Abstract
Mammalian mucin-type O-glycosylation is initiated by a large family of ∼20 UDP-GalNAc:polypeptide α-N-acetylgalactosaminyltransferases (ppGalNAc Ts) that transfer α-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.
Highlights
Mucin-type O-glycosylation is one of the most common post-translational modifications of secreted and membrane-associated proteins
It has been shown that the presence of the lectin domain of ppGalNAc T2 significantly shifts the preferred sites of glycosylation on glycopeptide substrates [23, 24, 28, 29], other studies have demonstrated that the catalytic domain of ppGalNAc T10 is responsible for its near absolute glycopeptide specificity [29, 30]
Studies utilizing P-VIII with ppGalNAc T1, T2, and T10 proved successful yielding random glycopeptides showing GalNAc glycosylation at the central Thr residue and no detectable Ser-O-GalNAc glycosylation [30, 62] in the random X regions based on the Edman sequencing of the lectin-isolated glycopeptide product
Summary
10.0 ϫ 109 10.0 ϫ 109 10.0 ϫ 109 nition of sites of mucin-type O-glycosylation based on peptide sequence. There is a need for characterizing the peptide substrate specificity of each isoform to further elucidate their specific targets and mechanism of action This information is critical for our understanding of the biological roles and significance of the ppGalNAc T family of transferases and mucin-type O-glycosylation in general. We extend our studies to three additional members of the family, ppGalNAc T3, T5, and T12, with potential roles in human disease, utilizing two previously reported random peptide substrates and an additional new substrate capable of obtaining preferences for neighboring nonglycosylated Ser residues (Table 1) With these substrates, unique substrate preference data for all amino acid residues except Thr, Trp, and Cys have been obtained for the following six mammalian ppGalNAc Ts: T1, T2, T3, T5, T10, and T12. We further demonstrate that for several of the transferases, these preferences can be used to predict isoform-specific glycosylation patterns consistent with previously reported experimental data
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