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

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 6

Increased VKORC1L1 levels in the absence of Vkorc1 rescues thrombin activity, carboxylation, and survival.

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Increased VKORC1L1 levels in the absence of Vkorc1 rescues thrombin acti...
(A) Thrombin activities in WT, Vkorc1–/–, Vkorc1–/–;APOE-Vkorc1l173, and APOE-Vkorc1l173 P5 pups were measured using a Coagucheck and are shown as international normalized ratio (INR) (n = 3-6; mean ± SEM). The limit of detection of the Coagucheck ranges from 0.8 to 8 and is depicted by dashed lines. (B) The global carboxylation profile in livers from WT, Vkorc1–/–, Vkorc1–/–;APOE-Vkorc1l173, and APOE-Vkorc1l173 P5 pups was analyzed by Western blot using α-Gla antibody. β-Actin was used as a loading control. Arrows show VKD proteins, while asterisks indicate nonspecific bands. (C) PT and GGCX carboxylation in WT, Vkorc1–/–, Vkorc1–/–;APOE-Vkorc1l173, and APOE-Vkorc1l173 P5 animals were measured by α-Gla immunoprecipitation followed by Western blot analysis using α-PT and α-GGCX antibodies. β-Actin was used as a loading control. Input represents 2% of total protein extract. (D) VKOR activity was measured in livers from WT, Vkorc1–/–, Vkorc1–/–;APOE-Vkorc1l173, and APOE-Vkorc1l173 P5 pups (n = 4; mean ± SEM; unpaired, 2-tailed Student’s t test was used to compare Vkorc1–/– and Vkorc1–/–; APOE-Vkorc1l173 livers; ***P < 0.001). (E) Kaplan-Meier survival curves were determined from birth to 5 months of age for WT, Vkorc1–/–, Vkorc1–/–;APOE-Vkorc1l173, and APOE-Vkorc1l173 mice (n = 6–14; Mantel-Cox test; ***P < 0.001). (F) The INR was determined for WT and Vkorc1–/–;APOE-Vkorc1l173 mice at 16 weeks of age (n = 4–7, mean ± SEM; unpaired, 2-tailed Student’s t test; **P < 0.01). For Western blot, duplicates represent biological replicates and the image is representative of 2 independent experiments.