Substrate specificity and catalytic efficiency of aldo-keto reductases with phospholipid aldehydes

Abstract

Phospholipid oxidation generates several bioactive aldehydes that remain esterified to the glycerol backbone ('core' aldehydes). These aldehydes induce endothelial cells to produce monocyte chemotactic factors and enhance monocyte-endothelium adhesion. They also serve as ligands of scavenger receptors for the uptake of oxidized lipoproteins or apoptotic cells. The biochemical pathways involved in phospholipid aldehyde metabolism, however, remain largely unknown. In the present study, we have examined the efficacy of the three mammalian AKR (aldo-keto reductase) families in catalysing the reduction of phospholipid aldehydes. The model phospholipid aldehyde POVPC [1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine] was efficiently reduced by members of the AKR1, but not by the AKR6 or the ARK7 family. In the AKR1 family, POVPC reductase activity was limited to AKR1A and B. No significant activity was observed with AKR1C enzymes. Among the active proteins, human AR (aldose reductase) (AKR1B1) showed the highest catalytic activity. The catalytic efficiency of human small intestinal AR (AKR1B10) was comparable with the murine AKR1B proteins 1B3 and 1B8. Among the murine proteins AKR1A4 and AKR1B7 showed appreciably lower catalytic activity as compared with 1B3 and 1B8. The human AKRs, 1B1 and 1B10, and the murine proteins, 1B3 and 1B8, also reduced C-7 and C-9 sn-2 aldehydes as well as POVPE [1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphoethanolamine]. AKR1A4, B1, B7 and B8 catalysed the reduction of aldehydes generated in oxidized C(16:0-20:4) phosphatidylcholine with acyl, plasmenyl or alkyl linkage at the sn-1 position or C(16:0-20:4) phosphatidylglycerol or phosphatidic acid. AKR1B1 displayed the highest activity with phosphatidic acids; AKR1A4 was more efficient with long-chain aldehydes such as 5-hydroxy-8-oxo-6-octenoyl derivatives, whereas AKR1B8 preferred phosphatidylglycerol. These results suggest that proteins of the AKR1A and B families are efficient phospholipid aldehyde reductases, with non-overlapping substrate specificity, and may be involved in tissue-specific metabolism of endogenous or dietary phospholipid aldehydes.

Scheme 1. Synthesis of sn-2-substituted phosphatidylcholines

Scheme 2. Synthesis of sn-2-substituted ethanolamine

Figure 1. AKR1B8 catalyses the reduction of phospholipid aldehydes

Reagent POVPC, POHyPC or PONyPC (50 μg) were incubated without or with recombinant AKR1B8 (100 μg) and NADPH (150 μM) in 0.1 M potassium phosphate (pH 6.0) for 3 h at 30 °C. The lipids were extracted in chloroform/methanol/water and analysed by ESI⁺–MS. Mass spectrum shows that reagent POVPC, POHyPC and PONyPC correspond to molecular ions with m/z 594.2 (A), 622.2 (C) and 650.5 (E) respectively, which are increased by 2 Da upon reduction with AKR1B8, indicating the formation of 1-palmitoyl-2-(5-hydroxyvaleroyl) phosphatidylcholine (PHVPC; B; m/z 596.3), 1-palmitoyl-2-(7-hydroxyheptanoyl)-sn-glycero-3-phosphocholine (PHHyPC; D, m/z 624.2) and 1-palmitoyl-2-(9-hydroxynonanoyl)-sn-glycero-3-phosphocholine (PHNyPC; F; m/z 652.5).

Figure 2. AKR1 proteins catalyse the reduction of phospholipid aldehydes generated from the oxidation of PAPC

PAPC (50 μg) was oxidized in air for 24 h and incubated without or with 100 μg of the AKR1 proteins, AKR1A4, 1B1, 1B7 and 1B8, and 150 μM NADPH in 0.1 M potassium phosphate (pH 6.0) for 3 h at 30 °C. The lipids were extracted in methanol/chloroform/water and analysed by ESI⁺–MS. As shown in (A), oxidation of PAPC generates phospholipid aldehydes at m/z 594, 620 and 650 corresponding to POVPC, POHPC, and PHOOPC respectively and PGPC (m/z 610.5). Incubation of this mixture with the AKR1A4 (B), AKR1B1 (C), AKR1B7 (D) or AKR1B8 (E) resulted in the reduction of these phospholipid aldehydes to their corresponding alcohols, as evinced by a 2 Da increase in their m/z values.

Figure 3. AKR1B8 catalyses the reduction of aldehydes generated from the oxidation of pPAPC, 1-alkyl-PAPC (1-O-hexadecyl-2-arachidonoyl-phosphocholine), PAPG and PAPA

Aliquots of pPAPC, 1-alkyl-PAPC, PAPG and PAPA (50 μg each) were oxidized in air for 24–72 h and resuspended in 0.1 M potassium phosphate buffer (pH 6.0) containing 0.15 mM NADPH and either left untreated or incubated with 100 μg of recombinant AKR1B8 for 3 h at 30 °C. The phospholipids were extracted and analysed by ESI–MS. Mass spectrum shows that oxidation of pPAPC (A) resulted in the formation of phospholipid aldehydes, pPOVPC (m/z 578.5), pPOHPC (m/z 604.4) and pPHOOPC (m/z 634.5), and pPGPC (m/z 594.5). The phospholipid aldehydes were reduced by AKR1B8 (B) to pPHVPC (m/z 580.5), pPHHPC (606.5) and pPDHOPC (636.7). Likewise, oxidation of 1-alkyl-PC (C) resulted in the formation of alkyl POVPC (m/z 580.5), alkyl PGPC (m/z 596.5), alkyl POHPC (m/z 606.5) and alkyl PHOOPC (m/z 636.5); oxidation of PAPG (E) resulted in the formation of POVPG (m/z 581.5), PGPG (m/z 597.5), POHPG (m/z 607.4) and PHOOPG (m/z 637.6), and oxidation of PAPA (G) resulted in the formation of POVPA (m/z 507.4), PGPA (m/z 523.4), POHPA (m/z 533.5) and PHOOPA (m/z 563.5). AKR1B8 efficiently catalysed the reduction of phospholipid aldehydes generated from the oxidation of 1-alkyl-PAPC (D), PAPG (F) and PAPA (H) to their corresponding alcohols, as evident by an increase of 2 Da in their m/z values. Oxidation of PAPG also resulted in the formation of a strong ion with m/z 613.5 (n=9), which disappeared upon reduction. Structural identity of this peak has not yet been established.