Statistical analysis of the protein environment of N-glycosylation sites: implications for occupancy, structure, and folding

Abstract

We recently reported statistical analysis of structural data on glycosidic linkages. Here we extend this analysis to the glycan-protein linkage, and the peptide primary, secondary, and tertiary structures around N-glycosylation sites. We surveyed 506 glycoproteins in the Protein Data Bank crystallographic database, giving 2592 glycosylation sequons (1683 occupied) and generated a database of 626 nonredundant sequons with 386 occupied. Deviations in the expected amino acid composition were seen around occupied asparagines, particularly an increased occurrence of aromatic residues before the asparagine and threonine at position +2. Glycosylation alters the asparagine side chain torsion angle distribution and reduces its flexibility. There is an elevated probability of finding glycosylation sites in which secondary structure changes. An 11-class taxonomy was developed to describe protein surface geometry around glycosylation sites. Thirty-three percent of the occupied sites are on exposed convex surfaces, 10% in deep recesses and 20% on the edge of grooves with the glycan filling the cleft. A surprisingly large number of glycosylated asparagine residues have a low accessibility. The incidence of aromatic amino acids brought into close contact with the glycan by the folding process is higher than their normal levels on the surface or in the protein core. These data have significant implications for control of sequon occupancy and evolutionary selection of glycosylation sites and are discussed in relation to mechanisms of protein fold stabilization and regional quality control of protein folding. Hydrophobic protein-glycan interactions and the low accessibility of glycosylation sites in folded proteins are common features and may be critical in mediating these functions.

Enhancing the secretion of recombinant proteins by engineering N‐glycosylation sites

“…14,15 The N-glycosylation outcome of DcR3 mutants has also shown similar conclusion. Thus, it becomes critical to select a proper N-glycosylation site for attaching the glycan chain effectively and functionally.…”

“…What determines the occupation of a potential N-glycosylation site in the expressed protein is still not completely clear, although many factors have been postulated. 14,15 Table 2 shows the discrepancy between the prediction and the actual expression results of our experiments. Only 5 of 15 predictions are accurately consistent with the actual outcome.…”

“…It has been recognized that a proline residue located in or around the N-glycosylation sequon will lead to the nonoccupation of the site. 14,15 Serine has also been suggested to reduce the occupancy of the sequons. 14,15 Although amino acid residue composition of the N-glycosylation sequons may be important in the efficiency of the occupation by glycan, our mutagenesis results (Table 2) suggest the location of the N-glycosylation sequon at the N-or C-terminal could compensate for the requirements for amino acid composition.…”

“…14,15 Serine has also been suggested to reduce the occupancy of the sequons. 14,15 Although amino acid residue composition of the N-glycosylation sequons may be important in the efficiency of the occupation by glycan, our mutagenesis results (Table 2) suggest the location of the N-glycosylation sequon at the N-or C-terminal could compensate for the requirements for amino acid composition. Table 2 also suggests that the occupation of an N-glycosylation sequon by an oligosaccharide chain can not always be correctly predicted by algorithms such as NetNGlyc 1.0 Server of Technical University of Denmark.…”

EVOLUTIONAL AND FUNCTIONAL ANALYSIS OF A SERINE PROTEASE IN Spodoptera litura

“…Primary sequence analysis identified that SlSP1 contained several important sites, such as putative phosphorylation, N-glycosylation positions. Glycosylation is considered as a character of SP (McBride et al, 2000), which may stabilize protein structure and resist against protease inhibitors (Petrescu et al, 2004). The multiple sequence alignments revealed that SlSP1 included three absolute conserved catalytic triad HDS (position 84, 127, 229) and six highly conserved cysteine residues (position 66, 82, 194, 211, 223, and 247) putatively forming three disulfide bonds.…”