Human Pancreatic Phospholipase Research Articles

Autoproteolytic Activation of a Symbiosis-regulated Truffle Phospholipase A2

Fungal phospholipases are members of the fungal/bacterial group XIV secreted phospholipases A(2) (sPLA(2)s). TbSP1, the sPLA(2) primarily addressed in this study, is up-regulated by nutrient deprivation and is preferentially expressed in the symbiotic stage of the ectomycorrhizal fungus Tuber borchii. A peculiar feature of this phospholipase and of its ortholog from the black truffle Tuber melanosporum is the presence of a 54-amino acid sequence of unknown functional significance, interposed between the signal peptide and the start of the conserved catalytic core of the enzyme. X-ray diffraction analysis of a recombinant TbSP1 form corresponding to the secreted protein previously identified in T. borchii mycelia revealed a structure comprising the five α-helices that form the phospholipase catalytic module but lacking the N-terminal 54 amino acids. This finding led to a series of functional studies that showed that TbSP1, as well as its T. melanosporum ortholog, is a self-processing pro-phospholipase A(2), whose phospholipase activity increases up to 80-fold following autoproteolytic removal of the N-terminal peptide. Proteolytic cleavage occurs within a serine-rich, intrinsically flexible region of TbSP1, does not involve the phospholipase active site, and proceeds via an intermolecular mechanism. Autoproteolytic activation, which also takes place at the surface of nutrient-starved, sPLA(2) overexpressing hyphae, may strengthen and further control the effects of phospholipase up-regulation in response to nutrient deprivation, also in the context of symbiosis establishment and mycorrhiza formation.

Peptide derived from the lipid binding domain of Group IB human pancreatic phospholipase A 2 possesses antibacterial activity

Group 1B human pancreatic secretory phospholipase A 2 (hp-sPLA 2), a digestive enzyme synthesized by pancreatic acinar cells and present in pancreatic juice, do not have antibacterial activity towards Escherichia coli. Our earlier results suggest that the N-terminal first ten amino acid residues of hp-sPLA 2 constitute major portion of the membrane binding domain of full-length enzyme and is responsible for the precise orientation of enzyme on the membrane surface by inserting into the lipid bilayers (Pande et al. (2006) Biochemistry, 45,12436–12447). In this study we report the antibacterial properties of a peptide (AVWQFRKMIK-CONH 2; N10 peptide), which corresponds to the N-terminal first ten amino acid residues of hp-sPLA 2, against E. coli. Full-length hp-sPLA 2, which contains this peptide sequence as N-terminal α-helix, did not showed detectable antibacterial activity. Presence of physiological concentration of salt or preincubation of N10 peptide with soluble anionic polymer inhibits the antibacterial activity indicating the importance of electrostatic interaction in binding of peptide to bacterial membrane. Addition of peptide resulted in destabilization of outer as well as inner cytoplasmic membrane of E. coli suggesting bacterial membranes to be the main target of action. N10 peptide exhibits strong synergism with lysozyme and potentiates the antibacterial activity of lysozyme. The peptide was inactive against human erythrocyte. Our result shows for the first time that a peptide fragment of hp-sPLA 2 possesses antibacterial activity towards E. coli and at subinhibitory concentration and can potentiate the antibacterial activity of membrane active enzyme. These observations suggest that N10 peptide may play an important role in the antimicrobial activity of pancreatic juice.

Changes in the Secondary and Tertiary Structures of Secreted Phospholipase A2 upon Activation

Activation of human pancreatic phospholipase A2 (PLA2) in the presence of DPPC/DPPG (7:3) vesicles was induced by a temperature shift from 4 to 38 oC. PLA2 activity was monitored by changes in fluorescence of bis-Pyrene-PC (2.5 mol % in the membranes), while simultaneous far- and near-UV circular dichroism (CD) spectra identified changes in the secondary and tertiary structures of the protein in real time. The 4-to-38 oC temperature shift caused dramatic changes in both bis-Pyrene-PC fluorescence and the protein CD spectra. The monomer fluorescence signal of bis-Pyrene-PC rapidly increased and the excimer signal decreased, demonstrating PLA2 activation. Drastic weakening in the α-helical CD signal of the protein, i.e., a 20% decrease in the n-π* transition intensity at 222 nm, was detected upon enzyme activation. The α-helical signal exhibited a significantly smaller change upon a similar temperature shift under non-catalytic conditions (1 mM EGTA), while little changes were detected in the absence of lipid. Strong changes in the tertiary structure during PLA2 activation were also identified. Initially, at 4 oC, the near-UV CD spectra showed a weak negative band around 280 nm. Upon a shift to 38 oC, strong positive CD bands rapidly developed around 250 and 280 nm, implying significant changes in the conformation and/or the microenvironment of Tyr and Trp side chains of PLA2, possibly accompanied with a global tertiary structure perturbation associated with deformation of the abundant disulfide bonds in the protein. These experiments provide new information on the structure-function relationship of PLA2 by near-simultaneous measurements of PLA2 activity and its secondary and tertiary structures.

Effect of Guggulsterone and Cembranoids ofCommiphora mukulon Pancreatic Phospholipase A2: Role in Hypocholesterolemia

Guggulsterone (7) and cembranoids (8-12) from Commiphora mukul stem bark resin guggul were shown to be specific modulators of two independent sites that are also modulated by bile salts (1-6) to control cholesterol absorption and catabolism. Guggulsterone (7) antagonized the chenodeoxycholic acid (3)-activated nuclear farnesoid X receptor (FXR), which regulates cholesterol metabolism in the liver. The cembranoids did not show a noticeable effect on FXR, but lowered the cholate (1)-activated rate of human pancreatic IB phospholipase A2 (hPLA2), which controls gastrointestinal absorption of fat and cholesterol. Analysis of the data using a kinetic model has suggested an allosteric mechanism for the rate increase of hPLA2 by cholate and also for the rate-lowering effect by certain bile salts or cembranoids on the cholate-activated hPLA2 hydrolysis of phosphatidylcholine vesicles. The allosteric inhibition of PLA2 by certain bile salts and cembranoids showed some structural specificity. Biophysical studies also showed specific interaction of the bile salts with the interface-bound cholate-activated PLA2. Since cholesterol homeostasis in mammals is regulated by FXR in the liver for metabolism and by PLA2 in the intestine for absorption, modulation of PLA2 and FXR by bile acids and selected guggul components suggests novel possibilities for hypolipidemic and hypocholesterolemic therapies.

Hyperamylasämie bei Anorexia nervosa und Bulimia nervosa: Indiz für eine Pankreaserkrankung?

Serum concentrations of amylase, lipase, human pancreatic lipase and phospholipase A were measured in 34 patients (average age 28.5 [18-64] years) with anorexia nervosa (9 patients) and bulimia (25 patients). Hyperamylasaemia was demonstrated in 13 of the 34 patients (38%). But in only two of them was there also an elevated concentration of lipase and human pancreatic lipase. Phospholipase A was normal in all. Only one patient with isolated hyperamylasaemia had cholecystolithiasis, which was asymptomatic. In none of the patients with raised enzyme values was there otherwise any evidence of pancreatic or biliary tract disease, of alcohol or drug abuse, hyperlipoproteinaemia or abnormal renal function. In the great majority of patients with anorexia nervosa or bulimia who had hyperamylasaemia, this could not be ascribed to pancreatic disease. Even in the two patients who also had an increase in lipase and pancreatic isolipase, there was no clinical and ultrasound evidence of pancreatic disease.

3D QSAR Study of Human PLA2 Inhibitors. A Modeling Approach to Select New and Specific Anti-Inflammatory Drugs

High concentrations of human non pancreatic secretory phospholipase A2 (hnps-PLA2) have been reported as inducing factors in different inflammatory diseases. Thus, hnps-PLA2 inhibitors would be potential drugs against disorders generated by high levels of this enzyme. The latter has been crystallized with different ligands and several classes of inhibitors are known, but the optimization of their therapeutic properties requires: (i) a better understanding of the inhibitorprotein interaction mechanism, and (ii) finding a strategy to predict the activity of new molecules. Approaches related to computational chemistry may help to resolve these two problems. An automated docking study was performed on a series of 188 competitive hnps-PLA2 inhibitors. The docking data were then used to establish 3D QSAR models by combining Comparative Molecular Field Analysis (CoMFA) and PLS modeling. The robustness and prediction power of the best model were assessed with help of cross-validation and test set procedures that delivered excellent scores. The search for the best inhibitors against hnps-PLA2 has to be associated with a high specificity of the molecules selected, minimizing possible human side effects. This requires keeping at an extremely low level the inhibitors activity against human pancreatic phospholipase A2 (hp-PLA2) which is in negligible concentration in all tissues except in pancreatic ones. Then, the above mentioned modeling procedure was applied also on a series of hp-PLA2 inhibitors and, once more, the 3D QSAR model thus generated showed an excellent robustness and prediction power. Finally, the combination of the two models generated on hnps-PLA2 and hp-PLA2 offered a global predictive tool able to select new strong anti-inflammatory drugs with negligible side effects, at least at pancreatic level.

Protein-based alignment in 3D QSAR of 26 indole inhibitors of human pancreatic phospholipase A 2

An automated docking procedure was applied on a series of 26 reversible and competitive indole inhibitors of human pancreatic phospholipase A 2 (hp-PLA 2). X-ray data of this enzyme are not available and the structure was then reconstructed exploiting its protein sequence and the crystallographic data of a bovine pancreatic source. The docking data were used to build a three-dimensional quantitative structure–activity relationship (3D QSAR) model, established using the comparative molecular field analysis (CoMFA) method. This model, joined to the previous one developed for the indole inhibitors of human non-pancreatic secretory phospholipase A 2 (hnps-PLA 2), an enzyme involved in inflammation processes, will allow for the selection of new strong anti-inflammatory drugs with negligible side effects, at least at the level of hp-PLA 2.

Elevations of group II phospholipase A2 concentrations in serum and amniotic fluid in association with preterm labor

Objective: The purpose of this study was to determine whether the elevation of secretory group II phospholipase A2 concentration in the serum and amniotic fluid in preterm labor is associated with intrauterine inflammation. Study Design: Serum and amniotic fluid were collected from women with preterm delivery (<37 weeks’ gestation; n = 38) and term delivery (n = 20). Phospholipase activity was measured with a highly sensitive system that was based on reverse-phase high-performance liquid chromatographic separation of 9-anthracenylmethyl derivatives of fatty acids released by phospholipase A2. The concentrations of immunoreactive isozymes (group I or II) of secretory phospholipase A2 were assayed with a radioimmunoassay kit with a monoclonal antibody against human pancreatic phospholipase A2 and splenic IIA phospholipase A2. Localization of immunoreactive group II phospholipase A2 in the amniotic membrane was determined by immunostaining visualized with the Vectastain ABC (Vector Laboratories, Inc, Burlingame, Calif) method. Results: Enzymatic activities of phospholipase A2 in the serum and amniotic fluid specimens obtained from patients in preterm labor with chorioamnionitis were significantly higher than those in specimens from patients in term labor. Significant elevations of phospholipase A2 activities were observed in patients with preterm labor without histologically evident chorioamnionitis. The activity of phospholipase A2 was clearly correlated with the concentration of the immunoreactive group II phospholipase A2. Group II phospholipase A2 was localized in amniotic cells obtained from patients with a pathologically determined diagnosis of chorioamnionitis. The predictive value for chorioamnionitis of the group II phospholipase A2 concentration was relatively higher than the predictive values of the concentrations of C-reactive protein and interleukins 6 and 8. Conclusion: Significant elevations of group II phospholipase A2 concentrations were detected in the serum and amniotic fluid of women with preterm labor. Group II phospholipase A2 concentration may be a useful indicator for preterm labor, and phospholipid metabolism is certainly activated both in preterm labor and in apparent inflammatory diseases. (Am J Obstet Gynecol 2000;183:1537-43.)

Identification of Two Secreted Phospholipases A2 in Human Epidermis

Phospholipases A2 are enzymes that catalyze the release of fatty acids from the sn-2 position of phospholipids. Fatty acids have been suggested to play a key role in the barrier function of the epidermis. The aim of this study was to identify and characterize the type of secretory phospholipase A2 expressed in human epidermis. We report the molecular cloning of two secretory phospholipase A2 in the human epidermis. The first enzyme is identical to human pancreatic type IB phospholipase A2. Western blots revealed a 14 kDa protein localized in the soluble fraction. The second phospholipase A2 is identical to human synovial type IIA enzyme and is localized in the membrane fraction. By semiquantitative reverse transcription-polymerase chain reaction performed on horizontal sections of the epidermis, we found that the mRNAs of both phospholipases A2 were expressed mainly in the basal layers of the epidermis. Our data thus provide evidence for the expression of two secretory phospholipases A2 in human epidermis. The different localization of these two secretory proteins strongly suggests that each enzyme might have a specific role in skin physiology and probably in the barrier function. Taken together, these data validate the reverse transcription-polymerase chain reaction technique performed on thin sections as a first approach to detect gene expression in different layers of the epidermis.

Distal Hereditary Motor Neuropathy Type II (Distal HMN Type II): Phenotype and Molecular Genetics.

The distal hereditary motor neuropathies (distal HMN) are clinically and genetically heterogeneous and are subdivided in seven subtypes according to the mode of inheritance, age at onset and clinical evolution. We studied a multigenerational Belgian pedigree with autosomal dominant distal HMN type II. The clinical phenotype closely resembles classical Charcot-Marie-Tooth (CMT) disease with an age at onset between 15 and 25 years. Linkage studies have shown that distal HMN II is not linked to the known CMT1 and CMT2 loci. A genome-wide search was performed and significant linkage was obtained between markers D12S86 and D12S340, suggesting that a gene causing distal HMN II is located on chromosome 12q24.3. The gene encoding the human pancreatic phospholipase A2 (PLA2A), which is expressed in peripheral nerves during degeneration, is a positional candidate gene. Because no disease-specific mutations were detected in the coding region, however, PLA2A is most likely not the disease causing gene. A yeast artificial chromosome (YAC) contig map spanning the candidate region has been constructed to isolate the gene responsible for distal HMN II. Positional and functional candidate genes are currently being screened for the presence of mutations in distal HMN II patients.

Bacterial expression and characterization of human pancreatic phospholipase A 2

Mammalian pancreatic phospholipases A 2 (PLA 2) have recently been implicated in cell surface receptor-mediated inflammation. As a first step toward understanding how human pancreatic PLA 2 (hp-PLA 2) interacts with membranes and other biological targets including cell-surface receptors, we constructed its bacterial expression vector which can be used for the mutagenesis and protein over-expression. The expression vector (pSH-hp) was constructed using a synthetic hp-PLA 2 gene whose transcription is controlled by T7 promoter. hp-PLA 2 was expressed as a mature protein in high concentration in Escherichia coli cells and formed inclusion body. The solubilization of inclusion body protein followed by the refolding and purification produced ca. 5 mg of pure protein from one liter of growth medium. Kinetic studies of recombinant human, bovine and porcine pancreatic PLA 2s using polymerized mixed liposomes and micelles as substrates showed that despite their highly homologous structures these mammalian pancreatic PLA 2s have distinct phospholipid head group specificity and different activity toward various lipid substrates.

Activation of MAP kinase cascade induced by human pancreatic phospholipase A 2 in a human pancreatic cancer cell line

We have found that the growth of human pancreatic cancer cells MIAPaCa-2, induced by human pancreatic phospholipase A 2 group I (hPLA 2-I), is mediated via its specific receptor but not via its catalytic property. The present study showed that the activation of mitogen-activated protein kinase (MAPK) cascade in MIAPaCa-2 cells is induced by hPLA 2-I: this digestive enzyme induced phosphorylation of MEK1/2, p44/42 MAPK and ATF-2, and the phosphorylation in the MAPK cascade was inhibited after the cells were pre-incubated with a selective inhibitor of MEK, PD98059. In addition, this inhibitor dose-dependently blocked the hPLA 2-I-induced MIAPaCa-2 proliferation, suggesting that activation of the MAPK cascade is essential for the hPLA 2-I-induced MIAPaCa-2 proliferation.

Mutation analysis of the human pancreatic phospholipase A 2 gene in a family with distal hereditary motor neuropathy type II linked to 12q24

Molecular genetic analysis in a Belgian family with distal hereditary motor neuropathy type II (distal HMN II), demonstrated significant linkage of markers located at chromosome 12q24. The candidate region, extending from D12S86 to D12S340, includes the gene encoding pancreatic phospholipase A 2 (PPLA2). PPLA2 is a candidate gene for distal HMN II in this family since it is expressed in the peripheral nervous system during nerve degeneration. We analyzed the sequences of the four coding exons of the PPLA2 gene in two patients affected with distal HMN II and in two unrelated healthy individuals of the pedigree. Two rare polymorphisms in exon 3 and one intronic three-base pair insertion were observed in both the patients as well as the control individuals. However, no disease specific mutation within the coding region of PPLA2 could be identified, suggesting that the PPLA2 gene is most likely not the disease causing gene for distal HMN II in this Belgian family.

Growth of human pancreatic cancer cells, induced by human pancreatic phospholipase A(2), is mediated via its specific receptor but not via its catalytic property

Human pancreatic phospholipase A(2) type I (hPLA(2)-I) has been found to stimulate the growth of human pancreatic cancer cell line, MIAPaCa-2, which has a receptor for PLA(2). In the present study, half-maximal inhibitory concentrations (IC(50)s) for the mature- and pro-form of hPLA(2)-I and certain eukaryotic and prokaryotic PLA(2)s were determined using ligand binding studies. The IC50 for the mature form was 3.5x10(-9) M compared to those for the pro-form and non-human PLA(2)s (over 5.0x10(-7) M), suggesting receptor specificity for mature hPLA(2)-I. Receptor binding was independent of Ca2+, which is required for PLA(2)'s digestive activity. Lysophospholipids, generated by PLA(2), showed no proliferative effect on the MIAPaCa-2 cells. Furthermore, MIAPaCa-2 cells treated with hPLA(2)-I did not release fatty acids. This implies that proliferation of these cells is mediated by binding of hPLA(2)-I to the specific receptor, not by its enzymatic activity. The hPLA(2)-I induced cell proliferation was blocked by preincubation of the enzyme with anti-hPLA(2)-I monoclonal antibody.

Association between a dimorphic site on chromosome 12 and clinical diagnosis of hypertension in three independent populations.

With the aim of identifying putative quantitative trait loci (QTLs) involved in the regulation of blood pressure, we have carried out association studies at a candidate genetic locus-a human pancreatic phospholipase A2 (PLA2) gene localized on chromosome 12. Positive associations were found between the presence of a Taq I dimorphic site localized in the first intron of this gene and hypertension in three sample populations (two from USA and one from Germany). These results indicate that a QTL implicated in determining an individual's genetic susceptibility to hypertension could be present within up to 30 cM of this human PLA2 gene.

Human pancreatic phospholipase A 2 stimulates the growth of human pancreatic cancer cell line

Phospholipase A 2 (PLA 2) from human pancreas, designated hPLA 2-I, functions as a digestive enzyme. Interestingly, the present study demonstrated that the mature form of hPLA 2-I stimulated the growth of a human pancreatic cancer cell line MIAPaCa-2, whereas the pro-form was ineffective. PLA 2s from Laticauda semifasciata fraction I, Crotalus adamanteus venom, Streptomyces violaceoruber and bee venom, showed no proliferative effect to the growth of MIAPaCa-2. The Scatchard plot analysis revealed that the MIAPaCa-2 cell had a specific binding site for the mature hPLA 2-I. The equilibrium binding constant ( K d) and the maximum binding capacity ( B max) were 2.6 nM and 0.4 fmol/10 6 cells, respectively. These results suggest that the mature hPLA 2-I, but not the pro-form, may function as a growth factor of pancreas carcinoma via the specific binding site.

TaqI dimorphic site in a human pancreatic phospholipase A2 (PLA2) gene localized on chromosome 12.

We report the identification of a TaqI dimorphic site in the first intron of a human phospholipase A2 gene that has been localized on chromosome 12. This single two-allele polymorphism was detected by DNA blot hybridization using a full-length, 584 bp human phospholipase A2 cDNA isolated from a human lung lambda gt10 cDNA library. Fragment lengths were detected at either 1.3 kb or 2.2 kb. The PIC value of this dimorphism was 0.369 in a random population of 353 Caucasians.

Hydrolysis of phosphatidylethanolamine by human pancreatic phospholipase A2. Effect of bile salts.

The 2-ester bond of 14C-2-arachidonyl phosphatidylethanolamine (PE) was hydrolyzed faster than that of 3H-2-arachidonyl phosphatidylcholine (PC) by human pancreatic phospholipase A2 (PLA2) with mixed PE-PC (1:9 w/w) liposomes of pure sonicated PE or PC as substrate. The PC portion of the mixed PE-PC liposomes was more readily attacked by PLA2 than the PC of pure PC liposomes. At different bile salt concentrations (sodium taurocholate (NaTC), 0-3 mM, and sodium taurodeoxycholate (NaTDC), 0-4 mM) the rates of hydrolysis varied with similar patterns for both phospholipids of the mixed liposomes. The rate of hydrolysis was optimal at a bile salt concentration of 0.75-1.5 mM NaTC and 1.0-2.0 mM NaTDC and decreased at higher concentrations. The pure PE substrate was efficiently hydrolyzed also without bile salts. This may have implications for the absorption of polyunsaturated phospholipid fatty acids in patients with bile salt deficiency. Separation of phospholipid classes from human bile by high-performance liquid chromatography and analysis of fatty acid composition indicated that PE contained 5.3% of the phospholipid arachidonic acid and 9.8% of the docosahexaenoic acid mass, but only 1.7% of the total phospholipid mass. Bile and dietary PE should not be overlooked as sources of arachidonic and docosahexaenoic acid for the small intestine.

Phosphatidylcholine as substrate for human pancreatic phospholipase A2. Importance of the physical state of the substrate.

The long-chain phosphatidylcholine/sodium cholate aqueous system as substrate for human pancreatic phospholipase A2 (PLA2) was investigated. At a constant phosphatidylcholine (PC) concentration of 8 mM, the enzyme activity increased with a decrease in cholate (C) concentration up to a PC/C ratio of approximately 0.8 and then rather abruptly decreased to lower values at a ratio above 1.5. At ratios between 0.8 and 1.5, an increasing lag phase in the PLA2 activity was seen, indicating a progressive decrease in substrate availability to the enzyme. Reaction mixtures with a PC/C ratio of up to 0.67 were optically clear solutions composed of mixed bile salt/PC micelles of increasing mixed micellar aggregate size. Ratios between 0.67 and 1.5 were characterized by an increase in turbidity (at 330 and 450 nm) due to increasing formation of vesicles or liposomes. Above a PC/C ratio of 1.5, a sharp increase in turbidity was seen due to increasing formation of bilayer structures other than vesicles. Pure vesicles obtained by dialysis of mixed micellar solutions were not hydrolyzed by the enzyme. Addition of bile salts reversed the inhibition which was accompanied by a decrease in turbidity. Phosphatidylcholine was preferred as substrate for human PLA2 when present in large mixed disc-like bile salt micelles. Vesicular or other types of lamellar liquid-crystalline phases of long-chain phosphatidylcholine did not serve as substrate for PLA2.

Characterization of recombinant human and rat pancreatic phospholipases A 2 secreted from Saccharomyces cerevisiae: Difference in proteolytic processing

An expression plasmid for human pancreatic phospholipase A 2 in Saccharomyces cerevisiae was constructed by insertion of cDNA encoding its preprophospholipase A 2 into a yeast expression vector pAM82. The resulting product secreted in the yeast culture medium was mainly prophospholipase A 2, which was the same as the natural proenzyme in all aspects examined, including the higher order structure. However, when the rat preprophospholipase A 2 cDNA was manipulated in the same manner, the active phospholipase A 2 of the intact mature form was secreted with the proenzyme being hardly detected in the medium. This unexpected favorable result would occur due to cleavage of rat phospholipase A 2 pro-peptide by a trypsin-like proteinase in S. cerevisiae. Based on this finding, we constructed a plasmid carrying the sequence coding for the prepro-peptide of rat pancreatic phospholipase A 2 behind the PHO5 promoter in the pAM82 vector, which leads to the secretion of heterologous proteins as their mature form. The use of this plasmid led to secretion of biologically active human pancreatic secretory trypsin inhibitor and a glutamic acid-specific endopeptidase from Staphylococcus aureus ATCC 12600, which are eukaryote and prokaryote proteins, respectively, in the culture medium of S. cerevisiae.