Sulfation of Tyr1680 of human blood coagulation factor VIII is essential for the interaction of factor VIII with von Willebrand factor.
The acidic region of the Factor VIII light chain was studied with regard to structural requirements for the formation of a functional von Willebrand factor (vWF)-binding site. Factor VIII mutants lacking the B domain, with additional deletions and an amino acid replacement within the sequence 1649-1689 were constructed using site-directed mutagenesis and expressed in Cos-1 cells. These mutants, which were recovered as single-chain molecules with similar specific activities, were compared in their binding to immobilized vWF. Deletion of amino acids 741-1648 or 741-1668 did not affect the binding of Factor VIII to vWF. However, a mutant with a deletion of residues 741-1689 was no longer capable of interacting with vWF. This indicates a role for residues within the sequence 1669-1689 in the formation of a vWF-binding site. When recombinant Factor VIII was expressed in the presence of chlorate, an inhibitor of protein sulfation, the resulting Factor VIII displayed strongly reduced binding to vWF. vWF binding was completely abolished when within the sequence 1669-1689 the tyrosine residue Tyr1680, which is part of a consensus tyrosine sulfation sequence, was replaced by phenylalanine. The Factor VIII sequence 1673-1689 was identified as a high affinity substrate for tyrosylprotein sulfotransferase (Km = 57 microM) in cell-free sulfation studies. It is concluded that sulfation of Tyr1680 is required for the interaction of Factor VIII with vWF. Two synthetic peptides that represent the sequence 1673-1689, but differ with respect to sulfation of Tyr1680 are shown to have vWF binding affinity that is considerably lower than the Factor VIII protein. Several models to accommodate our findings are discussed.
Leyte
A
A
Department of Blood Coagulation, Central Laboratory of The Netherlands Red Cross Blood Transfusion Service, Amsterdam.
van Schijndel
H B
HB
Niehrs
C
C
Huttner
W B
WB
Verbeet
M P
MP
Mertens
K
K
van Mourik
J A
JA
D001667:Binding, Competitive; D046508:Culture Techniques; D004247:DNA / Q000235:genetics; D005169:Factor VIII / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008715:Methionine; D009154:Mutation; D010957:Plasmids; D013464:Sulfuric Acids; D014162:Transfection; D014443:Tyrosine / Q000378:metabolism*; D014841:von Willebrand Factor / Q000235:genetics / Q000378:metabolism*
D013464:Sulfuric Acids; D014841:von Willebrand Factor; D014443:Tyrosine; D005169:Factor VIII; D004247:DNA; D008715:Methionine