Catalytic reduction of carbonyl groups in oxidized PAPC by Kvβ2 (AKR6)
Introduction
The β-subunits of the voltage gated potassium channel belong to the aldo-keto reductase (AKR) superfamily 6 (AKR6). The function of voltage-gated potassium channel (Kv) is essential for several physiological processes, including muscle contraction, neuronal excitation, and secretion. Despite extensive investigation, the exact biological role of Kvβ-subunit remains unclear. Identification of Kvβ substrates is important for understanding the biological role of Kv channels, how they are regulated, and how the activity of these channels could be therapeutically altered to treat diseases such as hypertension, epilepsy, and arrhythmias. Our previous studies have shown that Kvβ subunits bind to pyridine nucleotides with high affinity [1], [2]. Sequence analysis of Kvβs shows that the proteins belong to the AKR superfamily [3], however, their catalytic properties and substrate specificities remain poorly understood. Recent work by Weng et al. shows that these proteins display weak catalytic activity with model chemical substrates such as 4-cyanobenzaldehyde and 4-carboxybenzaldehyde [4]. In addition, investigations in our laboratory have shown that Kvβ can reduce both aldehydes and ketones, and that naturally occurring compounds such as POVPC (1-palmitoyl-2-oxovaleroyl-3-phosphatidylcholine), and PGJ2 have highest specific activity with Kvβ2 [5].
Products of lipid peroxidation, such as POVPC, however, are seldom generated in isolation. Lipid peroxidation generates several structurally similar aldehydes and ketones, several of which could also be potential Kvβ substrates. Hence, to identify which products of lipid peroxidation are reduced by Kvβ, we examined the activity of this protein with oxidized 1-palmitoyl-2-arachidonoyl-3-phosphotidyl choline (oxPAPC). The oxPAPC is a major component of minimally modified LDL (mmLDL) [6]. It has been detected in the atherosclerotic lesions of animals and humans [7], [8]. Oxidation of PAPC generates several highly reactive carbonyl compounds. Structurally, a typical oxidation product of PAPC contains a glycerol backbone with a palmitoyl group esterified at the sn1 position, an oxidized fatty acyl group at the sn2 position, and a phosphatidyl choline head group at the sn3 position (Fig. 1A). In a previous study, we have shown that aldose reductase (AKR1B1), and other members of AKR superfamily have high catalytic activity for products of oxPAPC that contain aldehyde groups [9].
To determine which of the oxidation products of PAPC are reduced by Kvβ, we preformed electrospray ionization mass spectrometric (ESI-MS) analysis of oxPAPC incubated with Kvβ2. Our results show that Kvβ2 efficiently reduces both aldehydes and ketones in oxidized phospholipids and therefore might play a regulatory role in sensing the carbonyl products that are generated during lipid oxidation.
Section snippets
Kvβ2 expression and purification
The C-terminal domain of rat Kvβ2 (amino acids 39–367) with a His tag at its N-terminus was expressed in the BL-21 strain of Escherichia coli, as described earlier [1]. The His-tagged protein was purified using Profinia Protein Purification System (Bio-Rad) with a 1 ml Bio-Scale Mini Profinity IMAC cartridge (Bio-Rad) according to manufacturer's instructions. The purified protein was suspended in 0.2 M potassium phosphate buffer (pH 7.4) immediately after the purification using a 10 ml Bio-Gel P-6
Air oxidation of PAPC and generation of lipid-carbonyls
As shown in Fig. 1, air oxidation of PAPC for 72 h resulted in the formation of oxPAPC containing several compounds with higher and lower m/z values than the parent compound (m/z 782). As previously reported, the major ion at m/z 594 was ascribed to POVPC (1-palmitoyl-2-oxo-valaroyl-3-phosphotidyl choline) compound 1 [9], whereas the ions at m/z 810 and 828 were epoxy isoprostane compounds 2 and 3: 1-palmitoyl-2-(5,6)- epoxyisoprostane E2-sn-glycero-3-phosphocholine (PEIPC) and
Discussion
This study demonstrates that Kvβ2 catalyzes the reduction of several structurally similar compounds in oxPAPC mixture, which suggests that Kvβ2 subunits can play a regulatory role in sensing the carbonyl products that are generated during lipid oxidation. Sequence homology studies indicate that Kvβ subunits belong to the AKR superfamily of proteins. The β-subunits of shaker potassium channel have β/α8 barrel structure with tight NADPH binding and have 20–25% homology to other AKRs [12]. Kvβ 1
Conflict of interest statement
None.
Acknowledgements
The authors thankfully acknowledge the research support provided by NIH (HL-54477, HL-59378, ES-11860; to AB), NCRR (RR024489), University of Louisville Clinical and Translational Science award 20017 and HL-089372 (to OAB), and American Heart Association Beginning Grant-in-Aid 0865466D (to SMT).
References (17)
- et al.
Binding of pyridine nucleotide coenzymes to the beta-subunit of the voltage-sensitive K+ channel
J. Biol. Chem.
(2001) - et al.
NADPH binding to beta-subunit regulates inactivation of voltage-gated K(+) channels
Biochem. Biophys. Res. Commun.
(2007) - et al.
Shaker K+ channel beta subunits belong to an NAD(P)H-dependent oxidoreductase superfamily
Cell
(1994) - et al.
Modulation of voltage-dependent Shaker family potassium channels by an aldo-keto reductase
J. Biol. Chem.
(2006) - et al.
Bioactive products of phospholipid oxidation: isolation, identification, measurement and activities
Free Radic. Biol. Med.
(2000) - et al.
Structural identification by mass spectrometry of oxidized phospholipids in minimally oxidized low density lipoprotein that induce monocyte/endothelial interactions and evidence for their presence in vivo
J. Biol. Chem.
(1997) - et al.
Aldose reductase-catalyzed reduction of aldehyde phospholipids
J. Biol. Chem.
(2004) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding
Anal. Biochem.
(1976)
Cited by (0)
- 1
These authors made equal contribution.