A comparison of the molecular species compositions of mammalian lung surfactant phospholipids.

While dipalmitoyl phosphatidylcholine (PC16:0/16:0) is essential for pulmonary surfactant function, roles for other individual molecular species of surfactant phospholipids have not been established. If any phospholipid species other than PC16:0/16:0 is important for surfactant function, then it may be conserved across animal species. Consequently, we have quantified, by electrospray ionisation mass spectrometry, molecular species compositions of phosphatidylcholine (PC), phosphatidylglycerol (PG) and phosphatidylinositol (PI) in surfactants from human, rabbit, rat and guinea pig lungs. While PC compositions displayed only relatively minor variations across the animal species studied, there were wide variations of PG and PI concentrations and compositions. Human surfactant PG and PI were enriched in the same three monounsaturated species (PG16:0/18:1, PG18:1/18:1 and PG18:0/18:1) with minimal amounts of PG16:0/16:0 or polyunsaturated species, while all animal surfactant PG contained increased concentrations of PG16:0/16:0 and PG16:0/18:2. Animal surfactant PIs were essentially monounsaturated except for a high content of PI18:0/20:4 (29%) in the rat. As these four surfactants all maintain appropriate lung function of the respective animal species, then all their varied compositions of acidic phospholipids must be adequate at promoting the processes of adsorption, film refinement, respreading and collapse characteristic of surfactant. We conclude that this effectively monounsaturated composition of anionic phospholipid molecular species is a common characteristic of mammalian surfactants.

The antibacterial properties of secreted phospholipases A(2).

There is a considerable body of evidence to support the antibacterial properties of the group IIa phospholipase A(2) as an important physiological function. This enzyme is able to act as an acute phase protein and may be part of the innate defence system of the body, acting in concert with other antibacterial proteins and peptides. The enzyme is most effective against Gram-positive bacteria whereas penetration of the lipopolysaccharide coat of Gram-negative bacteria requires bactericidal/permeability-increasing protein (BPI) as an additional permeabilizing factor. The global cationic nature of this protein (pI>10.5) appears to facilitate penetration of the anionic bacterial cell wall. In addition, the considerable preference of the enzyme for anionic phospholipid interfaces provides specificity toward anionic bacterial membranes as opposed to zwitterionic eucaryotic cell membranes.

Bacterial cell membrane hydrolysis by secreted phospholipases A(2): a major physiological role of human group IIa sPLA(2) involving both bacterial cell wall penetration and interfacial catalysis.

The ability of human group IIa secreted phospholipase A(2) (human sPLA(2)) to hydrolyse the phospholipid membrane of whole cell suspensions of Gram-positive bacteria is demonstrated in real time using a continuous fluorescence displacement assay. Micrococcus luteus is used as a model system and demonstrates an almost absolute specificity for this human enzyme compared with porcine pancreatic and Naja naja venom sPLA(2)s. This specificity is due to selective penetration of the highly cationic human sPLA(2)50%) phospholipid hydrolysis was observed and this was confirmed by electrospray mass spectrometry that allowed the identification of several molecular species of phosphatidylglycerol as the targets for hydrolysis. However, the bactericidal activity of the human enzyme under these assay conditions was low, highlighting the capacity of the organism to survive a major phospholipid insult. In addition to pure enzyme, the human sPLA(2) activity in tears was demonstrated using M. luteus as substrate. In comparison to M. luteus, cell suspensions of Staphylococcus aureus were highly resistant to hydrolysis by human sPLA(2) as well as to the pancreatic and venom enzymes. Treatment of this organism with the specific cell wall protease lysostaphin resulted in a dramatic enhancement in cell membrane phospholipid hydrolysis by all three sPLA(2)s. Overall, the results highlight the potential of the human sPLA(2) as a selective antimicrobial agent against Gram-positive bacteria in vivo because this enzyme is essentially inactive against mammalian plasma membranes. However, the enzyme will be most effective in combination with other antimicrobial agents that enhance the permeability of the bacterial cell wall and where potentiation of the effectiveness of other antibiotics would be expected.

Microsomal fatty acyl-CoA transacylation and hydrolysis: fatty acyl-CoA species dependent modulation by liver fatty acyl-CoA binding proteins.

arachidonoyl-CoA. In summary, the data established for the first time a role for both L-FABP and ACBP in microsomal phosphatidic acid biosynthesis. By preferentially stimulating microsomal transacylation of unsaturated long chain fatty acyl-CoAs while concomitantly exerting their differential protection from microsomal acyl-CoA hydrolase, L-FABP and ACBP can uniquely function in modulating the pattern of fatty acids esterified to phosphatidic acid, the de novo precursor of phospholipids and triacylglycerols. This may explain in part the simultaneous presence of these proteins in cell types involved in fatty acid absorption and lipoprotein secretion.

Binding of recombinant rat liver fatty acid-binding protein to small anionic phospholipid vesicles results in ligand release: a model for interfacial binding and fatty acid targeting.

A number of intracellular proteins bind to negatively charged phospholipid membranes, and this interfacial binding results in a conformational change that modulates the activity of the protein. Using a fluorescent fatty acid analogue, 11-[5-(dimethylamino)naphthalenesulfonyl]undecanoic acid (DAUDA), it is possible to demonstrate the release of this ligand from recombinant rat liver FABP in the presence of phospholipid vesicles that contain a significant proportion of anionic phospholipids. The ligand release that is observed with anionic phospholipids is sensitive to the ionic strength of the assay conditions and the anionic charge density of the phospholipid at the interface, indicating that nonspecific electrostatic interactions play an important role in the process. The stoichiometric relationship between anionic phospholipid and liver FABP suggests that the liver FABP coats the surface of the phospholipid vesicle. The most likely explanation for ligand release is that interaction of FABP with an anionic membrane interface induces a rapid conformational change, resulting in a reduced affinity of DAUDA for the protein. The nature of this interaction involves both electrostatic and nonpolar interactions as maximal release of liver FABP from phospholipid vesicles with recovery of ligand binding cannot be achieved with high salt and requires the presence of a nonionic detergent. The precise interfacial mechanism that results in the rapid release of ligand from L-FABP remains to be determined, but studies with two mutants, F3W and F18W, suggest the possible involvement of the amino-terminal region of the protein in the process. The conformational change linked to interfacial binding of this protein could provide a mechanism for fatty acid targeting within the cell.

Interfacial binding of secreted phospholipases A(2): more than electrostatics and a major role for tryptophan.

Secreted phospholipases A(2) have similar catalytic sites, but vastly different interfacial binding surfaces that modulate their binding affinity for different kinds of phospholipid vesicles by several orders of magnitude. The structure/function principles that dictate both the differential interfacial binding and the physiological function of these enzymes are beginning to be unraveled.

1,3-Dioxane-4,6-dione-5-carboxamide-based inhibitors of human group IIA phospholipase A: X-ray structure of the complex and interfacial selection of inhibitors from a structural library.

A library of 109 1,3-dioxane-4,6-dione-5-carboxamides was prepared by solution-phase methods as potential inhibitors of human group IIa phospholipase A2. Tight binding inhibitors were found by an interfacial affinity selection method. The crystal structure of the secreted phospholipase A2 containing one of the inhibitors was determined, and it reveals the inhibitor-calcium bidendate coordination.

Roles of Trp31 in high membrane binding and proinflammatory activity of human group V phospholipase A2.

Group V phospholipase A2 is a recently discovered secretory phospholipase A2 (PLA2) that has been shown to be involved in eicosanoid formation in inflammatory cells, such as macrophages and mast cells. We have demonstrated that human group V PLA2 (hsPLA2-V) can bind phosphatidylcholine (PC) membranes and hydrolyze PC substrates much more efficiently than human group IIa PLA2, which makes it better suited for acting on the outer plasma membrane (Han, S.-K., Yoon, E. T., and Cho, W. (1998) Biochem. J. 331, 353-357). In this study, we demonstrate that exogenous hsPLA2-V has much greater activity than does group IIa PLA2 to release fatty acids from various mammalian cells and to elicit leukotriene B4 formation from human neutrophils. To understand the molecular basis of these activities, we mutated two surface tryptophans of hsPLA2-V to alanine (W31A and W79A) and measured the effects of these mutations on the kinetic activity toward various substrates, on the binding affinity for vesicles and phospholipid-coated beads, on the penetration into phospholipid monolayers, and on the activity to release fatty acids and elicit eicosanoid formation from various mammalian cells. These studies show that the relatively high ability of hsPLA2-V to induce cellular eicosanoid formation derives from its high affinity for PC membranes and that Trp31 on its putative interfacial binding surface plays an important role in its binding to PC vesicles and to the outer plasma membrane.

Action of human group IIa secreted phospholipase A2 on cell membranes. Vesicle but not heparinoid binding determines rate of fatty acid release by exogenously added enzyme.

Human group IIa phospholipase A2 (hIIa-PLA2) is a highly basic protein that is secreted from a number of cells during inflammation and may play a role in arachidonate liberation and in destruction of invading bacteria. It has been proposed that rodent group IIa PLA2 is anchored to cell surfaces via attachment to heparan sulfate proteoglycan and that this interaction facilitates lipolysis. hIIa-PLA2 contains 13 lysines, 2 histidines, and 10 arginines that fall into 10 clusters. A panel of 26 hIIa-PLA2 mutants were prepared in which 1-4 basic residues in each cluster were changed to glutamate or aspartate (charge reversal). A detailed analysis of the affinities of these mutants for anionic vesicles and for heparin and heparan sulfate in vitro and of the specific activities of these proteins for hydrolysis of vesicles in vitro and of living cell membranes reveal the following trends: 1) the affinity of hIIa-PLA2 for heparin and heparan sulfate is modulated not by a highly localized site of basic residues but by diffuse sites that partially overlap with the interfacial binding site. In contrast, only those residues on the interfacial binding site of hIIa-PLA2 are involved in binding to membranes; 2) the relative ability of these mutants to hydrolyze cellular phospholipids when enzymes were added exogenously to CHO-K1, NIH-3T3, and RAW 264.7 cells correlates with their relative in vitro affinity for vesicles and not with their affinity for heparin and heparan sulfate. 3) The rates of exogenous hIIa-PLA2-catalyzed fatty acid release from wild type CHO-K1 cells and two mutant lines, one lacking glycosaminoglycan and one lacking heparan sulfate, were similar. Thus basic residues that modulate interfacial binding are important for plasma membrane fatty acid release by exogenously added hIIa-PLA2. Binding of hIIa-PLA2 to cell surface heparan sulfate does not modulate plasma membrane phospholipid hydrolysis by exogenously added hIIa-PLA2.

Tryptophan-containing mutant of human (group IIa) secreted phospholipase A2 has a dramatically increased ability to hydrolyze phosphatidylcholine vesicles and cell membranes.

Human nonpancreatic (group IIa) secreted phospholipase A2 (human sPLA2) is associated with a number of inflammatory disorders in which the extracellular concentrations of this enzyme can become highly elevated. It is probable that the enzyme normally acts as an acute-phase protein whose function is to facilitate the removal of infectious organisms or damaged host cells as part of the normal inflammatory response. The enzyme shows negligible activity with phosphatidylcholine (PC) vesicles and cell membranes, presumably reflecting the enzyme's lack of ability to bind productively to such condensed neutral interfaces. Mammalian pancreatic enzymes show modest activity with such interfaces and contain a unique tryptophan at position 3, which is part of the presumptive interfacial binding surface of these enzymes. Human sPLA2 does not contain tryptophan. The amphiphilic indole side chain of tryptophan is noted for its ability to penetrate the lipid interface of membranes, and tryptophan residues appear to be associated with the ability of lipases and phospholipases A2 to bind to and hydrolyze such interfaces. We have investigated in detail the properties of a V3W mutant of human sPLA2, which has a unique tryptophan on the interfacial binding surface of this enzyme. Although this enzyme shows a modest ( approximately 50%) reduction in activity when anionic substrates are used under standard assay conditions, the activity of the enzyme on phosphatidylcholine vesicles and cell membranes is dramatically increased compared with human sPLA2. This is particularly the case with small unilamellar vesicles of PC, where activity is enhanced over 250-fold compared to the almost zero activity expressed by human sPLA2. This enhanced activity is best explained by increased interfacial binding and activation of the V3W mutant and is not due to enhanced active-site binding and hydrolysis. The results highlight the important role that tryptophan residues can play in interfacial binding, particularly to condensed zwitterionic interfaces. The interfacial characteristics of the mutant human enzyme now resemble more closely the mammalian pancreatic enzymes that already have a tryptophan at position 3.

Monoacylglycerol binding to human serum albumin: evidence that monooleoylglycerol binds at the dansylsarcosine site.

The binding of monoacylglycerides of long-chain fatty acids to human serum albumin has been examined using monooleoylglycerol as the ligand. Binding was investigated using changes in tryptophan fluorescence and also the displacement of a variety of well-studied fluorescent ligands from human serum albumin (HSA). Monooleoylglycerol caused a decrease in fluorescence from tryptophan-214 when measured at 350 nm while oleic acid had no effect on fluorescence at this wavelength and did not compete with monooleoylglycerol. In contrast, oleic acid caused an increase in fluorescence at 330 nm whereas monooleoylglycerol did not affect fluorescence intensity at this wavelength. These results suggest that these two ligands do not bind to the same site on HSA. From competition studies using dansylglycine, dansylsarcosine, 11-(dansylamino)-undecanoic acid and 1-anilino-8-naphthalenesulfonic acid it was proposed that monooleoylglycerol binds at the dansylsarcosine site (site II) of HSA. Monooleoylglycerol was a competitive inhibitor of dansylsarcosine binding with a Kd of about 2.5 microM whereas oleic acid was not competitive with dansylsarcosine binding.

Inhibition of secreted phospholipases A2 by annexin V. Competition for anionic phospholipid interfaces allows an assessment of the relative interfacial affinities of secreted phospholipases A2.

The ability of annexins, particularly annexin 1 (lipocortin 1), to inhibit phospholipase A2 (PLA2) is well known and a substrate depletion mechanism is now widely accepted as the explanation for most inhibitory studies. In this investigation we have examined the substrate depletion mechanism of annexin V using a variety of phospholipid substrates and secreted PLA2's (sPLA2). The results suggest that the term interfacial competition best describes the inhibitory effect of annexin V although the overall inhibitory process remains one of substrate sequestration by the annexin. We have utilised the competitive nature of the interaction of enzyme and annexin V for a phospholipid interface as a means of quantifying the relative affinity of sPLA2's for anionic phospholipid vesicles. The results highlight the very high affinity of the human non-pancreatic sPLA2 for such vesicles (Kd<<10-(10) M) while the Naja naja venom PLA2 and porcine pancreatic sPLA2 showed lower affinities. Hydrolysis of mixed vesicles containing phosphatidylserine and phosphatidylcholine by the venom and pancreatic enzymes were differentially inhibited by annexin V. This difference must reflect the preference of both annexin V and the pancreatic enzyme for an anionic phospholipid interface. In contrast, the venom enzyme is able to readily hydrolyse phosphatidylcholine domains that would be minimally affected by annexin V. Annexin V was an effective inhibitor of cardiolipin hydrolysis by the pancreatic PLA2, however the inhibition was of a more complex nature than seen with other phospholipids tested. Overall the results highlight the ability of annexin V to inhibit phospholipid hydrolysis by sPLA2's by an interfacial competition (substrate depletion) mechanism. The effectiveness of annexin V as an apparent inhibitor depends on the nature of the enzyme and the phospholipid substrate.

Cardiolipin hydrolysis by human phospholipases A2. The multiple enzymatic activities of human cytosolic phospholipase A2.

The ability of mammalian phospholipases A2 (PLA2) to hydrolyse cardiolipin (diphosphatidylglycerol) was monitored with a fluorescent displacement assay which allows the use of natural phospholipid substrates. The mammalian enzymes used were porcine pancreatic (Group I) secretory PLA2 (sPLA2), human non-pancreatic (Group II) sPLA2 and human cytosolic PLA2 (cPLA2). High activity was observed with porcine pancreas sPLA2 whereas the human sPLA2 demonstrated only minimal activity with this substrate. In comparison, sPLA2 from Naja naja venom (Group I) also showed only modest activity with this substrate. Since many lipases possess PLA1 activity, a representative enzyme from Rhizopus arrhizus was also assessed for its ability to hydrolyse cardiolipin which proved to be a good substrate for this fungal lipase. In all cases dilysocardiolipin was the major product while some monolyso intermediate was detected after chromatographic separation. Human cPLA2 was unable to hydrolyse cardiolipin at a significant rate, however, both monolysocardiolipin and dilysocardiolipin, which are prepared by the PLA2-catalysed hydrolysis of cardiolipin, were good substrates providing a further example of the extensive lysophospholipase activity of this enzyme. Moreover, cardiolipin that was initially hydrolysed in situ with either excess porcine pancreatic PLA2 or R. arrhizus lipase (PLA1) was subsequently hydrolysed by human cPLA2. One explanation of this result is that human cPLA2 is able to hydrolyse both 1-acyl and 2-acyl-lysophospholipids. (c) 1998 Elsevier Science B.V.