Glycosylation of Proteins: A Computer Based Method for the Rapid Exploration of Comformational Space of N-Glycans

Inspection of protein databases suggests that as many as 70% of proteins have potential N-glycosylation sites. Unfortunately glycoproteins often refuse to crystallize and NMR techniques do not allow an unambiguous determination of the complete conformation of the sugar part. Therefore, time-consuming complex simulation methods are often used to explore the conformational space of N-glycans. The generation of a comprehensive data base describing the conformational space of larger fragments of N-glycans taking into account the effects of branching is presented. High-temperature molecular dynamics simulations of essential N-glycan fragments are performed until conformational equilibrium has been reached. Free energy landscapes are calculated for each glycosidic linkage. All possible conformations for each N-glycan fragment are automatically assigned, ranked according to their relative population and stored in a database. These values are recalled for the generation of a complete set of all possible conformations for a given N-glycan topology. The constructed conformations are ranked according to their energy content. Since this approach allows to explore the complete conformational space of a given N-glycan within a few minutes of CPU-time on a standard PC, it is well suited to be used as a Web-Based application. 1 Introduction Glycosylation is one of the most abundant forms of covalent protein and lipid modification. [1] There are two main types of protein glycosylation: N-glycosylation, in which the oligosaccharide is attached to an asparagine residue, and O-glycosylation, in which the oligosaccharide is attached to a serine or threonine residue. Glycoproteins usually exist as complex mixtures of glycosylated variants (glycoforms). Glycosylation occurs in the endoplasmic reticulum (ER) and Golgi compartments of the cell and involves a complex series of reactions catalyzed by membrane-bound glycosyltransferases and glycosidases. Many of these enzymes are exquisitely sensitive to other events taking place within the cell in which the glycoprotein is expressed. The populations of sugars attached to an individual protein will therefore depend on the cell type in which the glycoprotein is expressed and on the physiological status of the cell, and may be developmentally and disease regulated [2,3]. Inspection of protein databases suggests that as many as 70% of proteins have potential N-glycosylation sites (sequence motif ASN-X-[SER/THR] where X is not PRO) [4] The biological function of glycosylation is still not completely understood [1,5]. However, it is clear that glycoproteins are fundamental to many biological processes including fertilisation, immune defence, viral replications,