Emerging paradigms for the initiation of mucin-type protein O-glycosylation by the polypeptide GalNAc transferase family of glycosyltransferases.

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.

UDP-N-acetyl-α-D-galactosamine:polypeptide N-acetylgalactosaminyltransferases: completion of the family tree.

The formation of mucin-type O-glycans is initiated by an evolutionarily conserved family of enzymes, the UDP-N-acetyl-α-D-galactosamine:polypeptide N-acetylgalactosaminyltransferases (GalNAc-Ts). The human genome encodes 20 transferases; 17 of which have been characterized functionally. The complexity of the GalNAc-T family reflects the differential patterns of expression among the individual enzyme isoforms and the unique substrate specificities which are required to form the dense arrays of glycans that are essential for mucin function. We report the expression patterns and enzymatic activity of the remaining three members of the family and the further characterization of a recently reported isoform, GalNAc-T17. One isoform, GalNAcT-16 that is most homologous to GalNAc-T14, is widely expressed (abundantly in the heart) and has robust polypeptide transferase activity. The second isoform GalNAc-T18, most similar to GalNAc-T8, -T9 and -T19, completes a discrete subfamily of GalNAc-Ts. It is widely expressed and has low, albeit detectable, activity. The final isoform, GalNAc-T20, is most homologous to GalNAc-T11 but lacks a lectin domain and has no detectable transferase activity with the panel of substrates tested. We have also identified and characterized enzymatically active splice variants of GalNAc-T13 that differ in the sequence of their lectin domain. The variants differ in their affinities for glycopeptide substrates. Our findings provide a comprehensive view of the complexities of mucin-type O-glycan formation and provide insight into the underlying mechanisms employed to heavily decorate mucins and mucin-like domains with carbohydrate.

Glycopeptide-preferring polypeptide GalNAc transferase 10 (ppGalNAc T10), involved in mucin-type O-glycosylation, has a unique GalNAc-O-Ser/Thr-binding site in its catalytic domain not found in ppGalNAc T1 or T2.

Mucin-type O-gly co sy la tion is initiated by a large family of UDP-GalNAc:polypeptide alpha-N-acetylgalactosaminyltransferases (ppGalNAc Ts) that transfer GalNAc from UDP-GalNAc to the Ser and Thr residues of polypeptide acceptors. Some members of the family prefer previously gly co sylated peptides (ppGalNAc T7 and T10), whereas others are inhibited by neighboring gly co sy la tion (ppGalNAc T1 and T2). Characterizing their peptide and glycopeptide substrate specificity is critical for understanding the biological role and significance of each isoform. Utilizing a series of random peptide and glycopeptide substrates, we have obtained the peptide and glycopeptide specificities of ppGalNAc T10 for comparison with ppGalNAc T1 and T2. For the glycopeptide substrates, ppGalNAc T10 exhibited a single large preference for Ser/Thr-O-GalNAc at the +1 (C-terminal) position relative to the Ser or Thr acceptor site. ppGalNAc T1 and T2 revealed no significant enhancements suggesting Ser/Thr-O-GalNAc was inhibitory at most positions for these isoforms. Against random peptide substrates, ppGalNAc T10 revealed no significant hydrophobic or hydrophilic residue enhancements, in contrast to what has been reported previously for ppGalNAc T1 and T2. Our results reveal that these transferases have unique peptide and glycopeptide preferences demonstrating their substrate diversity and their likely roles ranging from initiating transferases to filling-in transferases.