Production of homogeneous glycoprotein with multisite modifications by an engineered N-glycosyltransferase mutant

Abstract

Naturally occurring N-glycoproteins exhibit glycoform heterogeneity with respect to N-glycan sequon occupancy (macroheterogeneity) and glycan structure (microheterogeneity). However, access to well-defined glycoproteins is always important for both basic research and therapeutic purposes. As a result, there has been a substantial effort to identify and understand the catalytic properties of N-glycosyltransferases, enzymes that install the first glycan on the protein chain. In this study we found that ApNGT, a newly discovered cytoplasmic N-glycosyltransferase from Actinobacillus pleuropneumoniae, has strict selectivity toward the residues around the Asn of N-glycosylation sequon by screening a small library of synthetic peptides. The inherent stringency was subsequently demonstrated to be closely associated with a critical residue (Gln-469) of ApNGT which we propose hinders the access of bulky residues surrounding the occupied Asn into the active site. Site-saturated mutagenesis revealed that the introduction of small hydrophobic residues at the site cannot only weaken the stringency of ApNGT but can also contribute to enormous improvement of glycosylation efficiency against both short peptides and proteins. We then employed the most efficient mutant (Q469A) other than the wild-type ApNGT to produce a homogeneous glycoprotein carrying multiple (up to 10) N-glycans, demonstrating that this construct is a promising biocatalyst for potentially addressing the issue of macroheterogeneity in glycoprotein preparation.

Keywords: N-linked glycosylation; glycoprotein; glycosyltransferase; post-translational modification (PTM); protein engineering.

Figure 1.

The residue preference within and surrounding the N-glycosylation sequon (−1, +1, and +3 positions). For each position at least five representative amino acids containing basic, acidic, hydroxyl, small hydrophobic, or aromatic side chains were evaluated. X presents variant amino acids. Contrasted to the mutant Q469A (0.4 μm), an ∼23-fold excess of the wild-type ApNGT (9.2 μm) was used in the assays due to its low efficiency.

Figure 2.

Putative peptide-binding sites of ApNGT. Shown is a comparative representation of the active sites of ApNGT (PDB ID 3Q3H) and XcOGT (PDB ID 2XGO). The extended loop (L_β10α19) connecting sheet β10 and helix α19 in ApNGT is highlighted in red (A). The potential acceptor binding groove of ApNGT is shown in surface representation and colored according to the electrostatic potential (blue in the positive regions and red in the negative regions). The electrostatic potential surface was calculated by using PyMOL (Schrödinger, LLC) (B). UDP complexed in the structure of ApNGT is shown as sticks.

Figure 3.

The relative activity of 18 ApNGT variants against a peptide (GGANVTKTIER) containing the optimal sequence ANVTK. Assays were performed in a total volume of 30 μl containing 1 mm various peptides, 20 mm UDP-Glc, 0.4 μm enzyme, and buffered with 100 mm Tris-HCl (pH 8.0) at 37 °C for 45 min. All assays were analyzed by RP-HPLC and carried out in duplicate.

Figure 4.

Twelve Asn-X-Ser/Thr sequons of HMW1ct acceptor (A) and the corresponding glycosylation reactions catalyzed by the mutant Q469A (B). Site-directed mutagenesis was used to generate 12 mutants (P1-P12) from a previously synthesized template carrying 12 inactive sequons with the Asn substituted by Ala. Each mutant has a single Asn-X-Ser/Thr sequon. Protein glycosylation was analyzed by MALDI-TOF mass spectrometry. Acceptor proteins and the potential products were labeled with gray lines and colored lines, respectively. The matrix adduct ion appears to be the addition of a matrix molecule to the protein ions with an accompanying loss of water (37, 38).

Figure 5.

Mass spectrometry analysis of protein (HMW1ct) glycosylation catalyzed by the wild-type ApNGT (red line) or the mutant Q469A (blue line). Various molar ratios of acceptor protein (HMW1ct possessing 12 Asn-X-Ser/Thr sequons (A, B, C, and D) or engineered HMW1ct with 7 (E) or 10 (F) Asn-X-Ser/Thr sequons) to enzyme (120:1, 30:1, or 10:1) were used in glycosylation reactions, which were incubated for 1 or 12 h. The numbers of added N-glucoses were labeled. The sequences of optimized N-glycosylation sites (P4, P10, and P12) are QVNLTAQ, NANVTGS, and EANVTIT, respectively. a.u., absorbance units.

Figure 6.

Amino acid preferences in occupied N-glycan sites. 1433 glycoproteins containing N-linked GlcNAc were retrieved from the PDB database by using a web-based glycan searching tool (Glycan Modeling and Stimulation). Total 2250 non-redundant N-glycan sites derived from these glycoproteins were identified by using pdb2linucs (39) and used to generate the sequence logo with WebLogo (40). Neighboring residues located downstream (positions +1 to +5) and upstream (positions −1 to −3) from the occupied asparagine residue (position 0) are shown. The size of each letter represents the residue prevalence at the putative position.