Processing and characterization of recombinant von Willebrand factor expressed in different cell types using a vaccinia virus vector.
The cloning of the cDNA encoding von Willebrand factor (vWF) has revealed that it is synthesized as a large precursor (pre-pro-vWF) molecule and it is now clear that the prosequence or vWAgII is responsible for the intracellular multimerization of vWF. We have cloned the complete vWF cDNA and expressed it using a recombinant vaccinia virus as vector. We have characterized the structure and function of the recombinant vWF (rvWF) secreted from five different cell types: baby hamster kidney (BHK), Chinese hamster ovary (CHO), human fibroblasts (143B), mouse fibroblasts (L) and primary embryonic chicken cells. Forty-eight hours after infection, the quantity of vWF antigen found in the cell supernatant varied from 3 to 12 U/dl depending on the cell type. By SDS-agarose gel electrophoresis, the percentage of high molecular weight forms of vWF varied from 39 to 49% relative to normal plasma for BHK, CHO, 143B and chicken cells but was less than 10% for L cells. In all cell types, the two anodic subbands of each multimer were missing. The two cathodic subbands were easily detected only in BHK and L cells. By SDS-PAGE of reduced samples, pro-vWF was present in similar quantity to the fully processed vWF subunit in L cells, present in moderate amounts in BHK and CHO and in very low amounts in 143B and chicken cells.(ABSTRACT TRUNCATED AT 250 WORDS)
Meulien
P
P
Transgène S.A., Strasbourg, France.
Nishino
M
M
Mazurier
C
C
Dott
K
K
Piétu
G
G
Jorieux
S
S
Pavirani
A
A
Girma
J P
JP
Oufkir
D
D
Courtney
M
M
D000818:Animals; D001792:Blood Platelets / Q000378:metabolism; D002478:Cells, Cultured; D003094:Collagen / Q000378:metabolism; D004247:DNA / Q000235:genetics; D005169:Factor VIII / Q000378:metabolism; D005822:Genetic Vectors; D006801:Humans; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014616:Vaccinia virus / Q000235:genetics; D014841:von Willebrand Factor / Q000235:genetics / Q000378:metabolism*
D011994:Recombinant Proteins; D014841:von Willebrand Factor; D005169:Factor VIII; D003094:Collagen; D004247:DNA