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.

Tyrosinase is the key enzyme in melanin biosynthesis, catalyzing multiple steps in this pathway. The mature glycoprotein is transported from the Golgi to the melanosome where melanin biosynthesis occurs. In this study, we have investigated the effects of inhibitors of N-glycan processing on the synthesis, transport, and catalytic activity of tyrosinase. When B16 mouse melanoma cells were cultured in the presence of N-butyldeoxynojirimycin, an inhibitor of the endoplasmic reticulum-processing enzymes ␣-glucosidases I and II, the enzyme was synthesized and transported to the melanosome but almost completely lacked catalytic activity. The cells contained only 2% of the melanin found in untreated cells. Structural analysis of the N-glycans from N-butyldeoxynojirimycin-treated B16 cells demonstrated that three oligosaccharide structures (Glc 3 Man 7-9 ) predominated. Removal of the glucose residues with ␣-glucosidases I and II failed to restore enzymatic activity, suggesting that the glucosylated N-glycans do not sterically interfere with the enzyme's active sites. The mannosidase inhibitor deoxymannojirimycin had no effect on catalytic activity suggesting that the retention of glucosylated N-glycans results in the inactivation of this enzyme. The retention of glucosylated N-glycans does not therefore result in misfolding and degradation of the glycoprotein, as the enzyme is transported to the melanosome, but may cause conformational changes in its catalytic domains.

Although the presence of an oligosaccharide may influence the physical and biological characteristics of a protein Glucosylated oligomannose N-linked oligosaccharides , a primary role for N-linked glycosylation (Glc x Man 9 GlcNAc 2 where x ⍧ 1-3) are not normally in many simple eukaryotes may have been to retain the found on mature glycoproteins but are involved in the protein in the ER so that it folds correctly. Oligosaccharide early stages of glycoprotein biosynthesis and folding as motifs, based on Glc The full role of the glucose residues during glycosylation Man 9 GlcNAc 2 , Glc 2 Man 9 GlcNAc 2 and Glc 1 Man 9 is not well understood. In vitro experiments have shown GlcNAc 2 conformations shows the glucose residues to that these residues produce a 10-fold increase in the be fully accessible for recognition. A more detailed apparent affinity constant of OST for the oligosaccharide analysis of the conformations allows potential recogni- (Breuer and Bause, 1995). The authors also suggest that tion epitopes on the glycans to be identified and can the recognition and binding of outer arm glucose residues form the basis for understanding the specificity of the induce conformational changes in the active site of OST, glucosidases and chaperones (such as calnexin) that influencing the association constant of the peptide substrate recognize these glycans, with implications for their (Breuer and Bause, 1995). mechanisms of action.The first stages of glycan processing involve sequential Keywords: glucosylated glycans/NMR/oligosaccharide removal of the terminal glucose residues from the precursor structure/protein folding N-glycan Glc 3 Man 9 GlcNAc 2 by α-glucosidase I and II. The Glc 1 Man 9 GlcNAc 2 glycan, formed after cleavage by α-glucosidase I and II, mediates interaction of the glycoprotein with the ER-resident chaperones calnexin

As seen many aspects of the statistics preformed on SAGS have a firm structural basis and the details of higher eukaryote OST structure, which are not yet known, will probably explain the other aspects of occupation signatures.