Could anionic LDL be a ligand for RAGE and TREM2 in addition to LOX-1 and thus exacerbate lung disease and dementia?

We recently highlighted the potential of protein glycation to generate anionic (electronegative) surfaces. We hypothesised that these anionic proteins are perceived by the innate immune system as arising from infection or damaged cell components, producing an inflammatory response within the lung involving the receptor RAGE. We now review two other pathologies linked to the innate immune response, cardiovascular disease and dementia that involve receptors LOX-1 and TREM2 respectively. Remarkable similarities in properties between RAGE, LOX-1 and TREM2 suggest that electronegative LDL may act as a pathogenic anionic ligand for all three receptors and exacerbate lung inflammation and dementia.

Age, obesity and hyperglycaemia: Activation of innate immunity initiates a series of molecular interactions involving anionic surfaces leading to COVID-19 morbidity and mortality.

Obesity and type 2 diabetes are major factors in COVID-19 causing a progression to excessive morbidity and mortality. An important characteristic of these conditions is poor glycaemic control leading to inappropriate chemical reactions and the production of glycated proteins in which positively charged lysine and arginine residues are neutralised. We propose that this protein glycation primes the inflammatory system as the presence of aspartate and glutamate residues in any glycated zwitterionic protein will thus increase its anionic characteristics. As a result, these macromolecules will be recognised by the innate immune system and identified as originating from infection or cell damage (sterile inflammation). Many proteins in the body exist to non-specifically target these anionic macromolecules and rely heavily on positively charged (cationic) binding-sites to produce a relatively non-specific interaction as the first step in the body's response. Proteins involved in this innate immunity are collectively referred to as damage associated molecular pattern molecules or pathogen associated molecular pattern molecules. A crucial player in this process is RAGE (Receptor for Advanced Glycation End products). RAGE plays a central role in the inflammatory response and on ligand binding stimulates many aspects of inflammation including the production of the key inflammatory mediator NF-κB, and the subsequent production of inflammatory cytokines. This process has the potential to show a positive feedback loop resulting in a dramatic response within the tissue. We propose that protein glycation primes the inflammatory system by generating negatively charged surfaces so that when a SARS-Cov-2 infection occurs within the lung the further release of negatively-charged macromolecules due to cell damage results in a potentially catastrophic inflammatory response resulting in the cytokine storm associated with COVID-19 morbidity and mortality. That part of the population who do not suffer from inflammatory priming (Phase 1), such as the young and the non-obese, should not be subjected to the catastrophic inflammatory response seen in others (Phase 2). This hypothesis further highlights the need for improved dietary intake to minimise the inflammatory priming resulting from poor glycaemic control.

Natural ligand binding and transfer from liver fatty acid binding protein (LFABP) to membranes.

Liver fatty acid-binding protein (LFABP) is distinctive among fatty acid-binding proteins because it binds more than one molecule of long-chain fatty acid and a variety of diverse ligands. Also, the transfer of fluorescent fatty acid analogues to model membranes under physiological ionic strength follows a different mechanism compared to most of the members of this family of intracellular lipid binding proteins. Tryptophan insertion mutants sensitive to ligand binding have allowed us to directly measure the binding affinity, ligand partitioning and transfer to model membranes of natural ligands. Binding of fatty acids shows a cooperative mechanism, while acyl-CoAs binding presents a hyperbolic behavior. Saturated fatty acids seem to have a stronger partition to protein vs. membranes, compared to unsaturated fatty acids. Natural ligand transfer rates are more than 200-fold higher compared to fluorescently-labeled analogues. Interestingly, oleoyl-CoA presents a markedly different transfer behavior compared to the rest of the ligands tested, probably indicating the possibility of specific targeting of ligands to different metabolic fates.

Catalytic and non-catalytic functions of human IIA phospholipase A2.

Group IIA phospholipase A2 (PLA2) is a low-molecular-mass secreted PLA2 enzyme that has been identified as an acute phase protein with a role in the inflammatory response to infection and trauma. The protein is possibly unique in being highly cationic and having a global distribution of surface arginine and lysine residues. This structure supports two functions of the protein. (1) An anti-bacterial role where the enzyme is targeted to the anionic cell membrane of Gram-positive bacteria and phospholipid hydrolysis assists in bacterial killing. (2) A proposed non-catalytic role in which the protein forms supramolecular aggregates with anionic phospholipid vesicles or debris. These aggregates are then internalized via interactions with cell surface heparin sulphate proteoglycans and macropinocytosis for disposal by macrophages.

A catalytically independent physiological function for human acute phase protein group IIA phospholipase A2: cellular uptake facilitates cell debris removal.

Human group IIA phospholipase A2 (IIA PLA2) is an acute phase protein first identified at high concentrations in synovial fluid from patients with rheumatoid arthritis. Its physiological role has since been debated; the enzyme has a very high affinity for anionic phospholipid interfaces but expresses almost zero activity with zwitterionic phospholipid substrates, because of a lack of interfacial binding. We have prepared the cysteine-containing mutant (S74C) to allow the covalent attachment of fluorescent reporter groups. We show that fluorescently labeled IIA was taken up by phorbol 12-myristate 13-acetate-activated THP-1 cells in an energy-dependent process involving cell surface heparan sulfate proteoglycans. Uptake concurrently involved significant cell swelling, characteristic of macropinocytosis and the fluorescent enzyme localized to the nucleus. The endocytic process did not necessitate enzyme catalysis, ruling out membrane phospholipid hydrolysis as an essential requirement. The enzyme produced supramolecular aggregates with anionic phospholipid vesicles as a result of bridging between particles, a property that is unique to this globally cationic IIA PLA2. Uptake of such aggregates labeled with fluorescent anionic phospholipid was dramatically enhanced by the IIA protein, and uptake involved binding to heparan sulfate proteoglycans on activated THP-1 cells. A physiological role for this protein is proposed that involves the removal of anionic extracellular cell debris, including anionic microparticles generated as a result of trauma, infection, and the inflammatory response, and under such conditions serum levels of IIA PLA2 can increase approximately 1000-fold. A similar pathway may be significant in the uptake into cells of anionic vector DNA involving cationic lipid transfection protocols.

The interaction of liver fatty-acid-binding protein (FABP) with anionic phospholipid vesicles: is there extended phospholipid anchorage under these conditions?

Liver FABP (fatty-acid-binding protein) binds a variety of non-polar anionic ligands including fatty acids, fatty acyl CoAs, lysophospholipids and bile acids. Liver FABP is also able to bind to anionic phospholipid vesicles under conditions of low ionic strength, and membrane binding results in the release of bound ligand. However, the molecular interactions involved in binding to the phospholipid interface and the mechanism of ligand release are not known. Ligand release could be due to a significant conformational change in the protein at the interface or interaction of a phospholipid molecule with the ligand-binding cavity of the protein resulting in ligand displacement. Two portal mutant proteins of liver FABP, L28W and M74W, have now been used to investigate the binding of liver FABP to anionic phospholipid vesicles, monitoring changes in fluorescence and also fluorescence quenching in the presence of brominated lipids. There is a large increase in fluorescence intensity when the L28W mutant protein binds to vesicles prepared from DOPG (dioleoyl-sn-phosphatidylglycerol), but a large decrease in fluorescence intensity when the M74W mutant binds to these vesicles. The Br(4)-phospholipid prepared by bromination of DOPG dramatically quenches both L28W and M74W, consistent with the close proximity of a fatty acyl chain to the tryptophan residues. The binding of liver FABP to DOPG vesicles is accompanied by only a minimal change in the CD spectrum. Overall, the results are consistent with a molecule of anionic phospholipid interacting with the central cavity of the liver FABP, possibly involving the phospholipid molecule in an extended conformation.

Probing phospholipid dynamics by electrospray ionisation mass spectrometry.

Recent advances in electrospray ionisation mass spectrometry (ESI-MS) have greatly facilitated the analysis of phospholipid molecular species in a growing diversity of biological and clinical settings. The combination of ESI-MS and metabolic labelling employing substrates labelled with stable isotopes is especially exciting, permitting studies of phospholipid synthesis and turnover in vivo. This review will first describe the methodology involved and will then detail dynamic lipidomic studies that have applied the stable isotope incorporation approach. Finally, it will summarise the increasing number of studies that have used ESI-MS to characterise structural and signalling phospholipid molecular species in development and disease.

Tryptophan insertion mutagenesis of liver fatty acid-binding protein: L28W mutant provides important insights into ligand binding and physiological function.

Liver fatty acid-binding protein (FABP) binds a variety of non-polar anionic ligands including fatty acids, fatty acyl CoAs, and bile acids. Previously we prepared charge reversal mutants and demonstrated the importance of lysine residues within the portal region in ligand and membrane binding. We have now prepared several tryptophan-containing mutants within the portal region, and one tryptophan at position 28 (L28W) has proved remarkably effective as an intrinsic probe to further study ligand binding. The fluorescence of the L28W mutant was very sensitive to fatty acid and bile acid binding where a large (up to 4-fold) fluorescence enhancement was obtained. In contrast, the binding of oleoyl CoA reduced tryptophan fluorescence. Positive cooperativity for fatty acid binding was observed while detailed information on the orientation of binding of bile acid derivatives was obtained. The ability of bound oleoyl CoA to reduce the fluorescence of L28W provided an opportunity to demonstrate that fatty acyl CoAs can compete with fatty acids for binding to liver FABP under physiological conditions, further highlighting the role of fatty acyl CoAs in modulating FABP function in the cell.

Effect of tryptophan insertions on the properties of the human group IIA phospholipase A2: mutagenesis produces an enzyme with characteristics similar to those of the human group V phospholipase A2.

An important characteristic of the human group IIA secreted phospholipase A(2) (IIA PLA(2)) is the extremely low activity of this enzyme with phosphatidylcholine (PC) vesicles, mammalian cell membranes, and serum lipoproteins. This characteristic is reflected in the lack of ability of this enzyme to bind productively to zwitterionic interfaces. Part of the molecular basis for this lack of activity is an absence of tryptophan, a residue with a known preference for residing in the interfacial region of zwitterionic phospholipid bilayers. In this paper we have replaced the eight residues that make up the hydrophobic collar on the interfacial binding surface of the enzyme with tryptophan. The catalytic and interfacial binding properties of these mutants have been investigated, particularly those properties associated with binding to and hydrolysis of zwitterionic interfaces. Only the insertion of a tryptophan at position 3 or 31 produces mutants that significantly enhance the activity of the human IIA enzyme against zwitterionic interfaces and intact cell membranes. Importantly, the ability of the enzyme mutants to hydrolyze PC-rich interfaces such as the outer plasma membrane of mammalian cells was paralleled by enhanced interfacial binding to zwitterionic interfaces. The corresponding double tryptophan mutant (V3,31W) displays a specific activity on PC vesicles comparable to that of the human group V sPLA2. This enhanced activity includes the ability to interact with human embryonic kidney HEK293 cells, previously reported for the group V enzyme [Kim, Y. J., Kim, K. P., Rhee, H. J., Das, S., Rafter, J. D., Oh, Y. S., and Cho, W. (2002) J. Biol. Chem. 277, 9358-9365].

Potentiation of tumor necrosis factor alpha-induced secreted phospholipase A2 (sPLA2)-IIA expression in mesangial cells by an autocrine loop involving sPLA2 and peroxisome proliferator-activated receptor alpha activation.

In rat mesangial cells, exogenously added secreted phospholipases A2 (sPLA2s) potentiate the expression of pro-inflammatory sPLA2-IIA first induced by cytokines like tumor necrosis factor-alpha (TNFalpha) and interleukin-1 beta. The transcriptional pathway mediating this effect is, however, unknown. Because products of PLA2 activity are endogenous activators of peroxisome proliferator-activated receptor alpha (PPAR alpha, we postulated that sPLA2s mediate their effects on sPLA2-IIA expression via sPLA2 activity and subsequent PPAR alpha activation. This study shows that various sPLA2s, including venom enzymes, human sPLA2-IIA, and wild-type and catalytically inactive H48Q mutant of porcine pancreatic sPLA2-IB, enhance the TNF alpha-induced sPLA2-IIA expression at the mRNA and protein levels. In cells transfected with luciferase sPLA2-IIA promoter constructs, sPLA2s are active only when the promoter contains a functional PPRE-1 site. The effect of exogenous sPLA2s is also blocked by the PPAR alpha inhibitor MK886. Interestingly, the expression of sPLA2-IIA induced by TNF alpha alone is also attenuated by MK886, by the sPLA2-IIA inhibitor LY311727, by heparinase, which prevents the binding of sPLA2-IIA to heparan sulfate proteoglycans, and by the specific cPLA2-alpha inhibitor pyrrolidine-1. Together, these data indicate that sPLA2-IIA released from mesangial cells by TNF alpha stimulates its own expression via an autocrine loop involving cPLA2 and PPAR alpha. This signaling pathway is also used by exogenously added sPLA2s including pancreatic sPLA2-IB and is distinct from that used by TNF alpha.

The crystal structure of the H48Q active site mutant of human group IIA secreted phospholipase A2 at 1.5 A resolution provides an insight into the catalytic mechanism.

The human group IIA secreted PLA(2) is a 14 kDa calcium-dependent extracellular enzyme that has been characterized as an acute phase protein with important antimicrobial activity and has been implicated in signal transduction. The selective binding of this enzyme to the phospholipid substrate interface plays a crucial role in its physiological function. To study interfacial binding in the absence of catalysis, one strategy is to produce structurally intact but catalytically inactive mutants. The active site mutants H48Q, H48N, and H48A had been prepared for the secreted PLA(2)s from bovine pancreas and bee venom and retained minimal catalytic activity while the H48Q mutant showed the maximum structural integrity. Preparation of the mutant H48Q of the human group IIA enzyme unexpectedly produced an enzyme that retained significant (2-4%) catalytic activity that was contrary to expectations in view of the accepted catalytic mechanism. In this paper it is established that the high residual activity of the H48Q mutant is genuine, not due to contamination, and can be seen under a variety of assay conditions including assays in the presence of Co(2+) and Ni(2+) in place of Ca(2+). The crystallization of the H48Q mutant, yielding diffraction data to a resolution of 1.5 A, allowed a comparison with the corresponding recombinant wild-type enzyme (N1A) that was also crystallized. This comparison revealed that all of the important features of the catalytic machinery were in place and the two structures were virtually superimposable. In particular, the catalytic calcium ion occupied an identical position in the active site of the two proteins, and the catalytic water molecule (w6) was clearly resolved in the H48Q mutant. We propose that a variation of the calcium-coordinated oxyanion ("two water") mechanism involving hydrogen bonding rather than the anticipated full proton transfer to the histidine will best explain the ability of an active site glutamine to allow significant catalytic activity.

The effect of charge reversal mutations in the alpha-helical region of liver fatty acid binding protein on the binding of fatty-acyl CoAs, lysophospholipids and bile acids.

Liver fatty acid binding protein (LFABP) is unique among the various types of FABPs in that it can bind a variety of ligands in addition to fatty acids. LFABP is able to bind long chain fatty acids with a 2:1 stoichiometry and the crystal structure has identified two fatty acid binding sites in the binding cavity. The presumed primary site (site 1) involves the fatty acid binding with the carboxylate group buried in the cavity whereas the fatty acid at site 2 has the carboxylate group solvent-exposed within the ligand portal region and in the vicinity of alpha-helix II. The alpha-helical region contains three cationic residues, K20, K31, K33 and modelling studies suggest that K31 on alpha-helix II could make an electrostatic contribution to anionic ligands binding to site 2. The preparation of three charge reversal mutants of LFABP, K20E, K31E and K33E has allowed an investigation of the role of site 2 in ligand binding, particularly those ligands with a bulky anionic head group. The binding of oleoyl CoA, lysophosphatidic acid, lysophosphatidylcholine, lithocholic acid and taurolithocholate 3-sulphate to LFABP has been studied using the alpha-helical mutants. The results support the concept that such ligands bind at site 2 of LFABP where solvent exposure allows the accommodation of their bulky anionic group.

Effect of charge reversal mutations on the ligand- and membrane-binding properties of liver fatty acid-binding protein.

Liver fatty acid-binding protein (FABP) is able to bind to anionic phospholipid vesicles under conditions of low ionic strength. This binding results in the release of ligand, the fluorescent fatty acid analogue 11-dansylaminoundecanoic acid (DAUDA), with loss of fluorescence intensity (Davies, J. K., Thumser, A. E. A., and Wilton, D. C. (1999) Biochemistry 38, 16932-16940). Using a strategy of charge reversal mutagenesis, the potential role of specific cationic residues in promoting interfacial binding of FABP to anionic phospholipid vesicles has been investigated. Cationic residues chosen included those within the alpha-helical region (Lys-20, Lys-31, and Lys-33) and those that make a significant contribution to the positive surface potential of the protein (Lys-31, Lys-36, Lys-47, Lys-57, and Arg-126). Only three cationic residues make a significant contribution to interfacial binding, and these residues (Lys-31, Lys-36, and Lys-57) are all located within the ligand portal region, where the protein may be predicted to exhibit maximum disorder. The binding of tryptophan mutants, F3W, F18W, and C69W, to dioleoylphosphatidylglycerol vesicles, containing 5 mol% of the fluorescent phospholipid dansyldihexadecanoylphosphatidylethanolamine, was monitored by fluorescence resonance energy transfer (FRET). All three mutants showed enhanced dansyl fluorescence due to FRET on addition of phospholipid to protein; however, this fluorescence was considerably greater with the F3W mutant, consistent with the N-terminal region of the protein coming in close proximity to the phospholipid interface. These results were confirmed by succinimide quenching studies. Overall, the results indicate that the portal region of liver FABP and specifically Lys-31, Lys-36, and Lys-57 are involved in the interaction with the interface of anionic vesicles and that the N-terminal region of the protein undergoes a conformational change, resulting in DAUDA release.

The antibacterial properties of secreted phospholipases A2: a major physiological role for the group IIA enzyme that depends on the very high pI of the enzyme to allow penetration of the bacterial cell wall.

The antibacterial properties of human group IIA secreted phospholipase A(2) against Gram-positive bacteria as a result of membrane hydrolysis have been reported. Using Micrococcus luteus as a model system, we demonstrate the very high specificity of this human enzyme for such hydrolysis compared with the group IB, IIE, IIF, V, and X human secreted phospholipase A(2)s. A unique feature of the group IIA enzyme is its very high pI due to a large excess of cationic residues on the enzyme surface. The importance of this global positive charge in bacterial cell membrane hydrolysis and bacterial killing has been examined using charge reversal mutagenesis. The global positive charge on the enzyme surface allows penetration through the bacterial cell wall, thus allowing access of this enzyme to the cell membrane. Reduced bacterial killing was associated with the loss of positive charge and reduced cell membrane hydrolysis. All mutants were highly effective in hydrolyzing the bacterial membrane of cells in which the cell wall was permeabilized with lysozyme. These same overall characteristics were also seen with suspensions of Staphylococcus aureus and Listeria innocua, where cell membrane hydrolysis and antibacterial activity of human group IIA enzyme was also lost as a result of charge reversal mutagenesis.