Residues F16-G33 and A784-N823 within platelet thrombospondin-1 play a major role in binding human neutrophils: evaluation by two novel binding assays.
The thrombospondin-1 (TSP1) structural requirements within its heparin-binding domain (HBD)(30 kd) or within the other domains of the molecule (450 kd) that interact with neutrophils (PMNs) have not been delineated. Synthetic peptides based on the HBD, a TSP1 proteolytic fragment lacking the HBD, a large C-terminal domain of TSP1 (210 kd), a TSP1 recombinant fragment (rTSP1(784-932)), and a monoclonal antibody directed against the TSP1 type 3 repeats (mAb D4.6) were utilized to map such structural requirements on TSP1. Synthetic peptides containing a heparin-binding motif and encompassing residues F16-G33 or A74-S95 of TSP1 competed quantitatively with iodine 125-labeled TSP1 for binding to heparinagarose beads. However, only F16-G33 was a competitor of TSP1 binding to PMNs, suggesting that the sequence F16-G33 within the HBD plays a role in PMN binding. The interaction site within the 450-kd fragment was further narrowed. A TSP1 -derived proteolytic fragment (210 kd), a recombinant TSP1 fragment (rTSP1(784-932)), and a type 3 repeat anti-TSP1 monoclonal antibody (mAb D4.6) competed for the binding of 125I-labeled TSP1 to PMNs. The N-terminal of rTSP1(784-932) and C-terminal sequence analysis of TSP1-210 kd delineated the structural requirements for the second binding region for PMNs-namely, residues A784-N823.
Majluf-Cruz
A
A
Sol Sherry Thrombosis Research Center, Department of Physiology, Temple University School of Medicine, Philadelphia, USA.
Manns
J M
JM
Uknis
A B
AB
Yang
X
X
Colman
R W
RW
Harris
R B
RB
Frazier
W
W
Lawler
J
J
DeLa Cadena
R A
RA
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D001665:Binding Sites / Q000187:drug effects / Q000276:immunology; D001792:Blood Platelets / Q000737:chemistry / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism; D002634:Chemotaxis, Leukocyte / Q000187:drug effects / Q000276:immunology; D005343:Fibrinolytic Agents / Q000378:metabolism / Q000494:pharmacology; D016650:Fluorescein-5-isothiocyanate; D005456:Fluorescent Dyes; D006493:Heparin / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D007457:Iodine Radioisotopes; D008969:Molecular Sequence Data; D009504:Neutrophils / Q000166:cytology / Q000378:metabolism*; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000494:pharmacology; D010449:Peptide Mapping; D017434:Protein Structure, Tertiary; D012680:Sensitivity and Specificity; D019700:Thrombospondin 1 / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D005343:Fibrinolytic Agents; D005456:Fluorescent Dyes; D007457:Iodine Radioisotopes; D010446:Peptide Fragments; D019700:Thrombospondin 1; D006493:Heparin; D016650:Fluorescein-5-isothiocyanate; D002118:Calcium