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VKOR paralog VKORC1L1 supports vitamin K–dependent protein carboxylation in vivo
Julie Lacombe, … , Kathleen L. Berkner, Mathieu Ferron
Julie Lacombe, … , Kathleen L. Berkner, Mathieu Ferron
Published January 11, 2018
Citation Information: JCI Insight. 2018;3(1):e96501. https://doi.org/10.1172/jci.insight.96501.
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Research Article Development Hematology

VKOR paralog VKORC1L1 supports vitamin K–dependent protein carboxylation in vivo

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Abstract

Vertebrates possess 2 proteins with vitamin K oxidoreductase (VKOR) activity: VKORC1, whose vitamin K reduction supports vitamin K–dependent (VKD) protein carboxylation, and VKORC1-like 1 (VKORC1L1), whose function is unknown. VKD proteins include liver-derived coagulation factors, and hemorrhaging and lethality were previously observed in mice lacking either VKORC1 or the γ-glutamyl carboxylase (GGCX) that modifies VKD proteins. Vkorc1–/– mice survived longer (1 week) than Ggcx–/– mice (midembryogenesis or birth), and we assessed whether VKORC1L1 could account for this difference. We found that Vkorc1–/–;Vkorc1l1–/– mice died at birth with severe hemorrhaging, indicating that VKORC1L1 supports carboxylation during the pre- and perinatal periods. Additional studies showed that only VKORC1 sustains hemostasis beyond P7. VKORC1 expression and VKOR activity increased during late embryogenesis and following birth, while VKORC1L1 expression was unchanged. At P0, most (>99%) VKOR activity was due to VKORC1. Prothrombin mRNA, protein, and carboxylation also increased during this period, as did mRNA levels of coagulation factors encoding genes F7, F9, and F10. VKORC1L1 levels in Vkorc1–/– mouse liver may therefore be insufficient for supporting carboxylation beyond day 7. In support of this conclusion, VKORC1L1 overexpression in liver rescued carboxylation and hemostasis in adult Vkorc1–/– mice. These findings establish that VKORC1L1 supports VKD protein carboxylation in vivo.

Authors

Julie Lacombe, Mark A. Rishavy, Kathleen L. Berkner, Mathieu Ferron

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Figure 1

Carboxylated vitamin K–dependent (VKD) proteins are present in Vkorc1–/– mice at birth, and hemizygosity at the Vkorc1l1 locus causes premature hemorrhaging and death of Vkorc1–/– mice.

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Carboxylated vitamin K–dependent (VKD) proteins are present in Vkorc1–/–...
(A) Prothrombin (PT) carboxylation in plasma from WT and Vkorc1–/– (c1–/–) mice at P0 and P7 was assessed by α-Gla antibody immunoprecipitation, followed by Western blot analysis with α-PT antibody. Input represents 4% of serum sample. (B) PT and GGCX carboxylation in e18.5, P0, and P7 livers from WT and Vkorc1–/– mice was tested using α-Gla immunoprecipitation and subsequent Western blot analysis with α-PT and α-GGCX antibodies. β-Actin was used as a loading control. Input represents 2% of total protein extract. (C) Representative picture showing WT, Vkorc1–/–, Vkorc1l1+/– (c1l1+/–), and Vkorc1–/–;Vkorc1l1+/– (c1–/–;c1l1+/–) P0 pups. Arrows indicate abdominal hemorrhages. (D) Shown are Kaplan-Meier survival curves for WT, Vkorc1+/– (c1+/–), Vkorc1+/–, Vkorc1–/–, and Vkorc1–/–;Vkorc1l1+/– mice (n = 7–15; Mantel-Cox test; **P < 0.01; ***P < 0.001). For Western blots, duplicates represent biological replicates and the image is representative of 2 independent experiments.

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