large family of ϳ20 UDP-GalNAc:polypeptide ␣-N-acetylgalactosaminyltransferases (ppGalNAc Ts) that transfer ␣-GalNAc from UDP-GalNAc to Ser and Thr residues of polypeptide acceptors. Characterizing the peptide substrate specificity of each isoform is critical to understanding their properties, biological roles, and significance. Presently, only the specificities of ppGalNAc T1, T2, and T10 and the fly orthologues of T1 and T2 have been systematically characterized utilizing random peptide substrates. We now extend these studies to ppGalNAc T3, T5, and T12, transferases variously associated with human disease. Our results reveal several common features; the most striking is the similar pattern of enhancements for the three residues C-terminal to the site of glycosylation for those transferases that contain a common conserved Trp. In contrast, residues N-terminal to the site of glycosylation show a wide range of isoform-specific enhancements, with elevated preferences for Pro, Val, and Tyr being the most common at the ؊1 position. Further analysis reveals that the ratio of positive (Arg, Lys, and His) to negative (Asp and Glu) charged residue enhancements varied among transferases, thus further modulating substrate preference in an isoform-specific manner. By utilizing the obtained transferase-specific preferences, the glycosylation patterns of the ppGalNAc Ts against a series of peptide substrates could roughly be reproduced, demonstrating the potential for predicting isoform-specific glycosylation. We conclude that each ppGalNAc T isoform may be uniquely sensitive to peptide sequence and overall charge, which together dictates the substrate sites that will be glycosylated.Mucin-type O-glycosylation is one of the most common post-translational modifications of secreted and membrane-associated proteins. Glycoproteins containing O-glycosylated mucin domains serve many important biological roles chiefly because of their unique biophysical and structural properties that include an extended peptide conformation and robust resistance to proteases. Consequently, glycoproteins containing O-glycosylated mucin domains function in the protection of the cell surface, the modulation of cell-cell interactions, in the inflammatory and immune response, in metastasis and tumorigenesis, and in protein sorting, targeting, and turnover (for examples see Refs.

Development of Isoform-specific Sensors of Polypeptide GalNAc-transferase Activity

“…4M). The presumed increase in glycosylation is consistent with the in vitro preference for a hydrophobic residue at this position (27). We were also fortunate that the FGF23-based T3 sensors showed strong isoform specificity and recapitulated the lectin domain-mediated mechanism by which T3 glycosylates Thr 178 in FGF23 3 thereby raising the possibility of screening for drugs targeting either the catalytic unit or the lectin domain of T3.…”

“…This was due to background activation stemming from any of a number of possibilities including inefficient O-glycosylation, highly efficient furin cleavage, or regulation by phosphorylation. Generally, peptide preferences of the GalNAc-transferases are sensitive to residues from Ϫ3 to ϩ3 relative to the glycosylation site with a preference for proline at positions ϩ1 and ϩ3 (27). Furthermore, as mentioned above, the residue at ϩ2 (Ser 180 ) impedes glycosylation when phosphorylated (31).…”

“…To improve the signal to noise ratio we decided to modify the FGF23 insert sequence with the goal of making it a better substrate of T3 glycosylation. Fortunately, extensive in vitro assays were previously used to assess glycosylation of various peptide sequences (27) and recent work identified a phosphorylation site (Ser 180 ) that negatively regulates glycosylation (31). Based on these results, we tested the single mutation H177V and the triple mutation H177V/S180G/A181P.…”

“…Therefore, we wanted to test a linker sequence that was entirely designed de novo. The design was based on previous in vitro analysis of the substrate specificity of T3 (27). The sequence, 1 RRAYRVTPGP 10 , contained a single potential glycan acceptor, Thr 7 , placed two residues C-terminal of an Arg 5 furin cleavage site in an attempt to maximize masking and thus decrease background sensor fluorescence.…”

“…Human embryonic kidney (HEK) cells were used in our experiments and they express all isoforms except T5, T9, T15, T17, T19, and T20 (Proteinatlas). In anticipation of the likelihood that T3 would also glycosylate Thr 226 we simultaneously tested a glycine substituted at position Ϫ1 (T225G) relative to the glycosylation site because glycine substitution at this position interferes with T3 but not T2-mediated glycosylation in vitro (27).…”