Dr. Robert Evans
King's College
University of London

Identification of putative receptor-binding regions of human transferrin and lactoferrin for mammalian and bacterial receptors

Infant mortality rates remain disproportionately higher in certain minority groups. Bacterial infections contribute to this high level of incidence and mortality. Iron uptake is an essential factor in pathogenicity of bacteria, being necessary for the bacterial cell multiplication. Understanding th different mechaisms that allow bacteria to provide iron inside the human host will help developing therapeutic strategies to combat infectious diseases.

In the vast majority of mammalian cell types, cellular acquisition of iron is from transferrin via a receptor-mediated endocytic pathway. Although iron is an essential element for most micro-organisms, it is not readily available in biological systems so that its acquisition is necessary for infection to occur. In recent years more and more micro-organisms, including meningococci and staphylococci, have been found to express surface receptors capable of scavenging the host's transferrin- and lactoferrin-bound iron, without internalisation of the iron-bound protein. The crystollagraphic structures of both hTf and hLf have been solved, however, the exact nature of the receptor-binding regions on the proteins for their mammalian and bacterial receptors have yet to be defined. No structural information is available for the mammalian and bacterial hTf and hLf receptors, although the crystal structures of FhuA and FepA of E. Coli hve been published very recently and show both receptors to comprise a 22-strand b barrel obstructed by a plug. As the meningococcal TbpA displays amino acid sequence homology with FhuA and FepA it is likely to have a similar structure. Work is therefore in progress within the group to predict a model for TbpA which will be used in our studies of its interaction with hTf.

It is now our intention to use protein mapping technology to identify putative receptor-binding regions on hTf and hLF. Two series of overlapping peptides, corresponding to the complete amino acid sequences of hTf and hLf, will be synthesized on cellulose membranes. The Peptide libraries will then be tested for their ability to bind transferrin- and lactotransferrin-receptor preparations. Peptides which display receptor-binding properties will be mapped on the 3-D structures of hTf and hLf. Subsequently, peptides corresponding to potential surface receptor-binding regions will be synthesized for competitive binding assays using the group’s surface plasmon resonance (SPR) facility. In the longer-term, the effects of mutations in the putative receptor receptor-binding regions of recombinant hTf and hLf will be investigated using SPR.

Information gained from these studies can be exploited to enhance transferrin-mediated targeting of molecules into cells and to develop therapeutic strategies to combat infectious diseases, especially as the meningococcal Tbps, which will be made available to use by our long-term collaborator, Dr. Andrew Gorringe (Centre for Applied Microbiology and Research, Salisbury), are candidate vaccines against serogroup B meningococcal disease. The results of the proposed project will complement studies within the group to characterise the iron-uptake system of N. meningitidis: the amino acid sequence of TbpA displays homology with FepA, the ferric enterobactin receptor, and FhuA, the ferrichrome receptor, and work is now in progress to predict a new model for TbpA based on the recently solved crystal structures of these receptors.