Production of homogeneous glycoprotein with multi-site modifications by an engineered N-glycosyltransferase mutant [Enzymology]

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 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 towards 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 (Gln469) of ApNGT that 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 can not only weaken the stringency of ApNGT but 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 homogeneous glycoprotein carrying multiple (up to ten) N-glycans, demonstrating this construct is a promising biocatalyst for potentially addressing the issue of macroheterogeneity in glycoprotein preparation.

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