Heterogeneity in the tyrosine sulfation of Chinese hamster ovary cell produced recombinant FVIII.
By the use of recombinant technology, several stable Chinese hamster ovary (CHO) cell lines expressing human FVIII were established. Thrombin treatment and SDS-PAGE analysis of the purified recombinant FVIII (rFVIII) revealed a striking difference from plasma-derived FVIII (pFVIII). A 43-kDa fragment of the FVIII heavy chain appears as a double band from rFVIII, while a single band from pFVIII is observed. All other fragments from the two samples appeared similar by SDS-PAGE. The heterogeneity is caused by incomplete tyrosine sulfation of one or more of the three potential tyrosine sulfation sites (Tyr718, Tyr719, Tyr723). To investigate if there is a general limitation and heterogeneity in the tyrosine sulfation of rFVIII, two other potential tyrosine sulfation sites on the FVIII light chain (Tyr1664, Tyr1680) were analyzed. The results show that both sites on the pFVIII light chain and on the rFVIII light chain are completely sulfated. The limitation of CHO cells to tyrosine sulfate rFVIII is therefore only restricted to a few sites. The two sulfated forms of rFVIII can easily be separated by ion-exchange chromatography, indicating the importance of the sulfate groups on the charge and/or conformation of FVIII. Both forms of rFVIII possess identical in vitro coagulation activity, von Willebrand factor binding, and thrombin activation profile. However, the difference in tyrosine sulfation may change other biological properties of FVIII.
Mikkelsen
J
J
Novo Nordisk A/S, DK-2820 Gentofte, Denmark.
Thomsen
J
J
Ezban
M
M
D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D005169:Factor VIII / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D005260:Female; D006026:Glycoside Hydrolases; D006801:Humans; D008970:Molecular Weight; D010053:Ovary; D010449:Peptide Mapping; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013917:Thrombin / Q000378:metabolism; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000032:analysis
D011994:Recombinant Proteins; C005662:tyrosine O-sulfate; D014443:Tyrosine; D005169:Factor VIII; D006026:Glycoside Hydrolases; D013917:Thrombin