Abstract
The coagulation factor VIII (FVIII) is the coagulation factor deficient in the X-chromosome-linked bleeding disorder hemophilia A. Previous transfection studies demonstrated that factor VIII was 10-100-fold less efficiently expressed than the homologous coagulation factor, factor V. To investigate the regulatory mechanisms of FVIII synthesis and secretion, we used the yeast two-hybrid system as an approach to search for proteins that associated with FVIII. The A2 domain (337-740 amino acids) of factor VIII (FVIII-A2) was used as a bait and phytanoyl-CoA alpha-hydroxylase (PAHX) was identified as a binding protein of FVIII-A2. PAHX had potential to interact with the residues 373-508 within the A2 domain, but not with A1 and A3 (the homologous domains of A2). The interaction between the A2 domain and PAHX was independent of the type 2 peroxisomal targeting signal (PTS2) of PAHX. Overexpression of PAHX in FVIII-produced cells decreased the expression of FVIII by about 70%. The elevated expression of von Willebrand factor had no effect on the suppression of FVIII secretion by PAHX. Expression of the green fluorescent PAHX fusion protein in SMMC-7721 cells affected the intracellular trafficking of FVIII-A2. These results suggested that the interaction between PAHX and FVIII-A2 was in part responsible for the low-level expression of factor VIII.
Highlights
Coagulation factor VIII (FVIII)1 is an essential protein in blood coagulation
These results suggested that the interaction between phytanoyl-CoA ␣-hydroxylase (PAHX) and FVIII-A2 was in part responsible for the low-level expression of factor VIII
Identification of the FVIII A2 Domain-associated Protein PAHX—The fusion protein of the FVIII-A2 domain (337–740 aa) and the LexA DNA-binding domain was used as a bait to screen a human fetal liver cell cDNA library in the yeast two-hybrid system
Summary
Coagulation factor VIII (FVIII)1 is an essential protein in blood coagulation. The deficiency in FVIII is responsible for hemophilia A (classic hemophilia), an X-chromosome-linked bleeding disorder. The A2 domain (337–740 amino acids) of factor VIII (FVIII-A2) was used as a bait and phytanoyl-CoA ␣-hydroxylase (PAHX) was identified as a binding protein of FVIII-A2. The elevated expression of von Willebrand factor had no effect on the suppression of FVIII secretion by PAHX.
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