Peptabodies as tools to test ligands isolated from phage-displayed peptide libraries.

We have previously isolated filamentous bacteriophages, expressing linear hexa-peptides and homing to bone marrow endothelium (BME), by panning in vivo of a phage-displayed peptide library in mice. Here, we used peptabody fusion proteins to test the binding capacity of the hexa-peptide SSLTTG to BME cells in vitro. To display this motif in a multimeric form, as originally presented on the bacteriophage, we expressed it N-terminally as a fusion with the peptabody cartilage oligomeric matrix assembly protein (COMP) pentamerization domain, either alone or followed by the N1 domain of the pIII phage coat protein. Binding of the peptabody constructs to the mouse BME cell line STR-10 was investigated by immunofluorescence using anti-COMP antibodies. Only peptabody fusion proteins co-expressing pIII-N1 exhibited binding to STR-10, regardless of the presence or absence of SSLTTG. These results indicate that the phage coat protein pIII-N1 domain is the principle determinant responsible for the binding of filamentous bacteriophages to cells of the reticulo-endothelial system (RES). Peptabodies expressing pIII-N1 did not bind to the osteoblast-like cell line MC3T3-E1, indicating that binding is mediated by receptors specifically expressed by BME cells in vivo. Polyinosinic acid (poly-I) was able to inhibit binding of bacteriophages and pIII-N1 expressing peptabodies to STR-10, confirming our previous studies showing that bacteriophages bind to scavenger receptors (SR) expressed by BME cells. In summary, the present study shows the usefulness of peptabodies as a general tool to test the binding capacity of peptide ligands identified by phage display.