Ferry Gilles, Giganti Adeline, Cogé Francis, Bertaux Fabien, Thiam Kader, Boutin Jean A
Pharmacologie Moléculaire et Cellulaire, Institut de Recherches Servier, 125 Chemin de Ronde, 78290 Croissy-sur-Seine, France.
FEBS Lett. 2007 Jul 24;581(18):3572-8. doi: 10.1016/j.febslet.2007.06.064. Epub 2007 Jul 3.
Autotaxin is a member of the phosphodiesterase family of enzymes, (NPP2). It is an important secreted protein found in conditioned medium from adipocytes. It also has a putative role in the metastatic process. Based on these observation, further validation of this potential target was necessary, apart from the classical biochemical ones. The construction of a knock out mouse strain for ATX was started. In this paper, we report the generation of a mouse line displaying an inactivated ATX gene product. The KO line was designed in order to generate a functional inactivation of the protein. In this respect, the threonine residue T210 was replaced by an alanine (T210A) leading to a catalytically inactive enzyme. If the experimental work was straight forward, we disappointedly discovered at the final stage that the breeding of heterozygous animals, ATX -/+, led to the generation of a Mendelian repartition of wild-type and heterozygous, but no homozygous were found, strongly suggesting that the ATX deletion is lethal at an early stage of the development. This was confirmed by statistical analysis. Although other reported the same lethality for attempted ATX-/- mice generation [van Meeteren, L.A., Ruurs, P., Stortelers, C., Bouwman, P., van Rooijen, M.A., Pradère, J.P., Pettit, T.R., Wakelam, M.J.O., Saulnier-Blache, J.S., Mummery, C.L., Moolenar, W.H. and Jonkers, J. (2006) Autotaxin, a secreted lysophospholipase D, is essential for blood vessel formation during development, Mol. Cell. Biol. 26, 5015-5022; Tanaka, M., Okudaira, S., Kishi, Y., Ohkawa, R., Isei, S., Ota, M., Noji, S., Yatomi, Y., Aoki, J., and Arai, H. (2006) Autotaxin stabilizes blood vessels and is required for embryonic vasculature by producing lysophosphatidic acid, J. Biol. Chem. 281, 25822-25830], they used more drastic multiple exon deletions in the ATX gene, while we chose a single point mutation. To our knowledge, the present work is the first showing such a lethality in any gene after a point mutation in an enzyme catalytic site.
自分泌运动因子是磷酸二酯酶家族（NPP2）的成员。它是一种在脂肪细胞条件培养基中发现的重要分泌蛋白。它在转移过程中也可能发挥作用。基于这些观察结果，除了经典的生化验证之外，进一步验证这个潜在靶点很有必要。于是开始构建自分泌运动因子基因敲除小鼠品系。在本文中，我们报告了一个显示自分泌运动因子基因产物失活的小鼠品系的产生。构建该基因敲除品系是为了使该蛋白功能失活。在这方面，苏氨酸残基T210被丙氨酸取代（T210A），从而产生一种催化失活的酶。如果实验工作进展顺利，我们却在最后阶段失望地发现，杂合动物（ATX -/+）的繁殖导致野生型和杂合子的孟德尔式分布，但未发现纯合子，这强烈表明自分泌运动因子的缺失在发育早期是致死的。这一点通过统计分析得到了证实。尽管其他研究报告了在尝试生成自分泌运动因子基因敲除小鼠时同样存在致死性[范·梅特伦，L.A.，鲁尔斯，P.，斯托特勒斯，C.，鲍曼，P.，范·鲁伊恩，M.A.，普拉德雷，J.P.，佩蒂特，T.R.，韦克拉姆，M.J.O.，索尔尼尔 - 布拉切，J.S.，穆默里，C.L.，穆勒纳尔，W.H.和琼克斯，J.（2006年）自分泌运动因子，一种分泌型溶血磷脂酶D，在发育过程中对血管形成至关重要，《分子细胞生物学》26卷，5015 - 5022页；田中，M.，奥久平，S.，岸，Y.，大川，R.，伊势，S.，太田，M.，能势，S.，矢富美，Y.，青木，J.和荒井，H.（2006年）自分泌运动因子通过产生溶血磷脂酸来稳定血管并对胚胎血管系统是必需的，《生物化学杂志》281卷，25822 - 25830页]，但他们在自分泌运动因子基因中使用了更剧烈的多个外显子缺失，而我们选择了单点突变。据我们所知，目前的这项工作是首次表明在酶催化位点进行单点突变后，任何基因会出现这种致死性。
