Structural and Mechanistic Insights into the Catalytic-Domain-Mediated Short-Range Glycosylation Preferences of GalNAc-T4

Abstract

Mucin-type O-glycosylation is initiated by a family of polypeptide GalNAc-transferases (GalNAc-Ts) which are type-II transmembrane proteins that contain Golgi luminal catalytic and lectin domains that are connected by a flexible linker. Several GalNAc-Ts, including GalNAc-T4, show both long-range and short-range prior glycosylation specificity, governed by their lectin and catalytic domains, respectively. While the mechanism of the lectin-domain-dependent glycosylation is well-known, the molecular basis for the catalytic-domain-dependent glycosylation of glycopeptides is unclear. Herein, we report the crystal structure of GalNAc-T4 bound to the diglycopeptide GAT*GAGAGAGT*TPGPG (containing two α-GalNAc glycosylated Thr (T*), the PXP motif and a "naked" Thr acceptor site) that describes its catalytic domain glycopeptide GalNAc binding site. Kinetic studies of wild-type and GalNAc binding site mutant enzymes show the lectin domain GalNAc binding activity dominates over the catalytic domain GalNAc binding activity and that these activities can be independently eliminated. Surprisingly, a flexible loop protruding from the lectin domain was found essential for the optimal activity of the catalytic domain. This work provides the first structural basis for the short-range glycosylation preferences of a GalNAc-T.

Figure 1

Biophysical characterization of GalNAc-T4. (a) Peptide glycosylation kinetics of GalNAc-T4 against (glyco)peptides 1–6 (see also Figure 4a). Michaelis–Menten kinetic values, K_m, V_max, and catalytic efficiency (V_max/K_m) for glycopeptides 3–6 were obtained from the nonlinear least-squares fit to the initial rate data, obtained as described in the Methods section and given in Table 2. Peptide substrates 1 and 2 are largely unglycosylated by GalNAc-T4. (b) Left panel: SPR sensogram for binding of glycopeptide 6 to GalNAc-T4. Right panel: Fitting of the SPR binding data giving a K_d of 70 ± 15 μM. (c) Mapping of substrate binding epitopes by saturation transfer difference (STD) NMR. The size of the colored spheres represents the normalized STD-NMR intensity (i.e., binding) observed for the indicated protons/residues. For sake of clarity, the STD response given for the indicated amino acid residues corresponds to the average of STD for all of the protons in the residue that could be accuracy measured. See Figures S4–S6 for the detailed STD-NMR enhancements of the identified residues/protons. Note that in addition to the GalNAc protons amino acid protons in the -T*TPGP- sequence also gave STD-NMR enhancements. (d) Representative 600 MHz ¹H NMR spectra of glycopeptide 6 (-T*--T*T-) at 877 μM in the presence of 13.5 μM GalNAc-T4, 75 μM UDP, and 75 μM MnCl₂ obtained at 298 K. The off resonance reference spectrum (labeled Off res) is displayed in blue, and the on resonance STD spectrum (labeled STD) is in red. Key proton resonances are labeled in the STD spectrum. Note the different STD responses for the identified GalNAc H2 protons of the glycosylated Thr3 and Thr11 found between 4.0 and 4.1 ppm of the STD spectrum.

Figure 2

Crystal structure of GalNAc-T4 in complex with UDP-Mn⁺² and glycopeptide 6. (a) Two different views of GalNAc-T4 in complex with 6. The catalytic and lectin domains are colored in gray, and the flexible linker and catalytic domain active site loop are depicted in red and yellow, respectively. The lectin domain flexible loop (LFL) is indicated by a black arrow. The GalNAc moiety of the Thr3-GalNAc and Thr11-GalNAc is shown as orange carbon atoms while the rest of the peptide is shown as green carbon atoms. The nucleotide is depicted as brown carbon atoms whereas the manganese atom is shown as a pink sphere. Inserted between the structures is a surface representation of GalNAc-T4 with the same orientation as the cartoon representation of the leftmost structure. (b) Electron density maps are F_O–F_C (blue) contoured at 2.0 σ for glycopeptide 6, UDP, and manganese ion. (c) Two different views of GalNAc-T2 in complex with MUC5AC-13 (PDB entry 5AJP(14)) again with an inserted surface representation between structures. Atom colors are the same as in part a above. (d) Close-up views of the GalNAc-T2-UDP-MUC5AC-13 and the GalNAc-T4-UDP-glycopeptide 6 complexes showing the bound glycopeptide and the catalytic domain active site flexible loop (in black). Note that flexible loop residues Trp331 in GalNAc-T2 and Trp334 in GalNAc-T4 adopt an “in” loop conformation.

Figure 3

Structural features of peptide, UDP, and lectin-domain-binding sites of GalNAc-T4. (a) View of the complete sugar nucleotide, peptide, and lectin-domain-binding sites of the GalNAc-T4-UDP-glycopeptide 6 complex. Upper panel: close-up view of bound glycopeptide. Lower panel: close-up view of the manganese binding site. The residues forming sugar-nucleotide, peptide, and lectin-domain-binding sites are depicted as black, yellow, and gray carbon atoms, respectively. UDP and the glycopeptide are shown as brown and green carbon atoms, respectively. Mn²⁺ and GalNAc moiety are depicted as a pink sphere and orange carbon atoms, respectively. Hydrogen bond interactions are shown as dotted green lines. Water molecules are depicted as red spheres. Note that we only show water-mediated interactions in which only one water molecule acts as a bridge between the residues. (b) View of the sugar nucleotide, glycopeptide, and lectin-domain-binding sites of the GalNAc-T2-UDP-MUC5AC-13 complex (PDB entry 4D0T). Colors are the same as above. (c) Modeled structure for a GalNAc-T4-UDP-GalNAc-glycopeptide 6 complex. The coordinates of the UDP-GalNAc were obtained by superposing the structure of GalNAc-T2 containing UDP-GalNAc (PDB entry 4D0T) with the GalNAc-T4-UDP-GalNAc-glycopeptide 6 complex. The structure shows that the Thr12 acceptor of glycopeptide 6 is close to the anomeric carbon of UDP-GalNAc.

Figure 4

Enzyme kinetics of wt and mutant GalNAc-T4 against the (glyco)peptide substrates in Table 1. Note that the left and right panels are plotted with different initial activity scales. Kinetic constants obtained from the plots are given in Table 2. (a) Wild-type GalNAc-T4 showing both long-range (left panel) and short-range (right panel) glycopeptide activities. (b) Lectin mutant (D549H) showing the loss of its long-range glycopeptide activity. (c) Catalytic mutant (T283S, Q285A) showing the partial loss of GalANc-T4’s short-range prior glycopeptide activity. (d) Lectin flexible link (LFL) mutant (P₄₆₃DNNP₄₆₇ to GGG) showing a partial loss of the short-range glycopeptide activity while possessing an intact catalytic domain. (e) Catalytic/LFL combined mutant (T283S, Q285A, D464A) showing a more complete loss of the short-range glycopeptide activity. (f) Lectin/catalytic combined mutant (T283S, Q285A, D459H) showing the near complete loss of both the long-range and short-range glycopeptide activities.