Characterization of recombinant murine GDE4 and GDE7, enzymes producing lysophosphatidic acid and/or cyclic phosphatidic acid

概要

Cyclic phosphatidic acid (cPA) and lysophosphatidic acid (LPA) are lipid mediators that stimulate common G protein-coupled receptors (GPCRs), and are involved in many cellular processes and diseases. Autotaxin (ATX) is a major enzyme to produce LPA from lysophosphatidylcholine (LPC). cPA that is produced by phospholipase D2 (PLD2) is also known as an antagonist of peroxisome proliferator-activated receptor γ (PPARγ). However, the enzymes or enzymes responsible for the in vivo production of cPA in mammals are not fully distinguished yet. GDE4 and GDE7 are the member of the mammalian glycerophosphodiester phosphodiesterase (GDE) family, that contains 7 proteins. We and other groups have reported that LPA is produced from lysophospholipids by GDE4 and GDE7 with their lysophospholipase D (lysoPLD) activity. However, the enzymatic properties including the substrate specificity and the requirement for divalent cations are not fully understood yet. In this study a detailed analysis of the enzyme activity of GDE4 and GDE7 was performed including the dependency of substrate specificities and divalent cations preferences. In addition, we show that GDE7 produces not only LPA but also cPA. A revealed novel substrate and divalent cation preferences.

Enzyme activity of the purified murine GDE4 and GDE7 were analyzed by the following two assay systems. Choline is one of the products derived from lysophosphocholine by GDE4 and GDE7. LPA and cPA are other products from lysophospholipids with various polar head groups by GDE4 and GDE7. The concentrations of choline, LPA and cPA were determined by an enzyme-coupled spectrophotometric assay and a mass spectrophotometric assay, respectively.

The effects of six different divalent cations including Co2+, Ni2+, Zn2+, Mg2+, Ca2+ and Mn2+, on the enzyme activity of GDE4 and GDE7 were tested. LysoPLD activity of GDE4 was observed in the presence of Mg2+ and Mn2+. Ca2+ and Mn2+ had a positive effect on the enzyme activity of GDE7. Also, the effects of the combination of two divalent cations out of the three divalent cations, that showed positive effects on either GDE4 or GDE7 were examined. The profiles of the combination of Mg2+and Ca2+ suggested the presence of three binding sites in GDE4. The profiles of the combination of Mn2+and Ca2+ suggested the presence of at least two binding sites in GDE7. Alkaline phosphatase (AP)and ATX are member of the AP superfamily. AP hydrolyzes phosphate monoesters. The active site of AP contains three divalent cations, two Zn2+and one Mg2+. However, ATX contains two divalent cation binding sites for Zn2+. The substrates for GDE4 are the same as that for ATX but the mode of divalent cations binding for GDE4 is likely to be similar to that of for AP.

The substrate specificities of GDE4 and GDE7 were analyzed in detail using choline- containing substrates including acyl-, alkyl- and alkenyl-LPCs by measuring the free choline. Among the 16:0 species of LPCs, judging from kcat/KM values, the preference of GDE4 and GDE7 was alkyl > acyl ≈ alkenyl and alkyl > acyl > alkenyl, respectively. However, they were clearly different from that of ATX (acyl-LPC 16:0 > alkyl-LPC 16:0 > alkenyl-LPC). These results suggest that alkyl-LPA is produced from alkyl- lysophospholipids by GDE4 and GDE7 in vivo. Acyl-LPC 16:0 was better substrate than the shorter and longer fatty acid-containing acyl-LPCs for GDE4 and GDE7. Among the acyl-LPC C18 species with different number of double bonds, the preference was LPC 18:1 > 18:0 > 18:2 for both GDE4 and GDE7.

Next, the enzymatic products were analyzed using liquid chromatography mass spectrometry (LC-MS). 1-Acyl-LPA 16:0, but not 2-acyl LPA 16:0, was detected as an enzymatic product of GDE4. These results indicated that GDE4 produced 1-acyl-LPA from 1- acyl-LPC, not 2-acyl-LPA from 2-acyl-LPC. In the case of GDE7, in addition to 1-acyl-LPA 16:0, cPA 16:0 was detected. These results clearly indicated that not only 1-acyl-LPA but also 1-acyl-cPA was produced from 1-acyl-LPC by GDE7.

Finally, various lysophospholipids with different polar head groups such as LPC, lysophosphatidylethanolamine (LPE) lysophosphatidylglycerol (LPG) and lysophosphatidylserine (LPS) were tested using LC-MS. Only LPA-producing lysoPLD activity was observed in the case of GDE4. Judging from kcat/KM values, the preference was LPG > LPC > LPS > LPE. In the case of GDE7, not only LPA- also, cPA-producing lysoPLD activity was observed. The preference of the substrates was LPS > LPC > LPG > LPE. This is the first report that LPG and LPS are preferred substrates for GDE4 and GDE7, respectively. However, LPC were also considerably processed. Thus, our observations suggest that these enzymes have broad substrate specificities toward lysophospholipids in vivo.

In this study, we revealed that cPA was largely produced by GDE7. A precise analysis of the dependency of divalent cations and substrate specificity for GDE4 and GDE7 would give us insight into the structures of their active sites. Further studies of GDE4 and GDE7 would be necessary to understand the physiological and pathological roles of LPA and cPA.