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 solid-phase 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.
Highlights
Pathogenic bacteria in the Neisseriaceae and Pasteurellaceae possess outer membrane proteins that bind transferrin from the host as the first step in the iron acquisition process
Studies of isogenic mutants that lack the expression of Tbp1 and/or Tbp2 have shown that both proteins play an important role in the effective binding of transferrin (6 –9)
The N-terminal and C-terminal lobes of Tf, which are similar in amino acid sequence and tertiary structure, are connected by an interdomain bridge that varies in length between different transferrin species
Summary
Transferrin; hTf, human Tf; bTf, bovine Tf; rTf, recombinant Tf; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; TBS, Tris-buffered saline; mAb, monoclonal antibody. Based on comparative amino acid sequence analysis, the Tbp polypeptide is predicted to be an integral outer membrane protein, while Tbp appears to be anchored to the outer membrane via an N-terminal lipid anchor [8, 10, 11] The specificity of these receptors for iron binding proteins of only their hosts is a characteristic feature of this mode of iron acquisition [12]. Protease cleavage may not necessarily result in dissociation of the proteolytic subfragments, and subsequent attempts at separating and isolating the resultant peptides (involving denaturing conditions and cleavage of disulfide bridges) may result in irreversible loss of conformation that is essential for the binding interaction These factors may be responsible for the lack of success in preliminary attempts at obtaining further subfragments of hTf that retain sufficient binding avidity for detection in the binding and affinity isolation. By combining domains of a host transferrin with domains from an alternate species, the regions required for interacting with the bacterial receptor complex can be identified
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
