Pradère Jean Philippe, Tarnus Evelyne, Grès Sandra, Valet Philippe, Saulnier-Blache Jean Sébastien
Inserm, U586, Unité de Recherches sur les Obésités, Toulouse, F-31432, France.
Biochim Biophys Acta. 2007 Jan;1771(1):93-102. doi: 10.1016/j.bbalip.2006.11.010. Epub 2006 Dec 6.
Autotaxin (ATX) is a lysophospholipase D involved in synthesis of lysophosphatidic acid (LPA). ATX is secreted by adipocytes and is associated with adipogenesis and obesity-associated diabetes. Here we have studied the mechanisms involved in biosynthesis and secretion of ATX by mouse 3T3-F442A adipocytes. We found that inhibition of N-glycosylation with tunicamycin or by double point deletion of the amino-acids N53 and N410 of ATX inhibit its secretion. In addition, N-glycosidase treatment and point deletion of the amino-acid N410 inhibits the lysophospholipase D activity of ATX. Analysis of the amino-acid sequence of mouse ATX shows the presence of a N-terminal signal peptide. Treatment with the signal peptidase inhibitor globomycin inhibits ATX secretion by adipocytes. Transfection in Cos-7 cells of site-directed deleted ATX shows that ATX secretion is dependent on the hydrophobic core sequence of the signal peptide, not on the putative signal peptidase cleavage site sequence. Analysis of the amino-acid sequence of mouse ATX also reveals the presence of a putative cleavage site by the protein convertase furin. Treatment of adipocytes with the furin inhibitor decanoyl-Arg-Val-Lys-Arg-chloromethylketone does not modified secretion or lysophospholipase D activity of ATX. Transfection in Cos-7 cells of site-directed deleted ATX shows that the furin recognition site is not required for secretion or lysophospholipase D activity of ATX. In conclusion, the present work demonstrates the crucial role of N-glycosylation in secretion and activity of ATX. The present work also confirms the crucial role signal peptidase in secretion of ATX by adipocytes.
自分泌运动因子（ATX）是一种参与溶血磷脂酸（LPA）合成的溶血磷脂酶D。ATX由脂肪细胞分泌，与脂肪生成和肥胖相关糖尿病有关。在此，我们研究了小鼠3T3-F442A脂肪细胞中ATX生物合成和分泌所涉及的机制。我们发现，用衣霉素抑制N-糖基化或通过删除ATX的N53和N410氨基酸的双点突变抑制其分泌。此外，N-糖苷酶处理和N410氨基酸的点突变抑制了ATX的溶血磷脂酶D活性。对小鼠ATX氨基酸序列的分析表明存在一个N端信号肽。用信号肽酶抑制剂球霉素处理可抑制脂肪细胞分泌ATX。在Cos-7细胞中转染定点缺失的ATX表明，ATX的分泌依赖于信号肽的疏水核心序列，而不是推定的信号肽切割位点序列。对小鼠ATX氨基酸序列的分析还揭示了存在一个由蛋白转化酶弗林蛋白酶切割的推定位点。用弗林蛋白酶抑制剂癸酰-精氨酸-缬氨酸-赖氨酸-精氨酸-氯甲基酮处理脂肪细胞不会改变ATX的分泌或溶血磷脂酶D活性。在Cos-7细胞中转染定点缺失的ATX表明，弗林蛋白酶识别位点对于ATX的分泌或溶血磷脂酶D活性不是必需的。总之，本研究证明了N-糖基化在ATX分泌和活性中的关键作用。本研究还证实了信号肽酶在脂肪细胞分泌ATX中的关键作用。
