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

Cynthia L Perrine,Anjali Ganguli,Peng Wu,Carolyn R Bertozzi,Timothy A Fritz,Jayalakshmi Raman,Lawrence A Tabak,Thomas A Gerken

doi:10.1074/jbc.m109.017236

Abstract

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.

Highlights

Several ppGalNAc T isoforms have been shown to be important for normal development or cellular processes
We have recently reported the use of oriented random peptide substrates, GAGA(X)nT(X)nAGAGK for determining the peptide substrate specificities of mammalian ppGalNAc T1, T2, and their fly orthologues [21, 38]
We extend this approach to the determination of the catalytic domain glycopeptide (Ser/Thr-O-GalNAc) substrate preferences for ppGalNAc T1, T2, and T10 employing two n ϭ 4 oriented random glycopeptide libraries (Table 1)

Summary

EXPERIMENTAL PROCEDURES

Enzymes and Reagents—Soluble recombinant bovine ppGalNAc T1 was a gift of Ake Elhammer (Kalamazoo, MI). Reactions consisted of 2– 4 mM UDP-GalNAc (3H- or 14C-labeled, 0.01 ␮Ci/ ␮l), ϳ0.24 mg/ml ppGalNAc T10, and 22– 45 mM random peptide substrates P-VI or P-VII, with final volumes of 53–200 ␮l. The biotin eluant was lyophilized and passed over Sephadex G-10 in 50 mM acetic acid buffer, pH 4.5; fractions were monitored for absorbance at 220 and 280 nm (see Fig. 2B). Glycosylation of Random Glycopeptide GP-II by ppGalNAc T1, T2, and T10—The glycosylation of random glycopeptide GP-II was performed for ppGalNAc T10 initially as described previously for P-VI and P-VII using 2 mM 3H-labeled UDP-GalNAc (0.01 ␮Ci/␮l) and ϳ10 mM random glycopeptide substrate GP-II. After separation on a Sephadex G-10 column, fractions were pooled based on radioactivity and lyophilized, and portions were Edman sequenced. The MALDI-TOF experiments (see Fig. S4) were performed on SCIEX prTOFTM 2000 mass spectrometer (PerkinElmer Life Sciences) with 2,5-dihydroxybenzoic acid utilized as the matrix

RESULTS

No of unique sequences

DISCUSSION