Lysosomal Phospholipase Research Articles

Determinants of pH profile and acyl chain selectivity in lysosomal phospholipase A2 [S

Lysosomal phospholipase A2 (LPLA2) is characterized by broad substrate recognition, peak activity at acidic pH, and the transacylation of lipophilic alcohols, especially N-acetyl-sphingosine. Prior structural analysis of LPLA2 revealed the presence of an atypical acidic residue, Asp13, in the otherwise hydrophobic active site cleft. We hypothesized that Asp13 contributed to the pH profile and/or substrate preference of LPLA2 for unsaturated acyl chains. To test this hypothesis, we substituted Asp13 for alanine, cysteine, or phenylalanine; then, we monitored the formation of 1-O-acyl-N-acetylsphingosine to measure the hydrolysis of sn-1 versus sn-2 acyl groups on a variety of glycerophospholipids. Substitutions with Asp13 yielded significant enzyme activity at neutral pH (7.4) and perturbed the selectivity for mono- and double-unsaturated acyl chains. However, this position played no apparent role in selecting for either the acyl acceptor or the head group of the glycerophospholipid. Our modeling indicates that Asp13 and its substitutions contribute to the pH activity profile of LPLA2 and to acyl chain selectivity by forming part of a hydrophobic track occupied by the scissile acyl chain.

B-13 progenitor-derived hepatocytes (B-13/H cells) model lipid dysregulation in response to drugs and chemicals.

Lipid dysregulation is a common hepatic adverse outcome after exposure to toxic drugs and chemicals. A donor-free rat hepatocyte-like (B-13/H) cell was therefore examined as an in vitro model for investigating mechanisms. The B-13/H cell irreversibly accumulated triglycerides (steatosis) in a time- and dose-dependent manner when exposed to fatty acids, an effect that was potentiated by the combined addition of hyperglycaemic levels of glucose and insulin. B-13/H cells also expressed the LXR nuclear receptors and exposure to their activators – T0901317 or GW3965 – induced luciferase expression from a transfected LXR-regulated reporter gene construct and steatosis in a dose-dependent manner with T0901317. Exposing B-13/H cells to a variety of cationic amphiphilic drugs – but not other hepatotoxins – also resulted in a time- and dose-dependent accumulation of phospholipids (phospholipidosis), an effect that was reduced by over-expression of lysosomal phospholipase A2. Through application of this model, hepatotoxin methapyrilene exposure was shown to induce phospholipidosis in both B-13 and B-13/H cells in a time- and dose-dependent manner. However, methapyrilene was only toxic to B-13/H cells and inhibitors of hepatotoxicity enhanced phospholipidosis, suggesting phospholipidosis is not a pathway in toxicity for this withdrawn drug. In contrast, pre-existing steatosis had minimal effect on methapyrilene hepatotoxicity in B-13/H cells. These data demonstrate that the donor free B-13 cell system for generating hepatocyte-like cells may be employed in studies of fatty acid- and LXR activator-induced steatosis and phospholipidosis and in the dissection of pathways leading to adverse outcomes such as hepatotoxicity.

Preferential hydrolysis of truncated oxidized glycerophospholipids by lysosomal phospholipase A2

Truncated oxidized glycerophospholipids (ox-PLs) are bioactive lipids resulting from oxidative stress. The catabolic pathways for truncated ox-PLs are not fully understood. Lysosomal phospholipase A2 (LPLA2) with phospholipase A and transacylase activities is a key enzyme in phospholipid homeostasis. The present study assessed whether LPLA2 could hydrolyze truncated ox-PLs. Incubation of LPLA2 with liposomes consisting of 1,2-O-octadecenyl-sn-glycero-3-phosphocholine (DODPC)/1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) or truncated oxidized phosphatidylcholine (ox-PC)/N-acetylsphingosine (NAS) under acidic conditions resulted in the preferential deacylation at the sn-1 position of the truncated ox-PCs. Additionally, the release of free fatty acid from the truncated ox-PCs preferentially occurred compared with the NAS-acylation. Incubation of LPLA2 with the liposomes consisting of DODPC/DOPC/truncated ox-PC/NAS resulted in the same preferential fatty acid release from the truncated ox-PC. The cationic amphiphilic drug, amiodarone, did not inhibit such fatty acid release, indicating that truncated ox-PCs partition from the lipid membrane into the aqueous phase and react with free LPLA2. Consistent with this mechanism, the hydrolysis of some truncated ox-PCs, but not DOPC, by LPLA2 was detected at neutral pH. Additionally, LPLA2-overexpressed Chinese hamster ovary cells efficiently catabolized truncated ox-PC and were protected from growth inhibition. These findings support the existence of a novel catabolic pathway for truncated ox-PLs via LPLA2.

Prolonged Ocular Inflammation in Endotoxin-Induced Uveitis in Lysosomal Phospholipase A2-Deficient Mice

ABSTRACTPurpose: The goal of present study was to elucidate the pathophysiological roles of lysosomal phospholipase A2 (LPLA2) in intraocular pressure (IOP) levels and ocular inflammation.Methods: C57BL/6 (wild-type) and LPLA2-deficient mice with C57BL/6 background were employed. The IOPs were compared between wild-type and LPLA2-deficient mice during their aging, after topical administration of antiglaucoma medications such as travoprost, dorzolamide, or timolol maleate, or after induction of endotoxin-induced uveitis (EIU) using lipopolysaccharide (LPS). Concerning the EIU, ocular inflammation was also evaluated by immunohistochemical analysis by the anti-glial fibrillary acidic protein (GFAP) antibody.Results: The LPLA2-deficient mice showed higher IOP levels than the wild-type mice until 2 months of age (P = 1.60E-06); in older mice there was no difference between the two groups. Significant differences in the IOP changes between groups in young mice were seen after administration of 0.5% timolol (P < 0.05). Upon induction of EIU by LPS, compared with wild-type mice (P < 0.05), IOPs were significantly elevated in LPLA2-deficient mice at maximum levels of the ocular inflammation (48 h). Immunohistochemical analysis indicated that LPLA2-deficient mice showed more prolonged expression of GFAP at the inner plexiform layer and inner nuclear layer by EIU than that found in the wild-type mice (P < 0.05).Conclusions: These results confirm that LPLA2 plays a significant role in the control of IOP during mouse ocular development or with ocular inflammation by facilitating the digestion of intraocular insoluble materials.

Lysosomal Lipases PLRP2 and LPLA2 Process Mycobacterial Multi-acylated Lipids and Generate T Cell Stimulatory Antigens

Complex antigens require processing within antigen-presenting cells (APCs) to form Tcell stimulatory complexes with CD1 antigen-presenting molecules. It remains unknown whether lipids with multi-acylated moieties also necessitate digestion by lipases to become capable of binding CD1 molecules and stimulate Tcells. Here, we show that the mycobacterial tetra-acylated glycolipid antigens phosphatidyl-myo-inositol mannosides (PIM) are digested to di-acylated forms by pancreatic lipase-related protein 2 (PLRP2) and lysosomal phospholipase A2 (LPLA2) within APCs. Recombinant PLRP2 and LPLA2 removed the sn1- and sn2-bound fatty acids from the PIM glycerol moiety, as revealed by mass spectrometry and nuclear magnetic resonance studies. PLRP2 or LPLA2 gene silencing in APCs abolished PIM presentation to Tcells, thus revealing an essential role of both lipases invivo. These findings show that endosomal lipases participate in lipid antigen presentation by processing lipid antigens and have a role in Tcell immunity against mycobacteria.

Fluctuation of lysosomal phospholipase A2 in experimental autoimmune uveitis in rats

Intraocular inflammation leads to oxidative stress and may generate lipid oxidation products. The present study was conducted to elucidate the pathophysiological roles of the lysosomal phospholipase A2 (LPLA2), a phospholipid-degrading enzyme, and the production of oxidized phospholipids (oxPLs) in autoimmune uveitis using a rat model. Lewis rats were immunized with a bovine interphotoreceptor retinoid-binding protein (bIRBP) peptide with complete Freund’s adjuvant (CFA) to induce experimental autoimmune uveitis (EAU). The aqueous humor (AH) and serum were collected every week for 4 weeks from the immunized rats. The LPLA2 activity of the AH and serum was detected using liposomes consisting of 1,2-dioleoylphosphatidylglycerol/N-acetylsphingosine as the substrate under acidic conditions. Immunohistochemical analysis was performed using antibodies against LPLA2 and oxPLs. The ocular inflammation was exacerbated at 2 weeks after immunization. The LPLA2 activity in the rat AH was increased by EAU induction, and was concomitant with the extent of inflammation in the anterior chamber (AC). In contrast, the LPLA2 activity in the rat serum was not influenced by EAU induction. At 2 weeks after immunization, immunoreactivity of LPLA2 was observed in infiltrated macrophages in the AC and vitreous cavity of the EAU rats. Furthermore, immunoreactivity of oxPLs was observed in the infiltrated macrophages of EAU rat eyes. These results demonstrated that the LPLA2 activity of the AH is augmented with the inflammation in the AC. The high expression of LPLA2 and production of oxPLs are found in the infiltrated macrophages in the acute inflammation of EAU rats. The present findings suggest the connection between LPLA2 activity and oxPL metabolism in the inflammation sites in the eye.

Polysorbates 20 and 80 Degradation by Group XV Lysosomal Phospholipase A2 Isomer X1 in Monoclonal Antibody Formulations

Decreases in polysorbate (PS80) content were observed while evaluating the long-term storage stability of Chinese hamster ovary–derived, purified monoclonal antibodies. It was determined that polysorbate had been enzymatically degraded; therefore, studies were performed to identify and characterize the protein(s) responsible. Polysorbate degrading activity was enriched from Chinese hamster ovary media leading to the identification of group XV lysosomal phospholipase A2 isomer X1 (LPLA2) by shotgun proteomics. Recombinant LPLA2 was over expressed, purified, and functional integrity confirmed against a diheptanoyl phosphatidylcholine substrate. Incubation of recombinantly produced LPLA2 with PS20 and PS80 resulted in hydrolysis of PS20 and PS80 monoester but a much slower rate was observed for higher-order PS80. Endogenous LPLA2 was detected and quantitated at less than 1 ppm in 3 formulated antibodies while LPLA2 was not detected (or less than 0.1 ppm) in a fourth formulated antibody. Furthermore, antibodies with detectable quantities of endogenous LPLA2 demonstrated polysorbate hydrolysis while in contrast the antibody without detectable LPLA2 did not show polysorbate hydrolysis. Comparison of polysorbate degradation products generated from the formulated antibody and samples of polysorbate incubated with recombinant LPLA2 resulted in similar elution profiles by liquid chromatography–mass spectrometry. These results suggest that LPLA2 may play a key role in polysorbate degradation in some antibody preparations.

Structural and Biochemical Studies on the Activation Mechanism of Lecithin:Cholesterol Acyltransferase

Coronary heart disease (CHD) is the leading cause of death in the United States and is strongly associated with low levels of high density lipoprotein‐cholesterol (HDL‐C), thus there is significant clinical interest in methods that raise HDL‐C in order to treat and prevent CHD. Lecithin:cholesterol acyltransferase (LCAT) esterifies cholesterol on high density lipoprotein (HDL), which promotes HDL maturation and reverse cholesterol transport, the process by which cholesterol is transported from arterial plaques to the liver. Mutations in LCAT are known to cause two genetic diseases. Fish eye disease (FED) is characterized by partial loss of LCAT activity, whereas familial LCAT deficiency (FLD) is characterized by complete loss of LCAT. Individuals with either FED or FLD have low HDL‐C levels and corneal opacities, while FLD patients ultimately develop renal failure. Consequently, administration of recombinant LCAT and LCAT‐activating compounds are being investigated as treatments to increase HDL in both CHD and FLD, and have already shown promise in animal models. However, improvement of these therapeutics is limited due to a lack of structural information on LCAT and its interactions with HDL. LCAT is 50% identical to lysosomal phospholipase A2 (LPLA2). We previously solved a 1.84 Å structure of LPLA2, as well as a low‐resolution structure of LCAT (8.7 Å), both of which are in an active “lid open” conformation. In addition, structures of LPLA2 bound to inhibitors that mimic substrate binding led to models for how LPLA2 and LCAT bind to phospholipids and subsequently transfer an acyl group to the appropriate acceptor. Here we present a new “closed” crystal structure of LCAT at 3.3 Å wherein a retractable lid appears to block substrate access to the active site. Interestingly, the lid interacts with residues on the catalytic core that are mutated in LCAT genetic disease and are hypothesized to be important for activation by apolipoprotein A‐I (ApoA‐I), the major protein component of HDL particles. We therefore hypothesized that ApoA‐I binding will compete with the lid and thereby stimulate LCAT activity. We have probed the role of the lid by examining disease‐causing and lid mutations for esterase activity on a soluble substrate, binding to synthetic HDLs, and transacylase activity on HDLs. Overall, our results confirm the importance of the retractable lid and its observed intramolecular contacts in proper HDL binding and cholesterol esterification. Overall, these studies are expected to guide the design of more potent small molecule LCAT activators as well as more catalytically efficient recombinant LCAT that could be used for enzyme replacement therapy.Support or Funding InformationThis work was supported by US National Institutes of Health (NIH) grant F037815, an American Heart Association (AHA) Postdoctoral Fellowship 15POST24870001 (K.A.M.), and a University of Michigan Chemical Biology Postdoctoral Fellowship (A.G.).

Augmentation of Lysosomal Phospholipase A2 Activity in the Anterior Chamber in Glaucoma

Aim: The lysosomal enzyme in the anterior chamber has a crucial role in the digestion of the insoluble materials in the aqueous humor (AH). The dysfunction of AH filtration in the trabecular meshwork (TM) causes increasing AH outflow resistance in the TM. Those insoluble objects, including phospholipids, should be digested in the TM for normal outflow. The present study was conducted to explore the involvement of lysosomal phospholipase A2 (LPLA2), a phospholipid-degrading enzyme, of the AH in glaucoma using clinical AH specimens.Materials and Methods: One hundred and twenty-five AH specimens were collected from patients. The measurement of LPLA2 activity in the AH was carried out using liposomes consisting of phosphatidylglycerol and N-acetylsphingosine (NAS). The correlation between the LPLA2 activity in the AH and ocular diseases was investigated.Results: The human AH showed both transacylation of NAS and the release of fatty acids under acidic conditions but not at a neutral pH, which is consistent with the known properties of LPLA2. The LPLA2 activity in the AH was not affected by age or systemic disease. A comparison between ocular diseases showed that the AH specimens obtained from patients with glaucoma had significantly higher LPLA2 activity than the other ocular disease groups.Discussion: The present findings suggest that the ascended level of LPLA2 activity in the AH of glaucoma patients is associated with the development of glaucoma.

Tuning Cerium(IV)-Assisted Hydrolysis of Phosphatidylcholine Liposomes under Mildly Acidic and Neutral Conditions.

With the goal of designing a lysosomal phospholipase mimic, we optimized experimental variables to enhance Ce(IV) -assisted hydrolysis of phosphatidylcholine (PC) liposomes. Our best result was obtained with the chelating agent bis-tris propane (BTP). Similar to the hydrolytic enzyme, Ce(IV) -assisted hydrolysis of PC phosphate ester bonds was higher at lysosomal pH (∼4.8) compared to pH 7.2. In the presence of BTP, the average cleavage yield at ∼pH 4.8 and 37 °C was: 67±1 %, 5.7-fold higher than at ∼pH 7.2 and roughly equivalent to the percent of phospholipid found on the metal-accessible exo leaflet of small liposomes. No Ce(IV) precipitation was observed. When BTP was absent, there was significant turbidity, and the amount of cleavage at ∼pH 4.8 (69±1 %) was 2.1-fold higher than the yield obtained at ∼pH 7.2. Our results show that BTP generates homogenous solutions of Ce(IV) that hydrolyze phosphatidylcholine with enhanced selectivity for lysosomal pH.

Structure and function of lysosomal phospholipase A2 and lecithin:cholesterol acyltransferase.

Lysosomal phospholipase A2 (LPLA2) and lecithin:cholesterol acyltransferase (LCAT) belong to a structurally uncharacterized family of key lipid metabolizing enzymes responsible for lung surfactant catabolism and for reverse cholesterol transport, respectively. Whereas LPLA2 is predicted to underlie the development of drug-induced phospholipidosis, somatic mutations in LCAT cause fish eye disease and familial LCAT deficiency. Here we describe several high resolution crystal structures of human LPLA2 and a low resolution structure of LCAT that confirms its close structural relationship to LPLA2. Insertions in the α/β hydrolase core of LPLA2 form domains that are responsible for membrane interaction and binding the acyl chains and head groups of phospholipid substrates. The LCAT structure suggests the molecular basis underlying human disease for most of the known LCAT missense mutations, and paves the way for rational development of new therapeutics to treat LCAT deficiency, atherosclerosis and acute coronary syndrome.

Lysosomal phospholipase A2: A novel player in host immunity to Mycobacterium tuberculosis

Phospholipases catalyze the cleavage of membrane phospholipids into smaller bioactive molecules. The lysosomal phospholipase A2 (LPLA2 ) is specifically expressed in macrophages. LPLA2 gene deletion in mice causes lysosomal phospholipid accumulation in tissue macrophages leading to phospholipidosis. This phenotype becomes most prominent in alveolar macrophages where LPLA2 contributes to surfactant phospholipid degradation. High expression of LPLA2 in alveolar macrophages prompted us to investigate its role in host immunity against the respiratory pathogen Mycobacterium tuberculosis, the causative agent of tuberculosis. Here we report that adaptive immune responses to M. tuberculosis were impaired in LPLA2 deficient mice. Upon aerosol infection with M. tuberculosis, LPLA2 deficient mice showed enhanced mycobacterial counts but less lung immunopathology and pulmonary inflammatory responses. Compromised T-cell priming in the lymph nodes was associated with impaired pulmonary T-cell recruitment and activation. Together with reduced Th1 type cytokine production, these results indicate that LPLA2 is indispensable for the induction of adaptive T-cell immunity to M. tuberculosis. Taken together, we identified an unexpected and novel function of a lysosomal phospholipid-degrading enzyme.

1-O-acylceramides are natural components of human and mouse epidermis

The lipid-rich stratum corneum functions as a barrier against pathogens and desiccation inter alia by an unbroken meshwork of extracellular lipid lamellae. These lamellae are composed of cholesterol, fatty acids, and ceramides (Cers) in an equimolar ratio. The huge class of skin Cers consists of three groups: group I, "classical" long and very long chain Cers; group II, ultra-long chain Cers; and group III, ω-esterified ultra-long chain Cers, which are esterified either with linoleic acid or with cornified envelope proteins and are required for the water permeability barrier. Here, we describe 1-O-acylceramides as a new class of epidermal Cers in humans and mice. These Cers contain, in both the N- and 1-O-position, long to very long acyl chains. They derive from the group I of classical Cers and make up 5% of all esterified Cers. Considering their chemical structure and hydrophobicity, we presume 1-O-acylceramides to contribute to the water barrier homeostasis. Biosynthesis of 1-O-acylceramides is not dependent on lysosomal phospholipase A2. However, glucosylceramide synthase deficiency was followed by a 7-fold increase of 1-O-acylceramides, which then contributed 30% to all esterified Cers. Furthermore, loss of neutral glucosylceramidase resulted in decreased levels of a 1-O-acylceramide subgroup. Therefore, we propose 1-O-acylceramides to be synthesized at endoplasmic reticulum-related sites.

Role of N-glycosylation of human lysosomal phospholipase A2 for the formation of catalytically active enzyme

To understand the role of N-glycosylation of lysosomal phospholipase A2 (LPLA2), four potential N-glycosylation sites in human LPLA2 (hLPLA2) were individually modified replacing asparagine (Asn) with alanine by site-direct mutagenesis. COS-7 cells transiently transfected with wild-type (WT) hLPLA2 gene produced catalytically active LPLA2. A single mutation at 273-, 289-, or 398-Asn partially reduced production of active LPLA2. A single mutation at 99-Asn and quadruple mutations at all four Asn sites resulted in a marked reduction of active LPLA2 and loss of active LPLA2, respectively. Western blot analysis using anti-hLPLA2 antibody showed that the LPLA2 expression level was similar between all transfectants. N-glycosidase F digestion revealed that multiple forms of LPLA2 found in individual transfectants are due to different N-glycans linked to the core protein. The LPLA2 activity in individual transfectants was mostly recovered in the soluble fraction and correlated to the quantity of LPLA2 detected in the soluble fraction. LPLA2 mutated at 99-Asn was mostly retained in the membrane fraction. The WT transfectants treated with tunicamycin markedly lost LPLA2 activity. These data indicate that the 99-Asn is the most critical N-glycosylation site for formation of native hLPLA2 in vivo and that the N-glycosylation of LPLA2 is crucial for biosynthesis of catalytically active hLPLA2.

Increase of lysosomal phospholipase A2 in aqueous humor by uveitis

This study was conducted to elucidate pathophysiological roles of the lysosomal phospholipase A2 (LPLA2), a phospholipid-degrading enzyme, of the aqueous humor (AH) in uveitis using an animal model and clinical specimens. Endotoxin-induced uveitis (EIU) was induced by subcutaneous injections of lipopolysaccharide from Escherichia coli to seven-week-old male Lewis rats. Inflammation of the anterior chamber (AC) was evaluated by measurement of the protein concentration of rat AH. The LPLA2 activity in the AH, serum and cerebrospinal fluid obtained from EIU rats was detected using liposomes consisting of 1,2-dioleoylphosphatidylglycerol/N-acetylsphingosine as the substrate under acidic conditions. Immunohistochemical analysis was performed using antibodies against CD11b and LPLA2. Sixty-five human AH specimens, in which 11 eyes had a history of chronic uveitis, were collected during patient cataract surgeries and used to determine LPLA2 activity. The LPLA2 activity in rat AH was significantly increased by EIU induction, and was correlated to the extent of inflammation in the AC. By contrast, the LPLA2 activity in rat serum or cerebrospinal fluid was not influenced by EIU induction. According to the immunohistochemistry, LPLA2 was found in CD11b positive cells in the AC of the EIU rats. In the clinical specimens, the AH obtained from the patients with a history of uveitis possessed significantly higher LPLA2 activity than that from the senile patients with cataract but without other ocular diseases. These results demonstrate that the LPLA2 activity in the AH is augmented with the inflammation in the AC and suggest that the LPLA2 in the AH participates in the inflammation process in the AC.

Is the bovine lysosomal phospholipase B-like protein an amidase?

The main function of lysosomal proteins is to degrade cellular macromolecules. We purified a novel lysosomal protein to homogeneity from bovine kidneys. By gene annotation, this protein is defined as a bovine phospholipase B-like protein 1 (bPLBD1) and, to better understand its biological function, we solved its structure at 1.9 Å resolution. We showed that bPLBD1 has uniform noncomplex-type N-glycosylation and that it localized to the lysosome. The first step in lysosomal protein transport, the initiation of mannose-6-phosphorylation by a N-acetylglucosamine-1-phosphotransferase, requires recognition of at least two distinct lysines on the protein surface. We identified candidate lysines by analyzing the structural and sequentially conserved N-glycosylation sites and lysines in bPLBD1 and in the homologous mouse PLBD2. Our model suggests that N408 is the primarily phosphorylated glycan, and K358 a key residue for N-acetylglucosamine-1-phosphotransferase recognition. Two other lysines, K334 and K342, provide the required second site for N-acetylglucosamine-1-phosphotransferase recognition. bPLBD1 is an N-terminal nucleophile (Ntn) hydrolase. By comparison with other Ntn-hydrolases, we conclude that the acyl moiety of PLBD1 substrate must be small to fit the putative binding pocket, whereas the space for the rest of the substrate is a large open cleft. Finally, as all the known substrates of Ntn-hydrolases have amide bonds, we suggest that bPLBD1 may be an amidase or peptidase instead of lipase, explaining the difficulty in finding a good substrate for any members of the PLBD family.

Role for lysosomal phospholipase A2 in iNKT cell-mediated CD1d recognition

Invariant natural killer T (iNKT) cells recognize self lipid antigens presented by CD1d molecules. The nature of the self-antigens involved in the development and maturation of iNKT cells is poorly defined. Lysophospholipids are self-antigens presented by CD1d that are generated through the action of phospholipases A1 and A2. Lysosomal phospholipase A2 (LPLA2, group XV phospholipase A2) resides in the endocytic system, the main site where CD1d antigen acquisition occurs, suggesting that it could be particularly important in CD1d function. We find that Lpla2(-/-) mice show a decrease in iNKT cell numbers that is neither the result of a general effect on the development of lymphocyte populations nor of effects on CD1d expression. However, endogenous lipid antigen presentation by CD1d is reduced in the absence of LPLA2. Our data suggest that LPLA2 plays a role in the generation of CD1d complexes with thymic lipids required for the normal selection and maturation of iNKT cells.

A fluorogenic phospholipid for the detection of lysosomal phospholipase A2 activity

Lysosomal phospholipase A2 (group XV PLA2, LPLA2) is a lysosomal enzyme linked to drug-induced phospholipidosis. We developed phospholipid “smart probes” based on the conversion of a quenched fluorogenic substrate to a fluorescent product. Due to the preference of LPLA2 for phosphatidylglycerol, three fluorogenic phosphatidylglycerols were synthesized. Two fluorogenic phosphatidylglycerols were conjugated with one FAM (fluorescein amidite) group and one DABCYL [4-(4-dimethylaminophenylazo)-benzoyl] group; the third substrate consisted of two FAM groups conjugated at the sn-1 and sn-2 positions. The sn-1 ester linkage was replaced with an amide linkage. 1-FAM-2-DABCYL-PG was degraded by recombinant LPLA2 and mouse serum but not by the serum obtained from LPLA2-deficient mice when 1,2-dioleoyl-PG/1-FAM-2-DABCYL-PG liposomes were used. The formation of 1-FAM-lyso-PG generated from 1-FAM-2-DABCYL-PG in the presence of LPLA2 was quantitatively determined by fluorescent measurements. The 1-FAM-2-DABCYL-PG incorporated into 1,2-dioleoyl-phosphatidylcholine/sulfatide liposomes was used to evaluate the effect of the cationic amphiphilic drugs amiodarone and fluoxetine on LPLA2 activity. The IC50 values of amiodarone and fluoxetine estimated by fluorescent measurement were 10 and 19μM, respectively. These results indicate that 1-FAM-2-DABCYL-PG is a specific substrate for LPLA2 and a useful reagent for the detection of LPLA2 activity from multiple sources.

Drug induced phospholipidosis: An acquired lysosomal storage disorder

There is a strong association between lysosome enzyme deficiencies and monogenic disorders resulting in lysosomal storage disease. Of the more than 75 characterized lysosomal proteins, two thirds are directly linked to inherited diseases of metabolism. Only one lysosomal storage disease, Niemann–Pick disease, is associated with impaired phospholipid metabolism. However, other phospholipases are found in the lysosome but remain poorly characterized. A recent exception is lysosomal phospholipase A2 (group XV phospholipase A2). Although no inherited disorder of lysosomal phospholipid metabolism has yet been associated with a loss of function of this lipase, this enzyme may be a target for an acquired form of lysosomal storage, drug induced phospholipidosis. This article is part of a Special Issue entitled Phospholipids and Phospholipid Metabolism.

Rituximab therapy in pulmonary alveolar proteinosis improves alveolar macrophage lipid homeostasis

RationalePulmonary Alveolar Proteinosis (PAP) patients exhibit an acquired deficiency of biologically active granulocyte-macrophage colony stimulating factor (GM-CSF) attributable to GM-CSF specific autoantibodies. PAP alveolar macrophages are foamy, lipid-filled cells with impaired surfactant clearance and markedly reduced expression of the transcription factor peroxisome proliferator-activated receptor gamma (PPARγ) and the PPARγ-regulated ATP binding cassette (ABC) lipid transporter, ABCG1. An open label proof of concept Phase II clinical trial was conducted in PAP patients using rituximab, a chimeric murine-human monoclonal antibody directed against B lymphocyte specific antigen CD20. Rituximab treatment decreased anti-GM-CSF antibody levels in bronchoalveolar lavage (BAL) fluid, and 7/9 patients completing the trial demonstrated clinical improvement as measured by arterial blood oxygenation.ObjectivesThis study sought to determine whether rituximab therapy would restore lipid metabolism in PAP alveolar macrophages.MethodsBAL samples were collected from patients pre- and 6-months post-rituximab infusion for evaluation of mRNA and lipid changes.ResultsMean PPARγ and ABCG1 mRNA expression increased 2.8 and 5.3-fold respectively (p ≤ 0.05) after treatment. Lysosomal phospholipase A2 (LPLA2) (a key enzyme in surfactant degradation) mRNA expression was severely deficient in PAP patients pre-treatment but increased 2.8-fold post-treatment. In supplemental animal studies, LPLA2 deficiency was verified in GM-CSF KO mice but was not present in macrophage-specific PPARγ KO mice compared to wild-type controls. Oil Red O intensity of PAP alveolar macrophages decreased after treatment, indicating reduced intracellular lipid while extracellular free cholesterol increased in BAL fluid. Furthermore, total protein and Surfactant protein A were significantly decreased in the BAL fluid post therapy.ConclusionsReduction in GM-CSF autoantibodies by rituximab therapy improves alveolar macrophage lipid metabolism by increasing lipid transport and surfactant catabolism. Mechanisms may involve GM-CSF stimulation of alveolar macrophage ABCG1 and LPLA2 activities by distinct pathways.