Glycosyltransferases in S-Layer Glycan Assembly

Glycosyltransferases play an important role in the assembly process of glycan chains of glycoproteins. In general, they are involved in the transfer of nucleotide-activated carbohydrate intermediates into growing glycan chains. While their mode of action is well-established for many eukaryotic glycoproteins, the data available from prokaryotic glycoproteins is still scarce. The aim of the proposed research project is the expression and functional characterization of the glycosyltransferases involved in the assembly and translocation of the S-layer glycan chains of the S-layer glycoprotein from Geobacillus stearothermophilus NRS 2004/3a. For this organism and also for related Aneurinibacillus thermoaerophilus strains, now the first completely sequenced slg (surface layer glycosylation) gene clusters are available. Although the glycan chains of G. stearothermophilus NRS 2004/3a S-layer protein SgsE consist only of L-rhamnose-containing homopolymers, different rhamnosyltransferases have been identified in the slg gene cluster by data base sequence comparison. Therefore, the characterization of their specificities is of major importance for the understanding of the entire glycan assembly process. Since L-rhamnose is not present in higher organisms, the corresponding transferases may be interesting canditates for the development of defense strategies against prokaryotes with rhamnose-containing polysaccharides. Motif search in the deduced amino acid sequences of other genes from the slg gene cluster of G. stearothermophilus NRS 2004/3a indicated the presence of different glycosyltransferase domains. These putative proteins might be involved in chain length determination and translocation process of the S-layer glycans across the cytoplasmic membrane. The presence of an ATP-dependent ABC-2 transporter system implies growth of the S- layer glycan at the non-reducing end of the chain. Since the investigated Gram-positive bacteria of interest have not been transformable so far, the expression and functional characterization of the putative gene products will be performed in heterologous systems such as Escherichia coli and Lactococcus lactis. In addition, exploratory experiments are planned for a modification of the genuine glycan structure of the S layer glycoprotein of G. stearothermophilus NRS 2004/3a. The proposed investigations are expected to provide eventually foundations for the production of modified S-layer glycoproteins by "carbohydrate engineering" that are useful for nanobiotechnological and biomedical applications.

Glycosyltransferases play an important role in the assembly process of glycan chains of glycoproteins. In general, they are involved in the transfer of nucleotide-activated carbohydrate intermediates into growing glycan chains. While their mode of action is well-established for many eukaryotic glycoproteins, the data available from prokaryotic glycoproteins is still scarce. The aim of the proposed research project is the expression and functional characterization of the glycosyltransferases involved in the assembly and translocation of the S-layer glycan chains of the S-layer glycoprotein from Geobacillus stearothermophilus NRS 2004/3a. For this organism and also for related Aneurinibacillus thermoaerophilus strains, now the first completely sequenced slg (surface layer glycosylation) gene clusters are available. Although the glycan chains of G. stearothermophilus NRS 2004/3a S-layer protein SgsE consist only of L-rhamnose-containing homopolymers, different rhamnosyltransferases have been identified in the slg gene cluster by data base sequence comparison. Therefore, the characterization of their specificities is of major importance for the understanding of the entire glycan assembly process. Since L-rhamnose is not present in higher organisms, the corresponding transferases may be interesting canditates for the development of defense strategies against prokaryotes with rhamnose-containing polysaccharides. Motif search in the deduced amino acid sequences of other genes from the slg gene cluster of G. stearothermophilus NRS 2004/3a indicated the presence of different glycosyltransferase domains. These putative proteins might be involved in chain length determination and translocation process of the S-layer glycans across the cytoplasmic membrane. The presence of an ATP-dependent ABC-2 transporter system implies growth of the S- layer glycan at the non-reducing end of the chain. Since the investigated Gram-positive bacteria of interest have not been transformable so far, the expression and functional characterization of the putative gene products will be performed in heterologous systems such as Escherichia coli and Lactococcus lactis. In addition, exploratory experiments are planned for a modification of the genuine glycan structure of the S layer glycoprotein of G. stearothermophilus NRS 2004/3a. The proposed investigations are expected to provide eventually foundations for the production of modified S-layer glycoproteins by "carbohydrate engineering" that are useful for nanobiotechnological and biomedical applications.