Production and Characterization of Chimeric Transferrins for the Determination of the Binding Domains for Bacterial Transferrin Receptors

Abstract

Pathogenic bacteria in the Neisseriaceae and Pasteurellaceae possess outer membrane proteins that specifically bind transferrin from the host as the first step in the iron acquisition process. As a logical progression from prior studies of the ligand-receptor interaction using biochemical approaches, we have initiated an approach involving the production of recombinant chimeric transferrins to further identify the regions of transferrin involved in receptor binding. In order to prepare bovine/human hybrids, the bovine transferrin gene was cloned, sequenced, and compared with the existing human transferrin gene sequence. After identification of potential splice sites, hybrid transferrin genes were constructed using the polymerase chain reaction-based approach of splicing by overlap extension. Five hybrid genes containing sequences from both bovine and human transferrin were constructed. Recombinant transferrins were produced in a baculovirus expression vector system and affinity-purified using concanavalin A-Sepharose. The recombinant proteins were analyzed for reactivity against polyclonal and monoclonal antibodies and assessed for binding to Neisseria meningitidis transferrin receptor proteins in solidphase binding assays and affinity isolation experiments. These experiments enabled us to localize the regions of human transferrin predominantly involved in binding to the N. meningitidis receptor to amino acid residues 346 -588. The construction of these chimeras provides unique tools for the investigation of transferrin binding to receptors from both human and bovine bacterial pathogens.

Iron Transport Systems in Neisseria meningitidis

“…This interaction is not dependent on the presence of carbohydrate chains on transferrin, indicating that it is a direct protein-protein interaction (108). Recombinant human-bovine transferrin hybrid proteins were designed to determine regions of contact with the transferrin receptor (127). Amino acid residues (aa) 346 to 588, within the carbox-yl-terminal end of transferrin, were found to be responsible for interacting with the meningococcal receptor; in addition, a region very near the carboxyl terminus has an influence on the avidity of the interaction (127).…”

Identification of human transferrin-binding sites within meningococcal transferrin-binding protein B

“…An amino acid stretch (residues 346 to 588) within the C lobe of the hTf was identified as being involved in the interaction. It was further demonstrated that the C tail of this C lobe influenced the avidity of binding to the bacterial receptor (30). No information was available on the regions involved within TbpB.…”

Peptide-Peptide Interactions between Human Transferrin and Transferrin-Binding Protein B from Moraxella catarrhalis

“…A prior study demonstrated that at least six regions on each hTf lobe bound to TbpB proteins from N. meningitidis and M. catarrhalis (34). This finding infers that each lobe of TbpB should possess six complementary binding regions.…”

“…Our results may also suggest that, in spite of the potential limitations, this approach could be used more extensively for the study of protein-protein interactions. It is noteworthy to mention that due to the number of sites involved in the interaction (34,35), alternative approaches, such as the use of chimeric proteins or site-directed mutagenesis, would not have been able to yield such detailed information.…”