The interdomain flexible linker of the polypeptide GalNAc transferases dictates their long-range glycosylation preferences

Abstract

The polypeptide GalNAc-transferases (GalNAc-Ts), that initiate mucin-type O-glycosylation, consist of a catalytic and a lectin domain connected by a flexible linker. In addition to recognizing polypeptide sequence, the GalNAc-Ts exhibit unique long-range N- and/or C-terminal prior glycosylation (GalNAc-O-Ser/Thr) preferences modulated by the lectin domain. Here we report studies on GalNAc-T4 that reveal the origins of its unique N-terminal long-range glycopeptide specificity, which is the opposite of GalNAc-T2. The GalNAc-T4 structure bound to a monoglycopeptide shows that the GalNAc-binding site of its lectin domain is rotated relative to the homologous GalNAc-T2 structure, explaining their different long-range preferences. Kinetics and molecular dynamics simulations on several GalNAc-T2 flexible linker constructs show altered remote prior glycosylation preferences, confirming that the flexible linker dictates the rotation of the lectin domain, thus modulating the GalNAc-Ts' long-range preferences. This work for the first time provides the structural basis for the different remote prior glycosylation preferences of the GalNAc-Ts.

Fig. 1

Modes of O-glycosylation found for the GalNAc-Ts. a–c These panels denote the three distinct modes of O-glycosylation performed by GalNAc-Ts: the neighboring glycosylation activity by the catalytic domain in a and b, and long-range lectin domain-mediated glycosylation activity in c. In c, two plausible models are suggested that might explain the different long-distance glycosylation preferences of GTs such as GalNAc-T3/T4/T16/T12. Oval-shaped figures in blue and green depict the lectin and catalytic domains, respectively. Peptides are indicated in blue and the black hexagon-shaped figure denotes the position of prior GalNAc moieties in the glycopeptides. Arrows indicate the positions of acceptor sites

Fig. 2

Biophysical characterization of GalNAc-T4 against peptides 1–3. a Peptide glycosylation kinetics of GalNAc-T4 against (glyco)peptides 1–3 (black, red, and blue symbols, respectively). Michaelis–Menten kinetic values, K_m, V_max, and catalytic efficiency (V_max/K_m) for monoglycopeptide 3 were obtained from the nonlinear least square fit to the initial rate data, obtained as described in the “Methods” section and given in Supplementary Table 2. Peptide substrates 1 and 2 are largely unglycosylated by GalNAc-T4. b STD-NMR-derived epitope mapping. The different colored spheres indicate the normalized STD signal (in percent) observed for each proton. See “Methods” section and Supplementary Fig. 1 for further details

Fig. 3

Structural characterization of the interaction between GalNAc-T4 and monoglycopeptide 3. a Two different views of GalNAc-T4 in complex with 3. The catalytic and lectin domains are colored in gray and the interdomain flexible linker is depicted in red. The catalytic domain flexible loop is depicted in yellow and is mostly disordered. The GlcNAc moiety of the Thr3-GalNAc is shown as orange carbon atoms while Thr3 is shown as green carbon atoms. b (left) Superposition of the GalNAc-T4-glycopeptide 3 (gray) and GalNAc-T2-MUC5AC-3-UDP-Mn⁺² (yellow) structures. The glycopeptide and the GalNAc moiety are shown in green and orange carbon atoms, respectively. The lectin α-subdomain GalNAc-binding residues, Asp458, and Asp459, of GalNAc-T2 and GalNAc-T4 are shown as sticks in black and gray carbon atoms, respectively. (right panel) Close-up view of the superposition between GalNAc-T2 and GalNAc-T4. Colors for peptides, GalNAc moiety, and proteins are identical as shown above. c–d Close-up view of the lectin α-subdomain GalNAc-binding site for both GalNAc-T4 (left) and T2 (right). The residues of both enzymes are depicted as gray carbon atoms. Hydrogen bond interactions are shown as dotted green lines. Electron density maps are F_O–F_C (blue) contoured at 2.2 σ for Thr3-GalNAc. Note that both GalNAc-binding sites are depicted in the same orientation for comparison. e Surface representation of GalNAc-T4 (model built with monoglycopeptide 3 and UDP/Mn⁺²), and GalNAc-T2 (with UDP/Mn⁺²/MUC5AC-13). Both are viewed from the same orientation as in b. Colors for the glycopeptide and the flexible loop are the same as above. f Superposition of the GalNAc-T4-glycopeptide 3-UDP-Mn⁺² (gray) and GalNAc-T2-MUC5AC-3-UDP-Mn⁺² (blue–white) structures. The glycopeptide and the GalNAc moiety in the structure of GalNAc-T2 and GalNAc-T4 are shown as spheres in blue/green and yellow/orange, respectively. UDP and Mn⁺² are shown as brown carbon atoms and as a pink sphere, respectively. g Superimposed structures of the 200 ns MD simulation trajectory for GalNAc-T4 in complex with glycopeptide 3/UDP/Mn⁺². GalNAc-T4 is depicted in a light gray surface view. The GalNAc moiety, the peptide backbone, UDP, and the acceptor Thr residues are shown as orange, green, brown, and blue carbon atoms, respectively

Fig. 4

Characterization of the GalNAc-T2 chimeras and mutants. a MD simulations in explicit water of chimera 2 (500 ns total simulation time; water molecules were removed for clarity). The 28 ns time-lapse snapshots show the dynamic events occurring during the lectin domain reorientation observed for this chimera. The GalNAc-T2 (far left) and GalNAc-T4 (far right) crystal structures are shown as references for the initial and final states. All structures are illustrated in a surface view with two different orientations. The lectin and catalytic domains, and the flexible linker of GalNAc-T2 and GalNAc-T4 are depicted as yellow/purple blue, orange/light gray, and red/deep teal, respectively. The colors used for the chimera 2 and GalNAc-T2 are the same except for the flexible linker of the former, which is pale green. Flexible linkers are also shown at the bottom in a cartoon- and sticks-like view. b Glycosylation time course plots of GalNAc-T2 and the GalNAc-T2 chimeras and mutants against (glyco)peptides 1–3 at substrate concentration of 1.4 mM. The obtained specific activities and selected substrate activity ratios are given in Supplementary Fig. 9. c Complete glycosylation kinetics (initial specific activity versus substrate concentration) for GalNAc-T2, the GalNAc-T2-triple mutant and the GalNAc-T2 chimera3 against substrate (glyco)peptides 1–3 (top panels) with plots of the obtained Michaelis–Menten kinetic parameters, K_m, V_max, catalytic efficiency (V_max/K_m), and catalytic efficiency ratios (monoglycopeptide 2 over 3) (bottom panels). Kinetic parameter values are summarized in Supplementary Table 2