Residues Comprising the Enhanced Aromatic Sequon Influence Protein N-Glycosylation Efficiency

Abstract

N-Glycosylation is an important co- and/or post-translational modification that occurs on the vast majority of the one-third of the mammalian proteome that traverses the cellular secretory pathway, regulating glycoprotein folding and functions. Previous studies on the sequence requirements for N-glycosylation have yielded the Asn-X-Ser/Thr (NXS/T) sequon and the enhanced aromatic sequons (Phe-X-Asn-X-Thr and Phe-X-X-Asn-X-Thr), which can be efficiently N-glycosylated. To further investigate the influence of sequence variation on N-glycosylation efficiency in the context of a five-residue enhanced aromatic sequon, we used the human CD2 adhesion domain (hCD2ad) to screen the i-2, i-1, i+1, and i+2 residues flanking Asn at the i position. We found that aromatic residues, especially Trp, and sulfur-containing residues at the i-2 position improved N-glycosylation efficiency, while positively charged residues such as Arg suppressed N-glycosylation. Thiol, hydroxyl, and aliphatic-based side chains at the i-1 position had higher N-glycosylation efficiency, and Cys, in particular, compensated for the negative effect of Arg at the i-2 position. Small residues and Ser at the i+1 position increased the likelihood of N-glycosylation, and Thr is better than Ser at the i+2 position. We devised an algorithm for prediction of N-glycosylation efficiency using the SAS software, employing the 120 sequences studied as a training set. We then introduced the optimized-enhanced aromatic sequons into other glycoproteins and observed an enhancement in N-glycan occupancy that was further supported by modeling the high-affinity interaction between the optimized sequence on hCD2ad and a human oligosaccharyltransferase (OST) subunit. The findings in this study provide useful information for enhancing or suppressing N-glycosylation at a site of interest and valuable data for a better understanding of OST-catalyzed N-glycosylation.

An Effective Neutralizing Antibody Against Influenza Virus H1N1 from Human B Cells

“…In the 32D6-Fab complex structure, the glycosylated N104 with a monosaccharide is observed on a SDNGT sequon to stabilize the protein structure of HA1 domain, along with the glycan contact with the side chains of K71, N81, and R238 (Fig. S2) 12 . Glycosylation at the residues N28 and N40 remain uncertain, whereas the other glycosylated residues 304, 498 and 557 were not included in the HA1 in this complex crystal.…”

An oligosaccharyltransferase from Leishmania major increases the N‐glycan occupancy on recombinant glycoproteins produced in Nicotiana benthamiana

“…For a recombinant elastase expressed in P. pastoris, a change of the sequon from N‐X‐S to N‐X‐T resulted in an increased glycosylation efficiency that was accompanied by higher production levels of the recombinant glycoprotein (Han et al ., ). By contrast, mutagenesis of flanking amino acids and generation of an optimized aromatic sequon with increased glycosylation efficiency negatively affected the secretion of IFN‐γ expressed in human cells and caused variability in protein expression of another glycoprotein (Huang et al ., ). Likewise, antibody engineering by generation of an aromatic sequon (FANST instead of the canonical QYNST) improved the thermal stability of the antibody, but reduced the affinity to specific Fcγ‐receptors (Chen et al ., ).…”

Germline VRC01 antibody recognition of a modified clade C HIV-1 envelope trimer and a glycosylated HIV-1 gp120 core

“…BLI experiments demonstrated the S278T mutation reduced the equilibrium dissociation constant by 10-fold relative to the wild type NLGS, mainly by decreasing association kinetics ( Figure 6A–D , Figure 5—figure supplement 2A ). The S278T mutation did not abrogate VRC01 GL -class antibody binding, despite an expected improvement in Asn276 glycosylation efficiency ( Huang et al, 2017 ). Moreover, semi-quantitative LC-MS/MS revealed (GlcNAc) 2 -(Man) 5 oligosaccharides were predominantly detected at position Asn276 in the S278T mutant, though some unglycosylated peptides were still present at low levels ( Figure 5A , Figure 2—figure supplement 1H,I ).…”

“…Whereas 82% of sequenced HIV-1 clades harbor a threonine residue at position 278 of gp120, 426c gp120 contains a serine at this position. Since recent reports suggested NXT NLGS sites are more efficiently glycosylated relative to NXS sites ( Huang et al, 2017 ), we compared the binding kinetics of full-length VRC01 GL -class IgGs, VRC01 GL and 12A21 GL , to HEK293 GnTI -/- -expressed 426c core and an 426c S278T core mutant ( Figure 1—figure supplement 1 and Figure 1—figure supplement 2 ). BLI experiments demonstrated the S278T mutation reduced the equilibrium dissociation constant by 10-fold relative to the wild type NLGS, mainly by decreasing association kinetics ( Figure 6A–D , Figure 5—figure supplement 2A ).…”

“…Since binding was reduced following introduction of the S278T mutation, these results collectively suggest that a significant fraction of the interactions observed by BLI between the 426c core (containing an intact Asn276 NXS NLGS) and VRC01 GL occurred with the unglycosylated Asn276 subspecies. This construct is expected to be less-frequently glycosylated at position Asn276 relative to the S278T mutant ( Huang et al, 2017 ). However, VRC01 GL -class IgG binding was also detected with the 426c S278T core ( Figure 6A–D , Figure 6—figure supplement 1A ), and EndoH treatment (which retains core GlcNAcs) of both the S278 and S278T constructs significantly improved IgG binding relative to their untreated counterparts ( Figure 6E–H , Figure 6—figure supplement 1A ).…”