Identification of sequences in human transferrin that bind to the bacterial receptor protein, transferrin-binding protein B.
Alignment of amino-acid sequences from the N-terminal and C-terminal halves of transferrin-binding protein B revealed an underlying bilobed nature with several regions of identity. Based on this analysis, purified recombinant fusion proteins of maltose-binding protein (Mbp) with intact TbpB, its N-terminal half or C-terminal half from the human pathogens Neisseria meningitidis and Moraxella catarrhalis were produced. Solid-phase binding assays and affinity isolation assays demonstrated that the N-terminal and C-terminal halves of TbpB could bind independently to human transferrin (hTf). A solid-phase overlapping synthetic peptide library representing the amino-acid sequence of hTf was probed with soluble, labelled Mbp-TbpB fusions to localize TbpB-binding regions on hTf. An essentially identical series of peptides from domains within both lobes of hTf was recognized by intact TbpB from both organisms, demonstrating a conserved TbpB-hTf interaction. Both halves of TbpB from N. meningitidis bound the same series of peptides, which included peptides from equivalent regions on the two hTf lobes, indicating that TbpB interacts with each lobe of hTf in a similar manner. Mapping of the peptide-binding regions on a molecular model of hTf revealed a series of nearly adjacent surface regions that nearly encircled each lobe. Binding studies with chimeric hTf/bTf transferrins demonstrated that regions in the C-lobe of hTf were preferentially recognized by the N-terminal half of TbpB. Collectively, these results provide evidence that TbpB consists of two lobes, each with distinct yet homologous Tf-binding regions.
Retzer
M D
MD
Department of Microbiology and Infectious Diseases, University of Calgary, Alberta, Canada. retzer@acs.calgary.ca
Yu
R H
RH
Schryvers
A B
AB
D002352:Carrier Proteins / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D033862:Iron-Binding Proteins; D008958:Models, Molecular; D001936:Moraxella catarrhalis / Q000737:chemistry; D009345:Neisseria meningitidis / Q000737:chemistry; D019151:Peptide Library; D011485:Protein Binding; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D014168:Transferrin / Q000737:chemistry* / Q000378:metabolism; D033882:Transferrin-Binding Protein B; D033863:Transferrin-Binding Proteins; D001616:beta-Galactosidase / Q000378:metabolism
D002352:Carrier Proteins; D033862:Iron-Binding Proteins; D019151:Peptide Library; D011993:Recombinant Fusion Proteins; D014168:Transferrin; D033882:Transferrin-Binding Protein B; D033863:Transferrin-Binding Proteins; D001616:beta-Galactosidase