Effect of GGCX mutations on the carboxylation of different VKD reporter proteins. (A) Distribution of naturally occurring mutations in the GGCX molecule. The 5-transmembrane domain topologic structure of GGCX illustrated is based on the model from reference 2 . (B) Carboxylation of the 3 VKD reporter proteins by selected GGCX mutations in HEK293 cells. The GGCX mutation was transiently expressed in GGCX-deficient HEK293 cells stably expressing FIXgla-PC, MGP, or BGP. Transfected cells were incubated with 5 µM vitamin K for 48 hours. Carboxylation efficiency of the reporter protein was determined in the cell culture medium by ELISA. Wild-type GGCX activity was normalized to 100%. Data are presented as the mean ± SD of 3 independent experiments (n = 3). *P < .001. (C) A heat-map representation of the carboxylation activity of the currently identified GGCX mutations for the 3 reporter proteins. Carboxylation activity was determined as described in panel B.