Interfacial kinetic and binding properties of mammalian group IVB phospholipase A2 (cPLA2beta) and comparison with the other cPLA2 isoforms

Abstract

The cytosolic (group IV) phospholipase A(2) (cPLA(2)s) family contains six members. We have prepared recombinant proteins for human α, mouse β, human γ, human δ, human ε, and mouse ζ cPLA(2)s and have studied their interfacial kinetic and binding properties in vitro. Mouse cPLA(2)β action on phosphatidylcholine vesicles is activated by anionic phosphoinositides and cardiolipin but displays a requirement for Ca(2+) only in the presence of cardiolipin. This activation pattern is explained by the effects of anionic phospholipids and Ca(2+) on the interfacial binding of mouse cPLA(2)β and its C2 domain to vesicles. Ca(2+)-dependent binding of mouse cPLA(2)β to cardiolipin-containing vesicles requires a patch of basic residues near the Ca(2+)-binding surface loops of the C2 domain, but binding to phosphoinositide-containing vesicles does not depend on any specific cluster of basic residues. Human cPLA(2)δ also displays Ca(2+)- and cardiolipin-enhanced interfacial binding and activity. The lysophospholipase, phospholipase A(1), and phospholipase A(2) activities of the full set of mammalian cPLA(2)s were quantified. The relative level of these activities is very different among the isoforms, and human cPLA(2)δ stands out as having relatively high phospholipase A(1) activity. We also tested the susceptibility of all cPLA(2) family members to a panel of previously reported inhibitors of human cPLA(2)α and analogs of these compounds. This led to the discovery of a potent and selective inhibitor of mouse cPLA(2)β. These in vitro studies help determine the regulation and function of the cPLA(2) family members.

FIGURE 1.

Specific activity of m-cPLA₂β on [¹⁴C]PAPC vesicles as a function of the mol % of PI(3,4,5)P₃ (200 μm total phospholipids). Filled circles, 0 μm Ca²⁺; open circles, 2 μm Ca²⁺; filled diamonds, 20 μm Ca²⁺.

FIGURE 2.

Activity of m-cPLA₂β on vesicles containing 23 mol % POPC, 40 mol % POPE, and 30 mol % POPS (200 μm total phospholipids) without PI(3,4,5)P₃ or vesicles containing 20 mol % POPC, 37 mol % POPE, 27 mol % POPS, and 10 mol % PI(3,4,5)P₃ in the absence or presence of 20 μm Ca²⁺. Vesicles also contained a trace of [¹⁴C]PAPC (100,000 dpm). Activity is expressed as percent of [¹⁴C]PAPC hydrolyzed.

FIGURE 3.

Specific activity of m-cPLA₂β on [¹⁴C]PAPC vesicles as a function of the mol % of CL in vesicles. Total phospholipid concentration was 200 μm. All reactions contained 20 μm Ca²⁺ and 2 μg of m-cPLA₂β at 37 °C for 20 min.

FIGURE 4.

Specific activity of m-cPLA₂β on [¹⁴C]PAPC/CL vesicles as a function of [Ca²⁺]. Top panel, vesicles contain 10 mol % CL; bottom panel, vesicles contain 30 mol % CL. In both cases reactions contained 200 μm total phospholipid and 1 μg of m-cPLA₂β at 37 °C for 10 min.

FIGURE 5.

Binding of m-cPLA₂β to vesicles. Binding reactions contained 200 μm total phospholipid as vesicles and 62 ng of enzyme in buffer with or without 20 μm free Ca²⁺. See “Experimental Procedures” for more information. Plotted is the amount of enzyme in the supernatant above pelleted vesicles expressed as a percentage of the amount of enzyme measured in a binding reaction that lacked vesicles. See “Experimental Procedures” for other details.

FIGURE 6.

Interfacial binding to vesicles. Top panel, SDS-PAGE analysis of m-cPLA₂β-C2 in the pelleted vesicle fraction: lane 1, PAPC vesicles + 0 μm Ca²⁺; lane 2, PAPC vesicles + 20 μm Ca²⁺; lane 3, PAPC, /10 mol % PI(3,4,5)P₃ vesicles + 0 μm Ca²⁺; lane 4, PAPC, 10 mol % PI(3,4,5)P₃ vesicles + 20 μm Ca²⁺. Bottom panel, SDS-PAGE analysis of h-cPLA₂α-C2 in the pelleted vesicle fraction: lane 1, PAPC vesicles + 0 μm Ca²⁺; lane 2, PAPC/10 mol % PI(3,4,5)P₃ vesicles + 0 μm Ca²⁺; lane 3, PAPC vesicles + 20 μm Ca²⁺; lane 4, PAPC/10 mol % PI(3,4,5)P₃ vesicles + 20 μm Ca²⁺. Data shown are representative of two independent SDS-PAGE analyses.

FIGURE 7.

Basic residues of m-cPLA₂β-C2. Basic residues mutated to asparagine are as follows: Arg-17/Arg-55 (red); Lys-24/Arg-49/Lys-52 (yellow); Arg-14/Arg-107/Lys-117 (magenta); Lys-78 (orange); His-44/His-82 (green); Arg-66/Arg-69/Arg-125 (blue); His-64/His-68 (gray); Lys-72 (pink). The Ca²⁺-binding surface loops are shown at the top panel. The left image is rotated ∼180° along the vertical axis to give the right image.

FIGURE 8.

Effect of CL and basic residue mutations on interfacial binding of m-cPLA₂β-C2 to vesicles. Top panel, SDS-PAGE analysis of the pelleted vesicle fraction: lane 1, m-cPLA₂β-C2 (5 μg) in the absence of vesicles; lane 2, PAPC, 10 mol % CL vesicles + 0 μm Ca²⁺; lane 3, PAPC, 10 mol % CL vesicles + 20 μm Ca²⁺; lane 4, PAPC, 20 mol % CL vesicles + 0 μm Ca²⁺; lane 5, PAPC, 20 mol % CL vesicles + 20 μm Ca²⁺; lane 6, PAPC, 30 mol % CL vesicles + 0 μm Ca²⁺; lane 7, PAPC, 30 mol % CL vesicles + 20 μm Ca²⁺. Middle panel, lane 1, wild type m-cPLA₂β-C2 (5 μg) in the absence of vesicles; lane 2, vesicle pellet fraction using PAPC, 30 mol % CL vesicles with wild type m-cPLA₂β-C2 + 0 μm Ca²⁺; lane 3, same as lane 2 but + 20 μm Ca²⁺; lane 4, same as lane 2 but with K24N/R49N/K52N mutant of m-cPLA₂β-C2 + 0 μm Ca²⁺; lane 5, same as lane 4 but + 20 μm Ca²⁺; lane 6, same as lane 2 but with the H44N/H82N mutant of m-cPLA₂β-C2 + 0 μm Ca²⁺; lane 7, same as lane 6 but + 20 μm Ca^2+; lane 8, same as lane 2 but with the K78N mutant of m-cPLA₂β-C2 + 0 μm Ca²⁺; lane 9, same as lane 8 but with 20 μm Ca²⁺. Bottom panel, binding of h-cPLA₂δ to vesicles (SDS-PAGE analysis of the pelleted vesicle fraction): lane 1, PAPC vesicles + 0 μm Ca²⁺; lane 2, PAPC vesicles + 20 μm Ca²⁺; lane 3, PAPC, 10% CL vesicles + 0 μm Ca²⁺; lane 4, PAPC, 10% CL vesicles + 20 μm Ca²⁺.