Genetic ´Carbohydrate Engineering´ of S-layer glycoproteins from Gram-positive eubacteria

Research project P 14209 Genetic "Carbohydrate Engineering" of S-Layer Glycoproteins Paul MESSNER 06.05.2000 The goal of the proposed research project is the production for the first time, of a bacterial S-layer glycoprotein which comprises an artificial glycan chain altered by molecular genetic methods. In addition, we propose to lay the groundwork for the subsequent design of genetically engineered bacterial glycoproteins comprising the glycan chains of capsular polysaccharides or mammalian proteoglycans. These are important target structures for use as innovative vaccines and biocompatible matrices. This work is scheduled for a duration of 3 years. The Gram-positive, thermophilic eu-bacteria Aneurinibacillus thermoaerophilus L420-91T and GS4-97, as well as Bacillus stearothermophilus NRS 2004/3a have been chosen as model organisms. The glycan chains of the S-layer glycoproteins from A. thermoaerophilus strains L420-91T and GS4-97 consist of identical repeats of four D-rhamnose units as a backbone. Two neighbouring D-rhamnose units are substituted with one 3-N-acetyl-D-fucosamine unit, each. The glycans of B. stearothermophilus, for comparison, comprise linear poly-L-rhamnan chains. The aim of the proposal is the specific modification of the S-layer glycoprotein glycan chain from A. thermoaerophilus by "carbohydrate engineering" to create an artificial S-layer neoglycoprotein comprising a linear D-rhamnan chain without the branching 3-N-acetyl-D-fucosamine units. Such experiments using prokaryotic glycoproteins have not been pursued so far. As an important prerequisite, cloning and sequencing of the gmd and rmd genes involved in the biosynthesis of GDP-D-rhamnose should be accomplished within the first year. In addition, assays for the biosynthetic enzymes of the 3-N-acetyl-D-fucosamine pathway need to be developed. In year two, the genes encoding the synthesis of the amino sugar should be cloned and sequenced. At this time the molecular characterization of the S-layer protein genes satA and satB of A. thermoaerophilus as well as sbsD of B. stearothermophilus should be finished. These data will be used to identify the position of the "S-layer glycan biosynthesis" cluster of A. thermoaerophilus in relation to the position of the S-layer protein genes satA/satB on a physical map. In year three, the glycan structures of the S-layer glycoproteins of A. thermoaerophilus will be modified by insertional mutagenesis so that, in the absence of 3-N-acetyl-D-fucosamine precursors, novel linear poly-D-rhamnan chains should be obtained instead of branched glycans. Bacillus stearothermophilus, however, should synthesize a non-glycosylated S-layer protein after equivalent mutagenesis. For specific applications of S- layer glycoproteins, the formation of uniform surface structures is of great importance. Therefore, the enzymes catalyzing the polymerization of the glycan repeating units will also be investigated in year three. Characterization of the topologyy of the glycosylation reactions will be accomplished by immunoelectron microscopy using antibody-labelled preparations. The proposed investigations are expected to provide important foundations for the production of specifically designed S-layer neoglycoproteins useful in biomedical and nanotechnological applications.

Gram-positive bacteria are frequently covered by regularly arranged protein crystals, the so-called surface layer (S- layer) proteins. In a few organisms these S-layer proteins are glycosylated which means that the glycan chains are covalently attached to the S-layer protein. In previous projects the chemical structure of S-layer glycan chains of selected strains has been determined. In the current project it was planned to determine the genes involved in the biosynthesis of these glycan chains and, in addition, to functionally characterize the overexpressed enzyme proteins. This task is required for developing strategies for specific "carbohydrate engineering", which means to influence and change the existing glycan structure of the S-layer glycoproteins by genetic means. A prerequisite of this endeavour is the complete analysis of S-layer glycan glycosylation clusters of several bacterial strains. Selected genes should be knocked out and new genes should be integrated into the organisms by using specific genetic methods. However, we experienced some problems with the transformability of the thermophilic bacteria in use which could not be solved yet. So we focused our efforts on cloning and sequencing of the genes from the glycan glycosylation cluster of the thermophilic Gram-positive bacteria in use and on functionally characterizing the encoded carbohydrate-processing enzyme proteins. In addition, a model for explaining the biosynthesis of S-layer glycoprotein glycans in Gram- positive bacteria was developed. "Carbohydrate engineering", the main topic of the project, is focused on a future application of glycosylated prokaryotic S-layer glycoproteins with defined carbohydrate structures or even S-layer neoglycoproteins with novel glycan structures in different areas of nanobiotechnology, biomimetics and biomedicine.