Mucin-type
O-gly co sy la tion is initiated by a large family of UDP-GalNAc:polypeptide α-
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.Mucin-type
O-glycosylation is a common post-translational modification of secreted and membrane-associated proteins.
O-Glycan biosynthesis is initiated by the transfer of GalNAc from UDP-GalNAc to the hydroxyl groups of serine or threonine residues in a polypeptide, catalyzed by a family of polypeptide
N-α-acetylgalactosaminyltransferases (ppGalNAc Ts).
5 To date, 16 mammalian members have been reported in the literature (
1–
16) with a total of at least 20 members currently present in the human genome data base. Multiple members of the ppGalNAc T family have also been identified in
Drosophila (
9,
10,
14),
Caenorhabditis elegans (
3,
8), and single and multicellular organisms (
17–
20). Several members show close sequence orthologues across species suggesting that the ppGalNAc Ts are responsible for biologically significant functions that have been conserved during evolution. For example, in
Drosophila four isoforms have close sequence orthologues to the mammalian transferases. Of the two that have been recently compared, nearly identical peptide substrate specificities have been observed between the fly and mammals, suggesting common but presently unknown functions preserved across these diverse species (
21).Recently, several ppGalNAc T isoforms have been shown to be important for normal development or cellular processes. For example, inactive mutations in the fly PGANT35A (the T11 orthologue in mammals) are lethal because of the disruption of the tracheal tube structures (
9,
10,
22), whereas mutations in PGANT3 alter epithelial cell adhesion in the
Drosophila wing blade resulting in wing blistering (
23). In humans, mutations in ppGalNAc T3 are associated with familial tumoral calcinosis, the result of the abnormal processing and secretion of the phosphaturic factor FGF23 (
24,
25). Human ppGalNAc T14 has been suggested to modulate apoptotic signaling in tumor cells by its glycosylation of the proapoptotic receptors DLR4 and DLR5 (
26), and very recently the specific
O-glycosylation of the TGFB-II receptor (ActR-II) by the GalNTL1 has been shown to modulate its signaling in development (
16).Historically, the major targets of the ppGalNAc Ts have been thought to be heavily
O-glycosylated mucin domains of membrane and secreted glycoproteins. Such domains typically contain 15–30% Ser or Thr, which are highly (>50%) substituted by GalNAc. One question in the field is as follows. How is this high degree of peptide core glycosylation achieved and is it related to the large number of ppGalNAc isoforms, some of which may even have specific mucin domain preferences? Interestingly, some members of the ppGalNAc T family are known to prefer substrates that have been previously modified with
O-linked GalNAc on nearby Ser/Thr residues, hence having so-called glycopeptide or filling-in activities,
i.e. ppGalNAc T7 and T10 (
8,
27–
29). Others simply possess altered preferences against glycopeptide substrates,
i.e. ppGalNAc T2 and T4 (
30–
33), or may be inhibited by neighboring glycosylation,
i.e. ppGalNAc T1 and T2 (
29,
34,
35). These latter transferases have been called early or initiating transferases, preferring nonglycosylated over-glycosylated substrates. Presently, little is known about which factors dictate the different peptide/glycopeptide specificities among the ppGalNAc Ts.The ppGalNAc Ts consist of an N-terminal catalytic domain tethered by a short linker to a C-terminal ricin-like lectin domain containing three recognizable carbohydrate-binding sites (
36). Because ppGalNAc T7 and T10 prefer to transfer GalNAc to glycopeptide acceptors, it has been widely assumed that their C-terminal lectin domains would play significant roles in this activity, as has been demonstrated for other family members (
27,
28,
32). Recently, Kubota
et al. (
37) solved the crystal structure of ppGalNAc T10 in complex with Ser-GalNAc specifically bound to its lectin domain. In this work (
37), the authors further demonstrated that a T10 lectin domain mutant indeed had altered specificity against GalNAc-containing glycopeptide substrates when the acceptor Ser/Thr site was distal from the pre-existing glycopeptide GalNAc site. However, it was also observed that the lectin mutant still possessed relatively unaltered glycopeptide activity when the acceptor Ser/Thr site was directly N-terminal of a pre-existing glycopeptide GalNAc site. Kubota
et al. (
37) therefore concluded that for ppGalNAc T10, both its lectin and indeed its catalytic domain must contain distinct peptide GalNAc recognition sites. In support of this, Raman
et al. (
33) have shown that the complete removal of the ppGalNAc T10 lectin domain only slightly alters its specificity against distal glycopeptide substrates while showing no difference in its ability to glycosylate residues directly N-terminal of an existing site of glycosylation. Thus, it seems that the catalytic domain of ppGalNAc T10 may have specific requirements for a peptide
O-linked GalNAc in at least the +1 position (toward the C terminus) of residues being glycosylated. As no systematic determination of the glycopeptide binding properties of the ppGalNAc Ts catalytic domain has been performed, it is unknown whether additional GalNAc peptide-binding sites exist in T10 or, for that matter, any of the other ppGalNAc Ts.We have recently reported the use of oriented random peptide substrates, GAGA(
X)
nT(
X)
nAGAGK (where
X indicates randomized amino acid positions and
n = 3 and 5) for determining the peptide substrate specificities of mammalian ppGalNAc T1, T2, and their fly orthologues (
21,
38). In the present work, 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 = 4 oriented random glycopeptide libraries (). Interestingly, ppGalNAc T10 displays few significant enhancements and specifically lacks the Pro residue enhancements observed for ppGalNAc T1 and T2. These findings further demonstrate the vast substrate diversity of the catalytic domains of the ppGalNAc T family of transferases.
TABLE 1
ppGalNAc transferase random substrates utilized in this workPVI, PVII, GP-I, and GP-II random (glyco)peptide substrates.
Peptide | Sequence | No. of unique sequences |
---|
| GAGAXXXXXTXXXXXAGAGK | |
P-VI | X = G, A, P, V, L, Y, E, Q, R, H | 10 × 109 |
P-VII | X = G, A, P, I, M, F, D, N, R, K | 10 × 109 |
|
| GAGAXXXXTXXXXAGAG | |
GP-I | X = G, A, P, V, I, F, Y, E, D, N, R, K, H, and Ser-O-α-GalNAc | 1.47 × 109 |
|
| GAGAXXXX(Thr-O-α-GalNAc)XXXXAGAG | |
GP-II | X = G, A, P, V, I, F, Y, E, D, N, R, K, H, S | 1.47 × 109 |
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