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

Abstract

Naturally occurring -glycoproteins exhibit glycoform heterogeneity with respect to-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 -glycosyltransferases, enzymes that install the first glycan on the protein chain. In this study we found that ApNGT, a newly discovered cytoplasmic-glycosyltransferase from , has strict selectivity toward the residues around the Asn of-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) -glycans, demonstrating that this construct is a promising biocatalyst for potentially addressing the issue of macroheterogeneity in glycoprotein preparation.

Semi-processive hyperglycosylation of adhesin by bacterial protein N-glycosyltransferases

“…The binding site of ApNGT was found to be flexible enough to allow several peptide binding modes ( Figure 5C, main binding modes in green and purple) near the postulated acceptor binding groove and making contacts with the proposed acceptor binding residues Phe39, His272, His277, and Gln469. 36,38 Our results suggest that the peptide-binding region in ApNGT is located on the solvent-exposed enzyme surface. In contrast, in hOGT the unfolded peptide binds in a groove that is located inside a superspiral formed by repeated TPR regions.…”

“…Several studies have focused on employing NGTs (and their engineered variants) in the biosynthesis of defined glycoproteins for biotechnological applications and vaccine development. 38,48,[53][54][55] The ApNGT mutant Q469A showed reduced product inhibition, and produced a more homogenously glycosylated HMW1ct, with up to 10 residues. Based on the central position of Q469 in both UDP-Glc and peptide binding as revealed by molecular modeling, we propose that Q469 may function as a 'processive switch', preventing the glycosylated product from leaving the binding site, and thereby increasing the association required for an additional round of catalysis.…”

“…Based on the central position of Q469 in both UDP-Glc and peptide binding as revealed by molecular modeling, we propose that Q469 may function as a 'processive switch', preventing the glycosylated product from leaving the binding site, and thereby increasing the association required for an additional round of catalysis. 38 Sequence alignment indicates a corresponding Gln residue in a conserved region in HiNGT (Gln495, Figure S22), but without more structural information, it is difficult to assess its involvement in the mechanism.…”

A cell-free biosynthesis platform for modular construction of protein glycosylation pathways

“…Importantly, NGTs are soluble enzymes that can be easily expressed functionally in the E. coli cytoplasm 22,25,26 . Because glycosylation systems using NGT for glycan-protein conjugation do not require protein transport across membranes or lipid-associated components, they have elicited great interest from the glycoengineering community for the production of recombinant protein therapeutics and vaccines 9,21,22,[26][27][28][29] . Several recent advances set the stage for this vision.…”

“…Several recent advances set the stage for this vision. First, the acceptor specificity of NGTs has been extensively studied using peptide and protein substrates 25,28 , glycoproteomic studies 25 , and the GlycoSCORES technique 26 . These studies revealed that NGTs modify N-X-S/T amino acid acceptor motifs resembling those in eukaryotic glycoproteins.…”