Viral transformation increases vitamin K-dependent gamma-carboxylation of glutamate.
Mammalian cells contain a microsomal vitamin K-dependent carboxylase activity which catalyzes the gamma-carboxylation of glutamate. While most cells have a limited ability to fully gamma-carboxylate proteins, it has been suggested that the ability of transformed cells to perform this complex post-translational modification may play a role in tumor biology. In this study, we examined the effect of transformation by adenovirus oncogenes on the ability of cells to efficiently gamma-carboxylate a vitamin K-dependent protein. Several morphologically transformed BHK-21 cell lines (BHK-Ad) were isolated following the chromosomal integration of the viral oncogenes E1A/E1B from human adenovirus type 12 (Ad12). The lines were capable of growing in soft agar and low serum and produced functional E1A as determined by promoter activation studies. Using a vector for the expression of the vitamin K-dependent recombinant human protein C (HPC), a regulator of the clotting cascade, Ad-transformed and nontransformed lines secreting rHPC were generated. The rHPC from the transformed and nontransformed cell lines displayed identical serine protease activities, and there were no apparent differences in the proteolytic processing of the proteins, although a minor difference in the proportion of each HPC glycoform was observed. However, the functional anticoagulant activity, which depends on the gamma-carboxyglutamic acid (Gla) content, was approximately 70% higher in the Ad-transformed lines. Approximately 90% of the rHPC from the Ad-transformed lines exhibited a calcium-dependent (high Gla) elution profile on anion-exchange resin, compared to only 15 to 26% from the nontransformed cell clones. By analyzing endogenous microsomal carboxylase, we determined that enzyme activity increased approximately 50% following transformation. Overall, our data demonstrate that transformation can increase the potential of a cell to efficiently gamma-carboxylate a protein and lend support to the suggested involvement of this post-translational modification in tumor cell function. Further, our results demonstrate a potential means of altering cells to enable full modification of vitamin K-dependent factors for structure/function studies and potentially for therapeutic use.
Berg
D T
DT
Department of Molecular Genetics, Lilly Corporate Center, Indianapolis, Indiana 46285.
McClure
D B
DB
Walls
J D
JD
Yan
S B
SB
Grinnell
B W
BW
D015055:1-Carboxyglutamic Acid / Q000378:metabolism*; D017456:Adenovirus Early Proteins; D000260:Adenoviruses, Human; D000818:Animals; D002118:Calcium / Q000502:physiology; D002461:Cell Line, Transformed; D002472:Cell Transformation, Viral / Q000235:genetics / Q000502:physiology*; D004279:DNA, Viral / Q000032:analysis; D006801:Humans; D008861:Microsomes / Q000201:enzymology; D009856:Oncogene Proteins, Viral / Q000235:genetics; D011486:Protein C / Q000378:metabolism* / Q000502:physiology; D011499:Protein Processing, Post-Translational / Q000502:physiology; D011994:Recombinant Proteins / Q000378:metabolism / Q000502:physiology; D014812:Vitamin K / Q000502:physiology*
D017456:Adenovirus Early Proteins; D004279:DNA, Viral; D009856:Oncogene Proteins, Viral; D011486:Protein C; D011994:Recombinant Proteins; D014812:Vitamin K; D015055:1-Carboxyglutamic Acid; D002118:Calcium