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.
Highlights
Occurring N-glycoproteins exhibit glycoform heterogeneity with respect to N-glycan sequon occupancy and glycan structure
The resolved crystal structure of NGT from Actinobacillus pleuropneumoniae (ApNGT) revealed that the N-glycosyltransferase contains an N-terminal ␣-helical domain fold and a C-terminal GT-B fold with two Rossmannlike domains
To compare the catalytic efficiency of the wild-type ApNGT and the mutant Q469A, which is the most efficient N-glycosyltransferase obtained in this study (Fig. 3), in protein glycosylation, a soluble fragment corresponding to amino acids 1205– 1536 of a mature HMW1 adhesin (HMW1ct) was employed as a protein acceptor
Summary
The residue preference within and surrounding the N-glycosylation sequon (position Ϫ1, ϩ1, and ϩ3) was investigated by screening three panels of synthetic peptide substrates in a systematic manner. Because the pair of residues locates above the UDP-binding site, the narrow entryway formed by them might block the access of amino acids with bulky side chains into the active site and plays a role in determining the residue preference surrounding the Asn of N-glycosylation sequons. The mutant Q469A and the wild-type ApNGT show a similar preference toward charged residues at all three positions, whereas the former exhibits better tolerance at both the Ϫ1 and ϩ1 positions for residues harboring the bulky side chain, such as Leu, Phe, and the acylated Lys at the Ϫ1 position as well as Ile and Leu at the ϩ1 position. The mutant Q469A exhibits better tolerance toward the Gln-X-Ser/Thr sequon (supplemental Fig. S2, A and B), which has been reported to be glycosylated by the wild-type ApNGT at an extremely low rate (Ͼ15,000-fold reduction in turnover rate as compared with Asn-X-Ser/Thr; Ref. 7).
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