Monoclonal antibodies that inhibit binding of propolypeptide of von Willebrand factor to collagen. Localization of epitopes.
We reported previously that bovine propolypeptide of von Willebrand factor (pp-vWF) binds to type I collagen. To determine the collagen-binding sites of pp-vWF we generated monoclonal antibodies (mAbs) against bovine pp-vWF. One mAb, designated TC8, very strongly inhibited the binding of pp-vWF to type I collagen; three other mAbs, designated TC2, TC6 and TC7, exhibited moderate inhibition. Competition between the mAbs for binding to intact pp-vWF revealed that the epitope for TC8 was structurally independent of that for TC6 and TC7. To determine directly the location of the epitope for each mAb on the bovine pp-vWF molecule, we tested the reactivity of mAbs by immunoblotting toward peptide fragments obtained by digestion with lysylendopeptidase. TC2 and TC8 recognized a fragment of mass 21 kDa, while TC6 and TC7 recognized a distinct fragment of 18 kDa. These two fragments were purified to homogeneity and their N-terminal amino acid sequences were determined. Comparing these sequences with the sequence of human pp-vWF, the locations of these fragments in the primary structure were estimated to be Phe570--Lys682 for the 21-kDa fragment and Glu281--Lys375 for the 18-kDa fragment. These data suggest that pp-vWF contains at least two collagen-binding sites which lie within or close to the regions between Phe570--Lys682 and Glu281--Lys375.
Fujisawa
T
T
Department of Biological Sciences, Faculty of Bioscience and Biotechnology, Tokyo Institute of Technology, Japan.
Takagi
J
J
Sekiya
F
F
Goto
A
A
Miake
F
F
Saito
Y
Y
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D003094:Collagen / Q000378:metabolism*; D000939:Epitopes / Q000032:analysis*; D005260:Female; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D014841:von Willebrand Factor / Q000276:immunology / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D000939:Epitopes; D014841:von Willebrand Factor; D003094:Collagen