Engineering the pattern of protein glycosylation modulates the thermostability of a GH11 xylanase

Abstract

Protein glycosylation is a common post-translational modification, the effect of which on protein conformational and stability is incompletely understood. Here we have investigated the effects of glycosylation on the thermostability of Bacillus subtilis xylanase A (XynA) expressed in Pichia pastoris. Intact mass analysis of the heterologous wild-type XynA revealed two, three, or four Hex(8-16)GlcNAc2 modifications involving asparagine residues at positions 20, 25, 141, and 181. Molecular dynamics (MD) simulations of the XynA modified with various combinations of branched Hex9GlcNAc2 at these positions indicated a significant contribution from protein-glycan interactions to the overall energy of the glycoproteins. The effect of glycan content and glycosylation position on protein stability was evaluated by combinatorial mutagenesis of all six potential N-glycosylation sites. The majority of glycosylated enzymes expressed in P. pastoris presented increased thermostability in comparison with their unglycosylated counterparts expressed in Escherichia coli. Steric effects of multiple glycosylation events were apparent, and glycosylation position rather than the number of glycosylation events determined increases in thermostability. The MD simulations also indicated that clustered glycan chains tended to favor less stabilizing glycan-glycan interactions, whereas more dispersed glycosylation patterns favored stabilizing protein-glycan interactions.

Keywords: Glycoside Hydrolases; Glycosylation; Mass Spectrometry (MS); Molecular Dynamics Simulation; Protein Design; Protein Engineering; Protein Thermostability.

FIGURE 1.

Amino acid sequence (A) and ribbon representation (B) of the crystal structure of xylanase A from B. subtilis showing the potential N-glycosylation sites. The sequence was modified to contain a C-terminal peptide with a polyhistidine. The underlined residues (EAE) are derived from the α-factor signal peptide. The numbers of amino acids correspond to the native sequence that does not include residues EAE.

FIGURE 2.

LC-MS of B. subtilis xylanase A expressed in P. pastoris before (A) and after (B) Endo H digestion. Intact mass analyses indicate three main glycoform clusters with different extents of glycosylation. These data are supported by Endo H digestion, where the protein mass (24,050 Da) plus four, three, or two HexNAc monosaccharides was observed. The inset shows SDS-PAGE from purified xylanase before and after Endo H digestion.

FIGURE 3.

Biochemical and molecular dynamics simulations results for wild-type XynA. A, temperature dependence of catalytic activities of wild-type XynA expressed either in E. coli (Wild-type_Ec) or P. pastoris (Wild-type_Pp). B, thermal inactivation profiles of the wild-type XynA at 55 °C. C, ribbon representations of MD simulations of the conformation of glycosylated XynA. The polypeptide portions of the molecules are represented by gray ribbons, whereas the glycan portions are represented as solid molecular surfaces in different colors according to their positions (glycosylation sites): red for the glycan at Asn²⁰, green for the glycan at Asn²⁵, blue for the glycan at Asn²⁹, magenta for the glycan at Asn¹⁴¹, and orange for the glycan at Asn¹⁸¹. D, IPE_{partial PG} computed as time-average for the protein-glycan interactions for each glycosylated position during the MD simulation. E, IPE for glycan-glycan intermolecular interactions (IPE_GG).

FIGURE 4.

IPE_Total computed as time-average for protein-glycan interactions along the MD simulation. Error bars, S.D.

FIGURE 5.

Molecular dynamics simulations and biochemical results for XynA mutant N181Q. A, ribbon representations of MD simulations of the conformation of glycosylated mutant. The polypeptide portions of the molecules are represented by gray ribbons, whereas the glycans portions are represented as solid molecular surfaces in different colors according to their positions (see the legend to Fig. 3). B, IPE_{partial PG} computed as time-average for the protein-glycan interactions for each glycosylated position during the MD simulation. C, IPE_GG. D, temperature dependence of catalytic activities of XynA mutant N181Q expressed in P. pastoris. E, thermal inactivation profile at 55 °C of the XynA mutant N181Q expressed in P. pastoris. Error bars, S.D.

FIGURE 6.

Thermal inactivation profiles at 55 °C of the wild type and the mutants. A, wild-type xylanase A expressed either in E. coli (Wild-type_Ec) or P. pastoris (Wild-type_Pp) and single mutants expressed in P. pastoris (see inset). B, results for double, triple, quadruple, and quintuple mutants. Error bars, S.D.

FIGURE 7.

Snapshots of MD simulations of the conformations of glycosylated mutants of xylanase A. The structures are ribbon representations of the average stabilized structures obtained every 0.5 ps from MD calculations at 328 K over the final 15 ns of the trajectory. The polypeptide portions of the molecules are represented by gray ribbons, whereas the glycan portions are represented as solid molecular surfaces in different colors according to their positions (glycosylation sites): red for the glycan at Asn²⁰, green for the glycan at Asn²⁵, blue for the glycan at Asn²⁹, magenta for the glycan at Asn¹⁴¹, and orange for the glycan at Asn¹⁸¹. These figures were generated using the PyMOL software (PyMOL Molecular Graphics System, version 1.5, Schrödinger, LLC).

FIGURE 8.

IPE_partial computed as time-average for the protein-glycan interactions for each glycosylated position during the MD simulation.

FIGURE 9.

IPEs per residue calculated from the time-averaged protein residue-glycan interactions during the MD simulation for the indicated mutants.