The euHCVdb suite of in silico tools for investigating the structural impact of mutations in hepatitis C virus proteins.

Hepatitis C is a viral infection of the liver that results in acute hepatitis and chronic liver disease, including cirrhosis and liver cancer. An estimated 170 million persons are chronically infected worldwide. The Hepatitis C virus is the pathogen agent responsible for hepatitis C. HCV is an enveloped RNA-positive virus of the flaviviridae family. The HCV genome shows remarkable sequence variability. This variability leads to the classification of HCV into 6 genotypes, numerous subtypes and HCV exists in each infected patient as quasi-species. The genotype may be linked to the severity of the disease and to the efficiency of the combination treatment with interferon and ribavirin. To date, no vaccine to prevent or cure HCV exists. Numerous HCV specific inhibitors have been designed and some are currently under clinical trials. However, resistances of HCV against these inhibitors have been identified. We developed the European Hepatitis C Virus Database (euHCVdb, http://euhcvdb.ibcp.fr/), a collection of functionally and structurally (3D-models) annotated HCV sequences integrated with sequence and structure analysis tools. We show below how the euHCVdb database is a useful in silico tool that can help drug design, combating resistance to drug treatment and understand structural biology of the HCV.

MAGOS: multiple alignment and modelling server.

UNLABELLED: MAGOS is a web server allowing automated protein modelling coupled to the creation of a hierarchical and annotated multiple alignment of complete sequences. MAGOS is designed for an interactive approach of structural information within the framework of the evolutionary relevance of mined and predicted sequence information. AVAILABILITY: The web server is freely available at http://pig-pbil.ibcp.fr/magos.

The living factory: in vivo production of N-acetyllactosamine containing carbohydrates in E. coli.

Scientific and commercial interest in oligosaccharides is increasing, but their availability is limited as production relies on chemical or chemo-enzymatic synthesis. In search for a more economical, alternative procedure, we have investigated the possibility of producing specific oligosaccharides in E. coli that express the appropriate glycosyltransferases. The Azorhizobium chitin pentaose synthase NodC (a beta(1,4)GlcNAc-oligosaccharide synthase), and the Neisseria beta(1,4)galactosyltransferase LgtB, were co-expressed in E. coli. The major oligosaccharide isolated from the recombinant strain, was subjected to LC-MS, FAB-MS and NMR analysis, and identified as betaGal(1,4)[betaGlcNAc(1,4)]4GlcNAc. High cell density culture yielded more than 1.0 gr of the hexasaccharide per liter of culture. The compound was found to be an acceptor in vitro for betaGal(1,4)GlcNAc alpha(1,3)galactosyltransferase, which suggests that the expression of additional glycosyltransferases in E. coli will allow the production of more complex oligosaccharides.

Fold recognition study of alpha3-galactosyltransferase and molecular modeling of the nucleotide sugar-binding domain.

The structure and fold of the enzyme responsible for the biosynthesis of the xenotransplantation antigen, namely pig alpha3 galactosyltransferase, has been studied by means of computational methods. Secondary structure predictions indicated that alpha3-galactosyltransferase and related protein family members, including blood group A and B transferases and Forssman synthase, are likely to consist of alternating alpha-helices and beta-strands. Fold recognition studies predicted that alpha3-galactosyltransferase shares the same fold as the T4 phage DNA-modifying enzyme beta-glucosyltransferase. This latter enzyme displays a strong structural resemblance with the core of glycogen phosphorylase b. By using the three-dimensional structure of beta-glucosyltransferase and of several glycogen phosphorylases, the nucleotide binding domain of pig alpha3-galactosyltransferase was built by knowledge-based methods. Both the UDP-galactose ligand and a divalent cation were included in the model during the refinement procedure. The final three-dimensional model is in agreement with our present knowledge of the biochemistry and mechanism of alpha3-galactosyltransferases.

Sequence-function relationships of prokaryotic and eukaryotic galactosyltransferases.

Galactosyltransferases are enzymes which transfer galactose from UDP-Gal to various acceptors with either retention of the anomeric configuration to form alpha1,2-, alpha1,3-, alpha1,4-, and alpha1, 6-linkages, or inversion of the anomeric configuration to form beta1, 3-, beta1,4-, and beta1-ceramide linkages. During the last few years, several (c)DNA sequences coding for galactosyltransferases became available. We have retrieved these sequences and conducted sequence similarity studies. On the basis of both the nature of the reaction catalyzed and the protein sequence identity, these enzymes can be classified into twelve groups. Using a sensitive graphics method for protein comparison, conserved structural features were found in some of the galactosyltransferase groups, and other classes of glycosyltransferases, resulting in the definition of five families. The lengths and locations of the conserved regions as well as the invariant residues are described for each family. In addition, the DxD motif that may be important for substrate recognition and/or catalysis is demonstrated to occur in all families but one.