Characterization of bitiscetin-2, a second form of bitiscetin from the venom of Bitis arietans : comparison of its binding site with the collagen-binding site on the von Willebrand factor A3-domain.
BACKGROUND
Bitiscetin, a heterodimeric snake venom protein purified from Bitis arietans, binds to the A1 domain of von Willebrand factor (VWF) and induces binding of this domain to platelet glycoprotein (GP) Ib. We previously purified a distinct form of dimeric bitiscetin (herein called bitiscetin-2) that also induces the VWF A1 domain-GPIb interaction, but does not bind to the A1 domain. Instead, it interacts with the collagen-binding A3 domain of VWF.


METHODS
In the current study we identify the amino terminal sequence of the bitiscetin-2 as DEGCLPDDSSRT, showing conclusively that the protein is distinct form the originally described bitiscetin. We further studied the interaction of bitiscetin-2 and VWF using DeltaA3 VWF and a series of 33 VWF point mutants previously prepared to map the collagen-binding site.


RESULTS
Our results confirm that DeltaA3 VWF, even though containing the A1-domain, is unable to interact with bitiscetin-2. Mutation of VWF-A3 residues Ile975, Asp979, Pro981, Ser1020 and His1023 reduces binding by 80% while mutation of residues Val980, Glu1001 and Arg1021 reduces binding by 30-60%. A 2- to 6-fold increase of binding is caused by mutation of residues Val985, Glu987, and Arg1016.


CONCLUSIONS
Nearly all of these mutations also affect collagen binding showing that the binding sites for bitiscetin-2 and collagen type III in the VWF-A3 domain closely overlap.
Obert
B
B
INSERM U770 and Faculté de médecine IFR93, Université Paris-Sud, 80 rue du Général Leclerc, 94276 Le Kremlin-Bicêtre, France.
Romijn
R A
RA
Houllier
A
A
Huizinga
E G
EG
Girma
J P
JP
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D003094:Collagen / Q000378:metabolism; D010455:Peptides / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019038:Platelet Glycoprotein GPIb-IX Complex / Q000378:metabolism; D017354:Point Mutation; D011485:Protein Binding / Q000235:genetics; D017434:Protein Structure, Tertiary; D012910:Snake Venoms; D014757:Viper Venoms / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017819:Viperidae; D014841:von Willebrand Factor / Q000737:chemistry* / Q000378:metabolism
D010455:Peptides; D019038:Platelet Glycoprotein GPIb-IX Complex; D012910:Snake Venoms; D014757:Viper Venoms; C513916:bitiscetin-2, Bitis arietans; D014841:von Willebrand Factor; C062186:bitistatin; D003094:Collagen