Heptose phosphate ligands

Lipopolysaccharides (LPS) located in the outer leaflet of the Gram-negative bacterial cell membrane are involved in many biomedically important host-microbe interactions. In numerous bacterial species, heptoses of the glycero- D-manno-configuration are frequently found constituents of the inner core region of LPS and constitute a relatively conserved structural domain with several potential cross-reactive epitopes and binding sites. Binding of these heptoses to core-reactive antibodies and mannose-binding lectins, respectively, has been described, but detailed analysis of the binding motifs involved in the recognition of the complex phosphorylated triheptoside core region, however, has not been accomplished thus far. In addition to the pyranose ring hydroxy groups, the side-chain entity of manno-configured heptoses provides additional attachments sites for specific interaction with proteins. The project is focussed to elucidate the structural and molecular basis of heptose and heptose- phosphate binding by selected components of the innate and adaptive immune system. Specifically, the binding of human lung-surfactant protein D (hSP-D), a member of the C-type lectin family, which regulates the innate immune response of the lung will be studied using synthetic phosphorylated heptosyl core oligosaccharides derived from the Escherichia coli J5- mutant. Compounds will be used to characterize the epitope specificity of the highly cross-reactive and protective monoclonal antibody WN1 222-5 binding to the LPS core region from E. coli, Salmonella enterica, Shigella and Citrobacter strains. In particular, the individual contribution of the phosphate groups at the 4 position of the heptose residues in the structurally conserved triheptoside unit will be addressed as well as the contribution of Kdo and parts of the lipid A region to the bioactive conformation of these epitopes. The selection of target compounds will focus on those oligosaccharides, which are not accessible from native bacterial sources. For the assembly of the complex oligosaccharides novel heptose trifluoroacetimidate donors will be elaborated and glycosylation efficiency will be explored using a continuous microfluid reactor. The compounds have been planned as allyl glycosides to be eventually equipped with thiol-containing spacer entities allowing for subsequent utilization for production of multivalent glycomaterials as well as for the preparation of neoglycoproteins. Binding studies will comprise immunochemical techniques, surface plasmon resonance, isothermal microcalorimetry, cocrystallization/soaking experiments of the ligands with the carbohydrate recognition domain of SP-D and the antibody WN1 222-5 and will be complemented by molecular modeling, TrNOE and STD-NMR experiments, respectively. Binding and crystallographic studies of native SP-D and selected mutants with synthetic oligosaccharides will be performed with E. Crouch (Washington State University, USA) and J. Head (Boston University, USA), whereas the work on mAb WN1 222-5 will be done in collaboration with S. Evans (Univ. Victoria, Canada) and H. Brade, S. Müller- Loennies (Research Center Borstel, Germany). The outcome of the project should contribute to a better understanding of protein binding to charged complex carbohydrates, provide the molecular basis for future vaccine development and contribute to improved therapies of clinically relevant infections including the treatment of sepsis.

The main results of the project have led to a deepened understanding of the interaction of bacterial cell-surface carbohydrates with proteins, thereby providing a rational basis for the development of vaccines and for improved therapies of clinically relevant infections. The outer leaflet of the Gram-negative bacterial cell membrane contains lipid-bound carbohydrates (lipopolysaccharides) exerting numerous interaction with components of the innate and adaptive immune system. Within the project, a structurally conserved region of lipopolysaccharide was addressed, containing higher-carbon sugars with 7 and 8 carbons, respectively (heptoses and 3-deoxy-octulosonic acid = Kdo), in order to define the binding to antibodies and lectins in molecular detail. Thus, phosphate-containing carbohydrate ligands were synthesized via an improved methodology, which then served as probes for structural studies (X-ray crystallography and nuclear magnetic resonance spectroscopy). Specifically, the binding of human lung-surfactant protein D (hSP-D), a member of the C-type lectin family, which regulates the innate immune response of the lung, was studied. It could be shown that bacterial heptoses harbor several potential binding sites (in the ring and side chain), which are recognized both by SP-D but also by bacterial lectins from Burkholderia. Whereas the binding by SP-D leads to an efficient aggregation and elimination of microbial pathogens, binding to Burkholderia lectins is relevant in the context of biofilm formation. A specific Burkholderia lectin may even be termed a super lectin since it displays two different sugar binding specificities allowing for the attachment to epithelial cells as well as growth of bacteria on cell surfaces. This work has been performed in close cooperation with groups in the US and in Europe (France, Czech Republic, Italy).Within the framework of cooperation with groups from Canada and Germany, a cross-reactive and protective antibody (WN1 222-5) could be crystallized with a bacterial dodecasaccharide. The crystal structure of the ligand in the binding site of the antibody closely resembles a related three-dimensional arrangement of lipopolysaccharide bound to the Toll like receptor 4, which now explains the neutralizing activity of the antibody due a competitive binding to the bacterial core region. Detailed binding studies are still ongoing. In summary it can be stated, that many Gram-negative bacteria carry a structurally and conformationally conserved inner carbohydrate domain, being accessible and involved in biomedically relevant interactions with carbohydrate-binding proteins.