Abstract
The screening of phage peptide libraries resulted in the identification of a sequence (named NW peptide, NWYLPWLGTNDW) that specifically binds to human monocytes and macrophages. Although the NW peptide can be used for the targeted delivery of therapeutics without knowledge of its receptor(s), the identification of-its binding partners will support future clinical applications-Here, we used the biotinylated NW peptide for cross-linking cell surface receptor(s) on live cells or as bait in pull-down assays with membrane proteins isolated from monocytes or human THP-1 cells differentiated into macrophages. Proteomic analysis of the captured proteins identified cell surface prohibitins (PHB1 and PHB2) and modified albumin as binding partners. Using flow cytometry and pull-down methods, we demonstrated that PHB1 and PHB2 interact directly with the NW peptide. Confocal imaging showed co-localization of the peptide with PHB1 on the surface of monocytes. Single replacement of either tryptophan or leucine with alanine completely inhibited binding, whereas the replacement of asparagine at position 1 or 10 and aspartic acid at position 11 with alanine did not affect the binding of the peptide variants. Neutral amino acid replacement of tryptophan at positions 2, 6, and 12 with tyrosine or phenylalanine also abolished the binding, implying that the indole ring of tryptophan is indispensable for the NW peptide to bind. Overall, the data suggest that membrane-associated prohibitins might be a useful target for the delivery of therapeutics to monocytes/macrophages and that tryptophan and leucine are key residues for peptide binding.