Tryptophan-Containing Mutant of Human (Group IIa) Secreted Phospholipase A<sub>2</sub> Has a Dramatically Increased Ability To Hydrolyze Phosphatidylcholine Vesicles and Cell Membranes

120

171

Mammals contain 9 -10 secreted phospholipases A 2 (sPLA 2 s) that display widely different affinities for membranes, depending on the phospholipid composition. The much higher enzymatic activity of human group X sPLA 2 (hGX) compared with human group IIA sPLA 2 (hGIIA) on phosphatidylcholine (PC)-rich vesicles is due in large part to the higher affinity of the former enzyme for such vesicles; this result also holds when vesicles contain cholesterol and sphingomyelin. The inclusion of anionic phosphatidylserine in PC vesicles dramatically enhances interfacial binding and catalysis of hGIIA but not of hGX. This is the result of the large number of lysine and arginine residues scattered over the entire surface of hGIIA, which cause the enzyme to form a supramolecular aggregate with multiple vesicles. Thus, high affinity binding of hGIIA to anionic vesicles is a complex process and cannot be attributed to a few basic residues on its interfacial binding surface, as is also evident from mutagenesis studies. The main reason hGIIA binds poorly to PC-rich vesicles is that it lacks a tryptophan residue on its interfacial binding surface, a residue that contributes to the high affinity binding of hGX to PC-rich vesicles. Results show that the lag in the onset of hydrolysis of PC vesicles by hGIIA is due in part to the poor affinity of this enzyme for these vesicles. Binding affinity of hGIIA, hGX, and their mutants to PC-rich vesicles is well correlated to the ability of these enzymes to act on the PC-rich outer plasma membrane of mammalian cells.

Section: Resultsmentioning

confidence: 66%

Section: Ibs Basic Residues Are Not the Critical Factor Governing Intmentioning

confidence: 99%

On the Binding Preference of Human Groups IIA and X Phospholipases A2 for Membranes with Anionic Phospholipids

Bezzine¹,

Bollinger²,

Singer³

et al. 2002

120

171

“…2C), which is considerably lower than the activities of hIBPLA 2 and hIIAPLA 2 , 24 Ϯ 5 and 52 Ϯ 8 mol/min/mg, respectively (shown are mean values and standard deviations from three experiments). Although hIIAPLA 2 shows little activity toward zwitterionic phosphatidylcholine membranes (30,31), it is more active than hIBPLA 2 toward micelles of short chain DHTPC. A possible explanation of this is that the extremely high excess positive charge of hIIAPLA 2 (ϳ15 excess cationic charges at pH 7.4, see TABLE ONE) prevents binding to zwitterionic membranes because of strong positive surface charge of membranes that rapidly builds up upon binding of a few PLA 2 molecules.…”

Section: Resultsmentioning

confidence: 94%

Evidence for the Regulatory Role of the N-terminal Helix of Secretory Phospholipase A2 from Studies on Native and Chimeric Proteins

Qin

Pande

Nemec

et al. 2005

The phospholipase A 2 (PLA 2 ) enzymes are activated by binding to phospholipid membranes. Although the N-terminal ␣-helix of group I/II PLA 2 s plays an important role in the productive mode membrane binding of the enzymes, its role in the structural aspects of membrane-induced activation of PLA 2 s is not well understood. In order to elucidate membrane-induced conformational changes in the N-terminal helix and in the rest of the PLA 2 , we have created semisynthetic human group IB PLA 2 in which the N-terminal decapeptide is joined with the 13 C-labeled fragment, as well as a chimeric protein containing the N-terminal decapeptide from human group IIA PLA 2 joined with a 13 C-labeled fragment of group IB PLA 2 . Infrared spectral resolution of the unlabeled and 13 C-labeled segments suggests that the N-terminal helix of membranebound IB PLA 2 has a more rigid structure than the other helices. On the other hand, the overall structure of the chimeric PLA 2 is more rigid than that of the IB PLA 2 , but the N-terminal helix is more flexible. A combination of homology modeling and polarized infrared spectroscopy provides the structure of membrane-bound chimeric PLA 2 , which demonstrates remarkable similarity but also distinct differences compared with that of IB PLA 2 . Correlation is delineated between structural and membrane binding properties of PLA 2 s and their N-terminal helices. Altogether, the data provide evidence that the N-terminal helix of group I/II PLA 2 s acts as a regulatory domain that mediates interfacial activation of these enzymes.Enzymes of the phospholipase A 2 (PLA 2 ) 2 family hydrolyze the sn-2 ester bond of diacylglycerophospholipids and thus initiate the biosynthesis of lipid-derived mediators, such as eicosanoids and platelet-activating factor, which are potent mediators of inflammation, allergy, and tumorigenesis (1-3). These enzymes are divided into several groups and subgroups based on their amino acid sequences, tissue distribution, and functional features (1, 2).PLA 2 s gain their full activity only when they bind to phospholipid micelles or membranes, an effect known as interfacial activation (4 -6). The molecular mechanism of interfacial activation has been the subject of debate over several decades. Initially, similarities of crystal structures of PLA 2 s with and without bound substrate mimics argued against conformational changes in the enzymes during interfacial activation (4). Later, the interfacially activated form of group IB PLA 2 was modeled by anion-mediated dimers (7), which revealed significant conformational changes compared with "inactive" monomeric forms, mainly involving the loop that has the functionally important Tyr 69 , and the presence of an assisting water molecule (8). Several spectroscopic studies also identified conformational changes in group IB and IIA PLA 2 s upon binding to phospholipid micelles or membranes. Fluorescence studies indicated that the N-terminal helix of porcine group IB PLA 2 becomes rigid upon enzyme-substrate complex formation at the m...

“…Tryptophan on the membrane binding surface of hGV is important for the high catalytic activity of this sPLA 2 on PCrich membranes (81), and addition of a tryptophan to the membrane binding surface of hGIIA renders this enzyme about 2 orders of magnitude more active on PC-rich membranes (80). We have shown recently that the presence of a tryptophan on the membrane binding surface of hGX and its absence on that of hGIIA is more important than the presence or absence of cationic residues (lysine and arginine) on the membrane binding surface of these sPLA 2 s for allowing high affinity binding to PC-rich membranes (23).…”

Section: Discussionmentioning

confidence: 99%

Interfacial Kinetic and Binding Properties of the Complete Set of Human and Mouse Groups I, II, V, X, and XII Secreted Phospholipases A2

Singer¹,

Ghomashchi²,

Calvez³

et al. 2002

313

506

Expression of the full set of human and mouse groups I, II, V, X, and XII secreted phospholipases A 2 (sPLA 2 s) in Escherichia coli and insect cells has provided pure recombinant enzymes for detailed comparative interfacial kinetic and binding studies. The set of mammalian sPLA 2 s display dramatically different sensitivity to dithiothreitol. The specific activity for the hydrolysis of vesicles of differing phospholipid composition by these enzymes varies by up to 4 orders of magnitude, and yet all enzymes display similar catalytic site specificity toward phospholipids with different polar head groups. Discrimination between sn-2 polyunsaturated versus saturated fatty acyl chains is <6-fold. These enzymes display apparent dissociation constants for activation by calcium in the 1-225 M range, depending on the phospholipid substrate. Analysis of the inhibition by a set of 12 active site-directed, competitive inhibitors reveals a large variation in the potency among the mammalian sPLA 2 s, with Me-Indoxam being the most generally potent sPLA 2 inhibitor. A dramatic correlation exists between the ability of the sPLA 2 s to hydrolyze phosphatidylcholine-rich vesicles efficiently in vitro and the ability to release arachidonic acid when added exogenously to mammalian cells; the group V and X sPLA 2 s are uniquely efficient in this regard.