A novel cobra venom metalloproteinase, mocarhagin, cleaves a 10-amino acid peptide from the mature N terminus of P-selectin glycoprotein ligand receptor, PSGL-1, and abolishes P-selectin binding.
Initial rolling of circulating neutrophils on a blood vessel wall prior to adhesion and transmigration to damaged tissue is dependent upon P-selectin expressed on endothelial cells and its specific neutrophil receptor, the P-selectin glycoprotein ligand-1 (PSGL-1). Pretreatment of neutrophils, HL60 cells, or a recombinant fucosylated soluble form of PSGL-1 (sPSGL-1.T7) with the cobra venom metalloproteinase, mocarhagin, completely abolished binding to purified P-selectin in a time-dependent and EDTA- and diisopropyl fluorophosphate-inhibitable manner consistent with mocarhagin selectively cleaving PSGL-1. A polyclonal antibody against the N-terminal peptide Gln-1-Glu-15 of mature PSGL-1 immunoprecipitated sPSGL-1.T7 but not sPSGL-1.T7 treated with mocarhagin, indicating that the mocarhagin cleavage site was near the N terminus. A single mocarhagin cleavage site between Tyr-10 and Asp-11 of mature PSGL-1 was determined by N-terminal sequencing of mocarhagin fragments of sPSGL-1.T7 and is within a highly negatively charged amino acid sequence 1-QATEYEYLDY decreases DFLPETEPPE, containing three tyrosine residues that are consensus sulfation sites. Consistent with a functional role of this region of PSGL-1 in binding P-selectin, an affinity-purified polyclonal antibody against residues Gln-1-Glu-15 of PSGL-1 strongly inhibited P-selectin binding to neutrophils, whereas an antibody against residues Asp-9-Arg-23 was noninhibitory. These combined data strongly suggest that the N-terminal anionic/sulfated tyrosine motif of PSGL-1 as well as downstream sialylated carbohydrate is essential for binding of P-selectin by neutrophils.
De Luca
M
M
Vascular Biology Laboratory, Baker Medical Research Institute, Prahran, Victoria, Australia.
Dunlop
L C
LC
Andrews
R K
RK
Flannery
J V
JV
Ettling
R
R
Cumming
D A
DA
Veldman
G M
GM
Berndt
M C
MC
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D004546:Elapid Venoms / Q000378:metabolism* / Q000494:pharmacology; D006801:Humans; D066298:In Vitro Techniques; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism*; D008666:Metalloendopeptidases / Q000378:metabolism* / Q000494:pharmacology; D008969:Molecular Sequence Data; D009504:Neutrophils / Q000187:drug effects / Q000378:metabolism; D019007:P-Selectin / Q000378:metabolism*; D013379:Substrate Specificity
D004546:Elapid Venoms; D008562:Membrane Glycoproteins; D019007:P-Selectin; C084882:P-selectin ligand protein; D008666:Metalloendopeptidases; C099092:mocarhagin