Characterization of a recombinant antihaemophilia-A factor (factor VIII-delta II) by matrix-assisted laser desorption/ionization mass spectrometry.
Factor VIII-delta II is a genetically engineered deletion variant of factor VIII, expressed by recombinant Chinese hamster ovary cells. This 1436-residues-long protein has a molecular mass, calculated from its sequence, of 164,954 Da and exhibits seven potential glycosylation sites. The glycoprotein, secreted as a single polypeptide chain, can be cleaved after Arg740 to generate a heavy-light chain complex of 90-80 kDa as revealed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. Due to its high mass range and excellent sensitivity, matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) has been chosen to play a key role in the precise determination of the molecular masses of recombinant factor VIII and the localization of the post-translational modifications within the protein. Native factor VIII-delta II displays a molecular mass of 178 kDa. The masses measured by MALDI for the heavy and light chains are respectively 89,900 Da and 87,100 Da. These mass values, found reproducible from batch to batch, are used to characterize factor VIII-delta II during the course of preclinical studies. The difference from the theoretical molecular molecular masses and the observation of broad molecular peaks suggest that recombinant FVIII-delta II has been effectively glycosylated by the host cell on both heavy and light chains. Similarly to plasma-derived factor VIII, the recombinant protein is proteolyzed by thrombin to generate the A1/A2/A3-C1-C2 trimer that is the active form of factor VIII in the coagulation pathway. MALDI-MS analysis of activated factor VIII-delta II suggested the presence of N-linked oligosaccharides in the proteolyzed light chain (A3-C1-C2 of 77,750 Da) and in the A1 domain (46,400 Da) of the heavy chain. By contrast, the similarity between the experimental and theoretical masses of the A2 domain indicated that its single potential glycosylation site has not been utilized.
Bihoreau
N
N
Laboratoire Français du Fractionnement et des Biotechnologies, Les Ulis, France.
Veillon
J F
JF
Ramon
C
C
Scohyers
J M
JM
Schmitter
J M
JM
D004789:Enzyme Activation; D005169:Factor VIII / Q000737:chemistry*; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods*; D006023:Glycoproteins / Q000737:chemistry; D006801:Humans; D011994:Recombinant Proteins / Q000737:chemistry; D017384:Sequence Deletion
D006023:Glycoproteins; D011994:Recombinant Proteins; D005169:Factor VIII