Figure 4.
V255M carboxylation in cells generates FIX with decreased clotting activity. (A) FLAG-tagged wild-type (WT) and V255M carboxylases were individually expressed in FIX 293 cells edited to eliminate endogenous carboxylase (−). M indicates molecular weight markers. (B) Cells cultured in the absence of vitamin K resulted in an uncarboxylated intracellular pool of FIX in ∼50-fold excess over the carboxylase, as determined by western blot analysis with anti-FIX and anti-FLAG antibodies and FIX and FLAG standards. Cells were then exchanged into serum-free media containing vitamin K (5 ng/ml) and harvested after 18 hours. During secretion, FIX was carboxylated in the endoplasmic reticulum (ER), and additional modifications occurred in the Golgi (eg, propeptide processing, sulfation, and aspartyl β-hydroxylation). Carboxylation was not obligatory for secretion, and secreted FIX could therefore be a mixture of forms with different degrees of carboxylation. (C) A panspecific anti-Gla antibody45 detected VKD protein in cell-spent media from FIX 293 cells but not the progenitor 293 cells, allowing the carboxylation of FIX expressed in 293 cells to be specifically analyzed. The FIX control was purified human plasma FIX (Enzyme Research Laboratories). (D) Media from cells cultured in the presence or absence of vitamin K (vit K) were analyzed in western blots using antibody against Gla45 or anti-FIX antibody that detects both carboxylated and uncarboxylated FIX.18 Lysates analyzed with anti–glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody indicated similar amounts of cellular material. (E) Media was quantitated for Gla and FIX content by comparison with purified plasma FIX (shown in panel D). Aliquots from the same media samples analyzed in the western blots were assayed for FIX activity in a clotting assay, as previously described.46,67 (F) Clotting activity and Gla content were compared and normalized to a ratio of 1 for FIX secreted from 293 cells expressing WT carboxylase. The ratio of clotting activity to Gla content revealed defective clotting in FIX carboxylated by the V255M mutant.

V255M carboxylation in cells generates FIX with decreased clotting activity. (A) FLAG-tagged wild-type (WT) and V255M carboxylases were individually expressed in FIX 293 cells edited to eliminate endogenous carboxylase (−). M indicates molecular weight markers. (B) Cells cultured in the absence of vitamin K resulted in an uncarboxylated intracellular pool of FIX in ∼50-fold excess over the carboxylase, as determined by western blot analysis with anti-FIX and anti-FLAG antibodies and FIX and FLAG standards. Cells were then exchanged into serum-free media containing vitamin K (5 ng/ml) and harvested after 18 hours. During secretion, FIX was carboxylated in the endoplasmic reticulum (ER), and additional modifications occurred in the Golgi (eg, propeptide processing, sulfation, and aspartyl β-hydroxylation). Carboxylation was not obligatory for secretion, and secreted FIX could therefore be a mixture of forms with different degrees of carboxylation. (C) A panspecific anti-Gla antibody45 detected VKD protein in cell-spent media from FIX 293 cells but not the progenitor 293 cells, allowing the carboxylation of FIX expressed in 293 cells to be specifically analyzed. The FIX control was purified human plasma FIX (Enzyme Research Laboratories). (D) Media from cells cultured in the presence or absence of vitamin K (vit K) were analyzed in western blots using antibody against Gla45 or anti-FIX antibody that detects both carboxylated and uncarboxylated FIX.18 Lysates analyzed with anti–glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody indicated similar amounts of cellular material. (E) Media was quantitated for Gla and FIX content by comparison with purified plasma FIX (shown in panel D). Aliquots from the same media samples analyzed in the western blots were assayed for FIX activity in a clotting assay, as previously described.46,67 (F) Clotting activity and Gla content were compared and normalized to a ratio of 1 for FIX secreted from 293 cells expressing WT carboxylase. The ratio of clotting activity to Gla content revealed defective clotting in FIX carboxylated by the V255M mutant.

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