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. 2014 Aug 15;9(8):e104078.
doi: 10.1371/journal.pone.0104078. eCollection 2014.

Vitamin K2 biosynthetic enzyme, UBIAD1 is essential for embryonic development of mice

Kimie Nakagawa  1 , Natsumi Sawada  1 , Yoshihisa Hirota  1 , Yuri Uchino  1 , Yoshitomo Suhara  2 , Tomoka Hasegawa  3 , Norio Amizuka  3 , Tadashi Okamoto  4 , Naoko Tsugawa  1 , Maya Kamao  1 , Nobuaki Funahashi  1 , Toshio Okano  1
Affiliations

Vitamin K2 biosynthetic enzyme, UBIAD1 is essential for embryonic development of mice

Kimie Nakagawa et al. PLoS One. .

Abstract

UbiA prenyltransferase domain containing 1 (UBIAD1) is a novel vitamin K2 biosynthetic enzyme screened and identified from the human genome database. UBIAD1 has recently been shown to catalyse the biosynthesis of Coenzyme Q10 (CoQ10) in zebrafish and human cells. To investigate the function of UBIAD1 in vivo, we attempted to generate mice lacking Ubiad1, a homolog of human UBIAD1, by gene targeting. Ubiad1-deficient (Ubiad1(-/-)) mouse embryos failed to survive beyond embryonic day 7.5, exhibiting small-sized body and gastrulation arrest. Ubiad1(-/-) embryonic stem (ES) cells failed to synthesize vitamin K2 but were able to synthesize CoQ9, similar to wild-type ES cells. Ubiad1(+/-) mice developed normally, exhibiting normal growth and fertility. Vitamin K2 tissue levels and synthesis activity were approximately half of those in the wild-type, whereas CoQ9 tissue levels and synthesis activity were similar to those in the wild-type. Similarly, UBIAD1 expression and vitamin K2 synthesis activity of mouse embryonic fibroblasts prepared from Ubiad1(+/-) E15.5 embryos were approximately half of those in the wild-type, whereas CoQ9 levels and synthesis activity were similar to those in the wild-type. Ubiad1(-/-) mouse embryos failed to be rescued, but their embryonic lifespans were extended to term by oral administration of MK-4 or CoQ10 to pregnant Ubiad1(+/-) mice. These results suggest that UBIAD1 is responsible for vitamin K2 synthesis but may not be responsible for CoQ9 synthesis in mice. We propose that UBIAD1 plays a pivotal role in embryonic development by synthesizing vitamin K2, but may have additional functions beyond the biosynthesis of vitamin K2.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Generation of Ubiad1 knockout mice.
(A) Schematic presentation of ubiad1 genome, targeting vector and disrupted Ubiad1 genome. (B) PCR genotyping of Ubiad1 +/+, Ubiad1 +/− and Ubiad1 −/− embryos. PCR genotyping of tail DNA of Ubiad1 +/+, Ubiad1 +/− and Ubiad1 −/− embryos. Lane 1, positive controls for Ubiad1 +/− allele. Lane 2, PCR bands of Ubiad1 +/− embryos. Lane 3, PCR bands of Ubiad1 +/+ embryos. Lane 4, PCR bands of Ubiad1 −/− embryos.
Figure 2
Figure 2. Developmental arrest in Ubiad1 knockout embryos.
(A) Morphology of E3.5 blastocysts. Blastocysts were cultured from in vitro fertilised embryos of Ubiad1 +/+, Ubiad1 +/− and Ubiad1 −/− mice. (B) Morphology of E7.5 embryos.
Figure 3
Figure 3. Ubiad1 and Coq2 expression and the biosynthesis of MK-4 and CoQ9 in ES cells derived from Ubiad1 +/+, Ubiad1 +/− and Ubiad1 −/− embryos.
(A) Morphology of Ubiad1 +/+, Ubiad1 +/− and Ubiad1 −/− ES cells. (B) Ubiad1 and Coq2 mRNA expression in Ubiad1 +/+, Ubiad1 +/− and Ubiad1 −/− ES cells. (C) The biosynthesis of MK-4-d7, 13C6-CoQ9 and 13C6-CoQ10 in Ubiad1 +/+, Ubiad1 +/− and Ubiad1 −/− ES cells. Mean ± s.e.m. Dunnett's test, *P<0.05. N.D.: not detected.
Figure 4
Figure 4. Morphological examination of Ubiad1 +/+, Ubiad1 +/− and Ubiad1 −/− embryos and weanling mice (postnatal day 1) from pregnant Ubiad1 +/− mice orally administered CoQ10.
(A) Morphology of Ubiad1 +/+, Ubiad1 +/− and Ubiad1 −/− embryos at E15.5 from CoQ10-supplemented pregnant Ubiad1 +/− mice. (B) HE staining of embryos at E15.5. (C) Immunohistochemical staining of UBIAD1 in Ubiad1 +/+, Ubiad1 +/− and Ubiad1 −/− embryos at E15.5. (D) Morphology of Ubiad1 +/+, Ubiad1 +/− and Ubiad1 −/− embryos at postnatal day 1. Ubiad1 +/+ and Ubiad1 +/− mice were born alive, but Ubiad1 −/− mice were stillborn. (E) Ubiad1 mRNA expression in the livers and brains of Ubiad1 +/+, Ubiad1 +/− and Ubiad1 −/− embryos at E15.5 from CoQ10-supplemented pregnant Ubiad1 +/− mice. Mean ± s.e.m. Dunnett's test, *P<0.05. N.D.: not detected. (F) UBIAD1 expression in the brains of Ubiad1 +/+, Ubiad1 +/− and Ubiad1 −/− embryos at E15.5 from CoQ10-supplemented pregnant Ubiad1 +/− mice.
Figure 5
Figure 5. Morphological examination of Ubiad1 +/+, Ubiad1 +/− and Ubiad1 −/− embryos from pregnant Ubiad1 +/− mice orally administered MK-4.
(A) Morphology of Ubiad1 +/+, Ubiad1 +/− and Ubiad1 −/− embryos at E15.5 from MK-4-supplemented pregnant Ubiad1 +/− mice. (B) HE staining of embryos at E15.5. (C) Morphology of Ubiad1 +/+, Ubiad1 +/− and Ubiad1 −/− embryos at E17.5 from MK-4-supplemented pregnant Ubiad1 +/− mice. (D) HE staining of embryos at E17.5.
Figure 6
Figure 6. MK-4 and CoQ9/CoQ10 biosynthetic mechanisms of UBIAD1 and COQ2 in mammals.
MD is released from PK in the intestine and converted to MK-4. 13C64-HB is prenylated to polyprenyl-4-HB by COQ2. Polyprenyl-4-HB is finally converted to CoQ9/CoQ10 by several enzymes.
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Grants and funding

This work was supported in part by a Grant-in-aid for Scientific Research (B) [grant number 23390022] to TO, a Grant-in-aid for Scientific Research (Young Scientists-B) [grant number 23790110] to KN from JSPS, the Naito Foundation Subsidy for Female Researchers after Maternity Leave to KN from the Naito Foundation and a Grant-in-aid for JSPS Fellows [grant number 24-7941] to YH. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.