Crystal structure of the complex formed between a group I phospholipase A2 and a naturally occurring fatty acid at 2.7 A resolution

This is the first evidence of a naturally bound fatty acid to a group I Phospholipase A(2) (PLA(2)) and also to a PLA(2) with Asp 49. The fatty acid identified as n-tridecanoic acid is observed at the substrate recognition site of PLA(2) hydrophobic channel. The complex was isolated from the venom of Bungarus caeruleus (Common Indian Krait). The primary sequence of the PLA(2) was determined using the cDNA method. Three-dimensional structure has been solved by the molecular replacement method and refined using the CNS package to a final R factor of 19.8% for the data in the resolution range from 20.0 to 2.7 A. The final refined model is comprised of 912 protein atoms, one sodium ion, one molecule of n-tridecanoic acid, and 60 water molecules. The sodium ion is located in the calcium-binding loop with a sevenfold coordination. A characteristic extra electron density was observed in the hydrophobic channel of the enzyme, into which a molecule of n-tridecanoic acid was clearly fitted. The MALDI-TOF measurements of the crystals had earlier indicated an increase in the molecular mass of PLA(2) by 212 Da over the native PLA(2). A major part of the ligand fits well in the binding pocket and interacts directly with His 48 and Asp 49. Although the overall structure of PLA(2) in the present complex is similar to the native structure reported earlier, it differs significantly in the folding of its calcium-binding loop.     

Cloning, sequence analysis and homology modeling of a novel phospholipase A2 from Heterometrus fulvipes (Indian black scorpion).

We report the cloning and sequencing of group III phospholipaseA(2) from Heterometrus fulvipes (HfPLA(2)), Indian black scorpion. The cDNA sequence codes for the mature portion of the group PLA(2) of 103 amino acids. The sequence has 85% identity with Mesobuthus tamulus (Indian red scorpion) PLA(2) and a 40% identity with bee venom PLA(2) and human group III PLA(2). Most of the essential features of group III PLA(2) like Ca(2+) binding loop and catalytic residues are conserved. Homology modeling was done with the known structure of group III bee venom PLA(2). All the secondary structural motifs and the disulfide bridges are as predicted. The variation like the replacement of aspartic acid residue with glutamic acid in the well known histidine-aspartic acid dyad is a rare feature. This is the first structural model report of an Indian black scorpion PLA(2).     

Sequence and crystal structure determination of a basic phospholipase A2 from common krait (Bungarus caeruleus) at 2.4 A resolution: identification and characterization of its pharmacological sites

This is the first phospholipase A2 (PLA2) structure from the family of kraits. The protein was isolated from Bungarus caeruleus (common krait) and the primary sequence was determined using cDNA approach. Three-dimensional structure of this presynaptic neurotoxic PLA2 from group I has been determined by molecular replacement method using the model of PLA2 component of beta2-bungarotoxin (Bungarus multicinctus) and refined using CNS package to a final R-factor of 20.1 % for all the data in resolution range 20.0-2.4 A. The final refined model comprises 897 protein atoms and 77 water molecules. The overall framework of krait phospholipase A2 with three long helices and two short antiparallel beta-strands is extremely similar to those observed for other group I PLA2s. However, the critical parts of PLA2 folding are concerned with its various functional loops. The conformations of these loops determine the efficiency of enzyme action and presence/absence of various pharmacological functions. In the present structure calcium-binding loop is occupied by a sodium ion with a 7-fold co-ordination. The conformation of loop 55-75 in krait PLA2 corresponds to a very high activity of the enzyme. A comparison of its sequence with multimeric PLA2s clearly shows the absence of critical residues such as Tyr3, Trp61 and Phe64, which are involved in the multimerization of PLA2 molecules. The protein shows anticoagulant and neurotoxic activities.     

Enzymatic activity and inhibition of the neurotoxic complex vipoxin from the venom of Vipera ammodytes meridionalis

Vipoxin from the venom of Vipera ammodytes meridionalis is an unique neurotoxic complex between a toxic phospholipase A2 and a highly homologous non-toxic protein inhibitor. It is an example of evolution of a catalytic and toxic function into inhibitory and non-toxic one. The activity of the V. ammodytes meridionalis toxin is 1.7 times higher than that of the closely related (92% sequence identity) neurotoxic complex RV4/RV7 from the venom of Vipera russelli formosensis The enhanced enzymatic activity of vipoxin is attributed to limited structural changes, in particular to the substitutions G54R and Q78K in the PLA2 subunit of the complex and to the T54R substitution in the inhibitor. Oleyloxyethylphosphocholine, aristolochic acid and vitamin E suppressed the enzymatic activity of vipoxin and its isolated PLA2 subunit. These compounds influence inflammatory processes in which PLA2 is implicated. The peptide Lys-Ala-Ile-Tyr-Ser, which is an integral part of the PLA2 components of the two neurotoxic complexes from V. ammodytes meridionalis and V. russelli formosensis (sequence 70-74) activated vipoxin increasing its PLA2 activity by 23%. This is in contrast to the inhibitory effect of the respective pentapeptides with 70-74 sequences on other group II PLA2s. Surprisingly, the same peptide inhibited 46% of the V. russelli formosensis PLA2 activity. The limited changes in the structure of the two highly homologous neurotoxins lead to considerable differences in their interaction with native peptides.     

PLA2R1 kills cancer cells by inducing mitochondrial stress

Little is known about the biological functions of the phospholipase A2 receptor (PLA2R1) except that it has the ability to bind a few secreted phospholipases A2 (sPLA2′s). We have previously shown that PLA2R1 regulates senescence in normal human cells. In this study, we investigated the ability of PLA2R1 to control cancer cell growth. Analysis of expression in cancer cells indicates a marked PLA2R1 decrease in breast cancer cell lines compared to normal or nontransformed human mammary epithelial cells. Accordingly, PLA2R1 ectopic expression in PLA2R1-negative breast cancer cell lines led to apoptosis, whereas a prosenescence response was predominantly triggered in normal cells. PLA2R1 structure–function studies and the use of chemical inhibitors of sPLA2-related signaling pathways suggest that the effect of PLA2R1 is sPLA2-independent. Functional experiments demonstrate that PLA2R1 regulation of cell death is driven by a reactive oxygen species (ROS)-dependent mechanism. While screening for ROS-producing complexes involved in PLA2R1 biological responses, we identified a critical role for the mitochondrial electron transport chain in PLA2R1-induced ROS production and cell death. Taken together, this set of data provides evidence for an important role of PLA2R1 in controlling cancer cell death by influencing mitochondrial biology.     

Structural analysis of secretory phospholipase A2 from Clonorchis sinensis: therapeutic implications for hepatic fibrosis

Hepatic fibrosis is a common complication of the infection by the parasite, Clonorchis sinensis. There is a high incidence of this disease in the Asian countries with an increased risk of conversion to cancer. A secretory phospholipase A(2) (PLA(2)) enzyme from the parasite is implicated in the pathology. This is an attractive drug target in the light of extensive structural characterization of this class of enzyme. In this study, the structure of the enzyme was modeled based on its sequence homology to the group III bee venom PLA(2). On analysis, the overall structure essentially is comprised of three helices, two sets of β-wings and an elongated C-terminal extension. The structure is stabilized by four disulfide bonds. The structure is comprised of a calcium binding loop, active site and a substrate binding hydrophobic channel. The active site of the enzyme shows the classical features of PLA(2) with the participation of the three residues: histidine-aspartic acid-tyrosine in hydrogen bond formation. This is an interesting variation from the house keeping group III PLA(2) enzyme of human which has a histidine-aspartic acid and phenylalanine arrangement at the active site. This difference is therefore an important structural parameter that can be exploited to design specific inhibitor molecules against the pathogen PLA(2). Likewise, there are certain unique structural features in the hydrophobic channel and the putative membrane binding surface of the PLA(2) from Clonorchis sinensis that not only help understand the mechanism of action but also provide knowledge for a targeted therapy of liver fibrosis caused by the parasite.     

Crystal structure of a myotoxic Asp49-phospholipase A2 with low catalytic activity: Insights into Ca2+-independent catalytic mechanism

A myotoxic Asp49-phospholipase A2 (Asp49-PLA2) with low catalytic activity (BthTX-II from Bothrops jararacussu venom) was crystallized and the molecular-replacement solution has been obtained with a dimer in the asymmetric unit. The quaternary structure of BthTX-II resembles the myotoxic Asp49-PLA2 PrTX-III (piratoxin III from B. pirajai venom) and all non-catalytic and myotoxic dimeric Lys49-PLA2S. Despite of this, BthTX-II is different from the highly catalytic and non-myotoxic BthA-I (acidic PLA2 from B. jararacussu) and other Asp49-PLA2S. BthTX-II structure showed a severe distortion of calcium-binding loop leading to displacement of the C-terminal region. Tyr28 side chain, present in this region, is in an opposite position in relation to the same residue in the catalytic activity Asp49-PLA2S, making a hydrogen bond with the atom O delta 2 of the catalytically active Asp49, which should coordinate the calcium. This high distortion may also be confirmed by the inability of BthTX-II to bind Na+ ions at the Ca2+-binding loop, despite of the crystallization to have occurred in the presence of this ion. In contrast, other Asp49-PLA2S which are able to bind Ca2+ ions are also able to bind Na+ ions at this loop. The comparison with other catalytic, non-catalytic and inhibited PLA2S indicates that the BthTX-II is not able to bind calcium ions; consequently, we suggest that its low catalytic function is based on an alternative way compared with other PLA2S.     

Phospholipase A(2) with platelet aggregation inhibitor activity from Austrelaps superbus venom: protein purification and cDNA cloning

Four phospholipase A(2) (PLA(2)) enzymes (Superbins a, b, c, and d) with varying platelet aggregation inhibitor activities have been purified from Austrelaps superbus by a combination of gel filtration, ion-exchange, and reversed-phase high-pressure liquid chromatography. Purity and homogeneity of the superbins have been confirmed by high-performance capillary zone electrophoresis and mass spectrometry. The electron spray ionization mass spectrometry data showed that their molecular masses range from 13,140 to 13,236 Da. Each of the proteins has been found to be basic and exhibit varying degrees of PLA(2) activity. They also displayed different platelet aggregation inhibitory activities. Superbin a was found to possess the most potent inhibitory activity with an IC(50) of 9.0 nM, whereas Superbin d was found to be least effective with an IC(50) of 3.0 microM. Superbins b and c were moderately effective with IC(50) values of 0.05 and 0.5 microM, respectively. The amino-terminal sequencing confirmed the identity of these superbins. cDNA cloning resulted in the identification of 17 more PLA(2) isoforms in A. superbus venom. It has also provided complete information on the precursor PLA(2). The precursor PLA(2) contained a 27-amino-acid signal peptide and 117- to 125-amino-acid PLA(2) (molecular mass ranging from 13,000 to 14,000 Da). Two of these PLA(2) enzymes resembled more closely (87%) Superbin a in structure. Two unique PLA(2) enzymes containing an extra pancreatic loop also have been identified among the isoforms.     

Crystal structure of a heterodimer of phospholipase A2 from Naja naja sagittifera at 2.3 A resolution reveals the presence of a new PLA2-like protein with a novel cys 32-Cys 49 disulphide bridge with a bound sugar at the substrate-binding site

The crystal structure of the phospholipase A2 (PLA2) heterodimer from Naja naja sagittifera reveals the presence of a new PLA2-like protein with eight disulphide bridges. The heterodimer is formed between a commonly observed group I PLA2 having seven characteristic disulfide bonds and a novel PLA2-like protein (Cys-PLA2) containing two extra cysteines at two highly conserved sites (positions 32 and 49) of structural and functional importance. The crystals of the heterodimer belong to tetragonal space group P41212 with cell dimensions, a = b = 77.7 A and c = 68.4 A corresponding to a solvent content of 33%, which is one of the lowest values observed so far in the PLA2 crystals. The structure has been solved with molecular replacement method and refined to a final R value of 21.6% [Rfree = 25.6%]. The electron density revealed the presence of cysteines 32 and 49 that are covalently linked to give rise to an eighth disulphide bridge in the PLA2-like monomer. A non-protein high-quality electron density was also observed at the substrate-binding site in the PLA2-like protein that has been interpreted as N-acetylglucosamine. The overall tertiary folds of the two monomers are similar having all features of PLA2-type folding. A zinc ion is detected at the interface of the heterodimer with fivefold coordination while another zinc ion was found on the surface of Cys-PLA2 with sixfold coordination. The conformations of the calcium-binding loops of both monomers are significantly different from each other as well as from those in other group I PLA2s. The N-acetylglucosamine molecule is favorably placed in the substrate-binding site of Cys-PLA2 and forms five hydrogen bonds and several van der Waals interactions with protein atoms, thus indicating a strong affinity. It also provides clue of the possible mechanism of sugar recognition by PLA2 and PLA2-like proteins. The formation of heterodimer seems to have been induced by zinc ion.     

Amino acid sequence of piratoxin-II, a myotoxic lys49 phospholipase A(2) homologue from Bothrops pirajai venom

The complete amino acid sequence of the 121 amino acid residues of piratoxin II, a phospholipase A(2) like myotoxin from Bothrops pirajai venom, is reported. PrTX-II is a basic protein with a molecular mass of 13740 Da, a calculated pI of 9.03, but an experimental pI of 8.4 +/- 0.2, showing sequence similarity with other bothropic (90-99%) or non-bothropic ( approximately 80%) Lys49 PLA(2)-like myotoxins. This similarity falls to approximately 70% when this sequence is aligned with that of Asp49 PLA(2). Due to the substitution of Asp49 by Lys49 and alterations in the calcium binding loop structure, as the replacement of Gly32 by Leu32, piratoxin-II shows no PLA(2) activity when assayed on egg yolk. Piratoxin-II showed the same primary structure as piratoxin-I, except that it has Lys116 for Leu116. Despite this slightly higher basicity at the C-terminal region, piratoxin-II was shown to be less myotoxic than piratoxin-I. The change Leu --> Lys induced an alteration of the molecule surface shape and probably of the environment charge high enough to slightly decrease the myotoxic activity. When aligned with B. jararacussu bothropstoxin-I and with B. asper Basp-II, piratoxin-II revealed a single (position 132) and a quintuple (positions 17, 90, 111, 120 and 132) amino acid substitution, respectively, suggesting a common evolutionary origin for these three myotoxins.     

Three-dimensional structure of a presynaptic neurotoxic phospholipase A2 from Daboia russelli pulchella at 2.4 A resolution

The phospholipase A(2 )from Daboia russelli pulchella (DPLA(2)) is the only known member of subclass II of group IIA. The three-dimensional structure of this presynaptic neurotoxic DPLA(2) enzyme has been determined at 2.4 A resolution. The structure was determined by the molecular replacement method using the model Crotalus atrox, and refined using X-PLOR to a final R-factor of 18.8 % for all data in the resolution range 20.0 A-2.4 A. The final refined model comprises 1888 atoms from two crystallographically independent protein molecules and 160 water oxygen atoms. The overall folding of DPLA(2), with three long helices and two short antiparallel beta-strands is grossly similar to those observed for other PLA(2)s. In the present structure, the calcium binding site is empty but the conformation of the calcium binding loop is similar to those observed in the calcium bound states. Two spatially adjacent regions of residues 55-61 (a typical beta-turn I) and 83-94 (a well defined loop) are remarkably different in conformation, electrostatic characteristics and inter-segmental interactions from those found in non-neurotoxic PLA(2)s. Yet another striking structural feature in DPLA(2 )pertains to the stretch of residues 53-77, which has a series of positively charged residues protruding outwardly. The above segment is presumed to be involved in the anticoagulant activity. A unique hydrophobic patch including residues Leu17, Ala18, Ile19, Pro20, Phe106 and Leu110 is found on the surface together with an equally emphatic region of -OH groups containing residues such as Ser21, Tyr22, Ser23, Ser24, Tyr25 and Tyr28. The interactions between two molecules of DPLA(2) in the asymmetric unit are remarkably different from those observed in the standard dimers and trimers of PLA(2)s, leaving the enzyme's active site fully exposed for enzyme-substrate reactions, it makes this structure one of the most favourable examples for structure-based drug design through soaking experiments.     

Phospholipase A2 as a target protein for nonsteroidal anti-inflammatory drugs (NSAIDS): crystal structure of the complex formed between phospholipase A2 and oxyphenbutazone at 1.6 A resolution

Phospholipase A(2) (PLA(2); EC 3.1.1.4) is a key enzyme involved in the production of proinflammatory mediators known as eicosanoids. The binding of the substrate to PLA(2) occurs through a well-formed hydrophobic channel. To determine the viability of PLA(2) as a target molecule for the structure-based drug design against inflammation, arthritis, and rheumatism, the crystal structure of the complex of PLA(2) with a known anti-inflammatory compound oxyphenbutazone (OPB), which has been determined at 1.6 A resolution. The structure has been refined to an R factor of 0.209. The structure contains 1 molecule each of PLA(2) and OPB with 2 sulfate ions and 111 water molecules. The binding studies using surface plasmon resonance show that OPB binds to PLA(2) with a dissociation constant of 6.4 x 10(-8) M. The structure determination has revealed the presence of an OPB molecule at the binding site of PLA(2). It fits well in the binding region, thus displaying a high level of complementarity. The structure also indicates that OPB works as a competitive inhibitor. A large number of hydrophobic interactions between the enzyme and the OPB molecule have been observed. The hydrophobic interactions involving residues Tyr(52) and Lys(69) with OPB are particularly noteworthy. Other residues of the hydrophobic channel such as Leu(3), Phe(5), Met(8), Ile(9), and Ala(18) are also interacting extensively with the inhibitor. The crystal structure clearly reveals that the binding of OPB to PLA(2) is specific in nature and possibly suggests that the basis of its anti-inflammatory effects may be due to its binding to PLA(2) as well.     

Crystal structure of the complex of the secretory phospholipase A2 from Daboia russelli pulchella with an endogenic indole derivative, 2-carbamoylmethyl-5-propyl-octahydro-indol-7-yl-acetic acid at 1.8 A resolution

Phospholipase A2 (PLA2) enzymes from snake venoms are approximately 14 kDa secretory proteins and catalyze the release of arachidonic acid which is the precursor of proinflammatory mediators such as prostaglandins, leukotrienes, thromboxanes and platelet-activating factors. The structure of the PLA2 enzyme purified from the venom of Daboia russelli pulchella was determined using molecular replacement method and refined to an R value of 18.3% for all the reflections to 1.8 A resolution. The structure contains two crystallographically independent molecules A and B which form an asymmetric homodimer. The Ca2+ ion was not detected in the present structure, however, a characteristic non-protein high quality electron density was observed at the substrate-binding site of molecule A which allowed a clear interpretation of a natural ligand identified as a derivative of indole, 2-carbamoylmethyl-5-propyl-octahydro-indol-7-yl)-acetic acid. The corresponding substrate-binding site in molecule B was empty. The ligand present in molecule A is involved in extensive interactions with the protein atoms including important catalytic residues such as Asp-49 and His-48. The results also show that the indole derivatives act as potent inhibitors of secretory group II PLA2 enzymes that can be further modified to be used as potential therapeutic agents.     

Structural analysis of phospholipase A2 from functional perspective. 1. Functionally relevant solution structure and roles of the hydrogen-bonding network

Bovine pancreatic phospholipase A2 (PLA2), a small (13.8 kDa) Ca2+-dependent lipolytic enzyme, is rich in functional and structural character. In an effort to examine its detailed structure-function relationship, we determined its solution structure by multidimensional nuclear magnetic resonance (NMR) spectroscopy at a functionally relevant pH. An ensemble of 20 structures generated has an average root-mean-square deviation (RMSD) of 0.62 +/- 0.08 A for backbone (N, Calpha, C) atoms and 0.98 +/- 0.09 A for all heavy atoms. The overall structure shows several notable differences from the crystal structure: the first three residues at the N-terminus, the calcium-binding loop (Y25-T36), and the surface loop (V63-N72) appear to be flexible; the alpha-helical conformation of helix B (E17-F22) is absent; helix D appears to be shorter (D59-V63 instead of D59-D66); and the hydrogen-bonding network is less defined. These differences were analyzed in relation to the function of PLA2. We then further examined the H-bonding network, because its functional role or even its existence in solution has been in dispute recently. Our results show that part of the H-bonding network (the portion away from N-terminus) clearly exists in solution, as evidenced by direct observation (at 11.1 ppm) of a strong H-bond between Y73 and D99 and an implicated interaction between D99 and H48. Analyses of a series of mutants indicated that the existence of the Y73.D99 H-bond correlates directly with the conformational stability of the mutant. Loss of this H-bond results in a loss of 2-3 kcal/mol in the conformational stability of PLA2. The unequivocal identification and demonstration of the structural importance of a specific hydrogen bond, and the magnitude of its contribution to conformational stability, are uncommon to the best of our knowledge. Our results also suggest that, while the D99.H48 catalytic diad is the key catalytic machinery of PLA2, it also helps to maintain conformational integrity.     

Catalytically inactive phospholipase A2 homologue binds to vascular endothelial growth factor receptor-2 via a C-terminal loop region

VEGF (vascular endothelial growth factor) regulates neovascularization through binding to its receptor KDR (kinase insert domain-containing receptor; VEGF receptor-2). We recently identified a catalytically inactive PLA(2) (phospholipase A(2)) homologue (KDR-bp) in the venom of eastern cottonmouth (Agkistrodon piscivorus piscivorus) as a third KDR-binding protein, in addition to VEGF(165) and tissue inhibitor of metalloproteinase-3. KDR-bp binds to the extracellular domain of KDR with a K(d) of 10(-8) M, resulting in specific blockade of endothelial cell growth induced by VEGF(165). Inactive PLA(2) homologues are widely distributed in the venoms of Viperidae snakes and are known to act as myotoxins. In the present study, we demonstrated that KDR-binding ability is a common characteristic for inactive PLA(2) homologues in snake venom, but not for active PLA(2)s such as neurotoxic and platelet aggregation-modulating PLA(2)s. To understand better the KDR and KDR-bp interaction, we resolved the binding region of KDR-bp using eight synthetic peptides designed based on the structure of KDR-bp. A synthetic peptide based on the structure of the C-terminal loop region of KDR-bp showed high affinity for KDR, but other peptides did not, suggesting that the C-terminal loop region of KDR-bp is involved in the interaction with KDR. The results of the present study provide insight into the binding of inactive PLA(2) homologues to KDR, and may also assist in the design of novel anti-KDR molecules for anti-angiogenic therapy.     

Purification, sequencing and structural analysis of two acidic phospholipases A2 from the venom of Bothrops insularis (jararaca ilhoa)

Bothrops snake venoms contain a variety of phospholipases (PLA(2)), some of which are myotoxic. In this work, we used reverse-phase HPLC and mass spectrometry to purify and sequence two PLA(2) from the venom of Bothrops insularis. The two enzymes, designated here as BinTX-I and BinTx-II, were acidic (pI 5.05 and 4.49) Asp49 PLA(2), with molecular masses of 13,975 and 13,788, respectively. The amino acid sequence and molecular mass of BinTX-I were identical to those of a PLA(2) previously isolated from this venom (PA2_BOTIN, SwissProt accession number ) while those of BinTX-II indicated that this was a new enzyme. Multiple sequence alignments with other Bothrops PLA(2) showed that the amino acids His48, Asp49, Tyr52 and Asp99, which are important for enzymatic activity, were fully conserved, as were the 14 cysteine residues involved in disulfide bond formation, in addition to various other residues. A phylogenetic analysis showed that BinTX-I and BinTX-II grouped with other acidic Asp49 PLA(2) from Bothrops venoms, and computer modeling indicated that these enzymes had the characteristic structure of bothropic PLA(2) that consisted of three alpha-helices, a beta-wing, a short helix and a calcium-binding loop. BinTX-I (30 microg/paw) produced mouse hind paw edema that was maximal after 1h compared to after 3h with venom (10 and 100 microg/paw); in both cases, the edema decreased after 6h. BinTX-1 and venom (40 microg/ml each) produced time-dependent neuromuscular blockade in chick biventer cervicis preparations that reached 40% and 95%, respectively, after 120 min. BinTX-I also produced muscle fiber damage and an elevation in CK, as also seen with venom. These results indicate that BinTX-I contributes to the neuromuscular activity and tissue damage caused by B. insularis venom in vitro and in vivo.     

Prevalence of Enhanced Granular Expression of Thrombospondin Type-1 Domain-Containing 7A in the Glomeruli of Japanese Patients with Idiopathic Membranous Nephropathy

Membranous nephropathy (MN) is a leading cause of nephrotic syndrome in adults. Autoantibodies against M-type phospholipase A2 receptor (PLA2R) and thrombospondin type-1 domain-containing 7A (THSD7A), which mainly belong to the IgG4 subclass, were reported as associated antibodies for the development of MN. Although PLA2R is a major target antigen for idiopathic MN, the prevalence of MN patients seropositive for PLA2R in Japan is lower than that in other countries. In this study, we conducted immunohistochemical analysis of the presence of THSD7A and PLA2R in renal specimens of MN patients to estimate the prevalence of THSD7A/PLA2R-related idiopathic MN in Japan. Enhanced granular expression of THSD7A and PLA2R was detected in 9.1% and 52.7%, respectively, of the patients with idiopathic MN. Although none of patients with secondary MN displayed enhanced granular expression of THSD7A, 5.4% of them had enhanced granular expression of PLA2R. In conclusion, the prevalence of enhanced granular expression of THSD7A in the glomeruli of Japanese patients with idiopathic MN was higher than the prevalence of MN patients seropositive for THSD7A in USA and Europe.     

The crystal structure of prokaryotic phospholipase A2

In this study, the x-ray crystal structures of the calcium-free and calcium-bound forms of phospholipase A(2) (PLA(2)), produced extracellularly by Streptomyces violaceoruber, were determined by using the multiple isomorphous replacement and molecular replacement methods, respectively. The former and latter structures were refined to an R-factor of 18.8% at a 1.4-A resolution and an R-factor of 15.0% at a 1.6-A resolution, respectively. The overall structure of the prokaryotic PLA(2) exhibits a novel folding topology that demonstrates that it is completely distinct from those of eukaryotic PLA(2)s, which have been already determined by x-ray and NMR analyses. Furthermore, the coordination geometry of the calcium(II) ion apparently deviated from that of eukaryotic PLA(2)s. Regardless of the evolutionary divergence, the catalytic mechanism including the calcium(II) ion on secreted PLA(2) seems to be conserved between prokaryotic and eukaryotic cells. Demonstrating that the overall structure determined by x-ray analysis is almost the same as that determined by NMR analysis is useful to discuss the catalytic mechanism at the molecular level of the bacterial PLA(2).     

Interaction mode of n-dodecylphosphorylcholine, a substrate analogue, with bovine pancreas phospholipase A2 as determined by X-ray crystal analysis.

Three-dimensional structure of a bovine pancreas phospholipase A2 (PLA2) crystal complexed with n-dodecylphosphorylcholine (n-C12PC), a substrate-type inhibitor, has been determined by the X-ray diffraction method. The present conventional R value is 0.275 at 2.3A resolution. The binding mode of n-C12PC to the PLA2 was clearly indicated, where the dodecyl chain was stably held by the hydrophobic contacts with the N-terminal region of PLA2 (Leu-2, Phe-5, and Ile-9), and the choline moiety was contacted with the hydrophobic space created by the side chains of Lys-53 and 56. The present result indicates that remarkable changes from the native PLA2 structure are caused at the N-terminal and middle (residues 60 to 70) regions by the binding of n-C12PC to the enzyme.     

Identification of a soluble form phospholipase A2 receptor as a circulating endogenous inhibitor for secretory phospholipase A2

Venomous snakes have various types of phospholipase A(2) inhibitory proteins (PLIs) in their circulatory system to protect them from attack by their own phospholipase A(2)s (PLA(2)s). Here we show the first evidence for the existence of circulating PLI against secretory PLA(2)s (sPLA(2)s) in mammals. In mouse serum, we detected specific binding activities of group IB and X sPLA(2)s, which was in contrast with the absence of binding activities in serum prepared from mice deficient in PLA(2) receptor (PLA(2)R), a type I transmembrane glycoprotein related to the C-type animal lectin family. Western blot analysis after partial purification with group IB sPLA(2) affinity column confirmed the identity of serum sPLA(2)-binding protein as a soluble form of PLA(2)R (sPLA(2)R) that retained all of the extracellular domains of the membrane-bound receptor. Both purified sPLA(2)R and the recombinant soluble receptor having all of the extracellular portions blocked the biological functions of group X sPLA(2), including its potent enzymatic activity and its binding to the membrane-bound receptor. Protease inhibitor tests with PLA(2)R-overexpressing Chinese hamster ovary cells suggested that sPLA(2)R is produced by cleavage of the membrane-bound receptor by metalloproteinases. Thus, sPLA(2)R is the first example of circulating PLI that acts as an endogenous inhibitor for enzymatic activities and receptor-mediated functions of sPLA(2)s in mice.