Cell-specific peptide binding by human neutrophils.
Analysis of peptide binding to human neutrophils (PMN) using phage display techniques has revealed cell-specific motifs reactive with the PMN surface. Phage libraries displaying either linear 9-mer or cyclic 10-mer and 6-mer peptides were incubated with normal human neutrophils followed by elution of bound phage with low pH (pH 2.2) and non-ionic detergent. Three rounds of selection generated several related peptide sequences that bound with high avidity to PMN. Using the linear 9-mer library, PMN-binding phage expressed peptides with the motif (G/A)PNLTGRW. The binding of phage bearing this motif was highly specific since no binding was observed on lymphocytes, fibroblasts, epithelial, or endothelial cells. Functional assays revealed that phage bearing the sequence FGPNLTGRW induced a pertussis toxin-sensitive increase in PMN cytosolic calcium analogous to that observed with Galphai coupled receptors. Other prominent motifs identified included phage bearing the consensus DLXTSK(M/L)X(V/I/L), where X represents a non-conserved position. Phage with this motif bound exclusively to a sub population of human PMN that comprised approximately 50% of the total and did not elicit a calcium response. The binding of such phage to PMN was prevented by co-incubation with competing peptides displaying identical or similar sequences (IC50 range from 0.6 micromol/L to 50 micromol/L for DLXTSK and GPNLTG, respectively). We speculate that these techniques will be useful in identifying functional cell-specific binding motifs and contribute to the development of new therapeutic and diagnostic strategies in human disease.
Department of Pathology, Brigham and Women's Hospital and Harvard Medical School, Boston, MA, USA.
L
L
Mazzucchelli
J B
JB
Burritt
A J
AJ
Jesaitis
A
A
Nusrat
T W
TW
Liang
A T
AT
Gewirtz
F J
FJ
Schnell
C A
CA
Parkos
D000595:Amino Acid Sequence; D001435:Bacteriophages; D002118:Calcium / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D009504:Neutrophils / Q000378:metabolism*; D019151:Peptide Library; D010455:Peptides / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D017421:Sequence Analysis
D019151:Peptide Library; D010455:Peptides; D002118:Calcium