Lysophospholipids are ubiquitous intermediates in a variety of metabolic and signalling pathways in eukaryotic cells. We have reported recently that lysoglycerophosphatidylcholine (lyso-GPCho) synthesis in the insect form of the ancient eukaryote Trypanosoma brucei is mediated by a novel phospholipase A1 (TbPLA1). In the present study, we show that despite equal levels of TbPLA1 gene expression in wild-type insect and bloodstream trypomastigotes, both TbPLA1 enzyme levels and lysoGPCho metabolites are approx. 3-fold higher in the bloodstream form. Both of these parasite stages synthesize identical molecular species of lysoGPCho. TbPLA1 null mutants in the bloodstream form of the parasite are viable, but are deficient in lysoGPCho synthesis, a defect that can be overcome by the expression of an ectopic copy of TbPLA1. The biochemical attributes of TbPLA1-mediated lysoGPCho synthesis were examined in vitro using recombinant TbPLA1. Although TbPLA1 possesses an active-site serine residue, it is insensitive to serine-modifying reagents, such as di-isopropyl fluorophosphate and PMSF, a characteristic shared by lipases that possess lid-sheltered catalytic triads. TbPLA1 does not require metal co-factors for activity, but it does require interfacial activation prior to catalysis. Results from size-exclusion chromatography and binding kinetics analysis revealed that TbPLA1 activation by Triton X-100/GPCho mixed micelle surfaces was not specific and did not require the pre-formation of a specific enzyme–substrate complex to achieve surface binding.
All eukaryotic organisms are thought to metabolize phospholipids by expressing a highly regulated and variable complement of the phospholipases A1, A2, C, D and B. In Trypanosoma brucei, a medically important protist that causes African sleeping sickness, the complement of cellular phospholipases has not been fully elucidated. The only identified phospholipase in T. brucei was GPI-PLC (glycosylphosphatidylinositol-phospholipase C), which specifically recognizes GPI membrane anchors [1–3]. However, a highly active PLA1 has recently been cloned in T. brucei . TbPLA1 (T. brucei PLA1) exhibits substrate preference towards GPCho (glycerophosphatidylcholine) and produces unsaturated lysoGPCho (lysoglycerophosphatidylcholine) metabolites in vivo, approx. 50% of which contain a long and highly unsaturated FA (fatty acid) chain, consisting of 22 carbon atoms . Bacteria secrete PLA1 [5,6], but TbPLA1, which has a bacterial origin, is localized to the cytosol of T. brucei . In the insect PCF (procyclic form) of T. brucei, TbPLA1-synthesized lysoGPCho metabolites have homoeostatic levels of approx. 156 pmol/108 cells .
LysoGPCho is an amphiphilic lipid metabolite derived from GPCho, whose levels are vitally managed by balancing synthesis with degradation. As in other eukaryotes, GPCho is the most abundant phospholipid in T. brucei, comprising approx. 57% and 48% of total cellular phospholipid in the PCF and the mammalian BSF (bloodstream form) respectively [7,8]. Unlike in other eukaryotes, however, how T. brucei cells regulate GPCho homoeostasis is not understood. In higher eukaryotes, GPCho is metabolized by one of a number of lipolytic reactions, the best characterized ones being the following: 1) saturated lysoGPCho is synthesized by a PLA2, of which one type deacylates GPCho-derived arachidonic acid for use in cell signalling ; 2) saturated lysoGPCho is synthesized by the action of LCAT (lecithin–cholesterol acyltransferase) ; 3) PLD catalyses the hydrolysis of GPCho to phosphatidic acid and choline ; or 4) PLB deacylates the fatty acyl moieties of GPCho to form glycerophosphocholine and free FAs [12–14], a degradation pathway which is stimulated by Sec14 [15,16]. Interestingly, T. brucei is thought to lack PLA2 activity  and analysis of its genome does not reveal putative PLA2 or PLD homologues . On the other hand, several LysoPLA (lysophospholipase A)/PLB homologues are present in the database, which may explain previous studies that reported their corresponding specific esterase activity [18–21]. Despite the possession of an LCAT homologue in T. brucei, this parasite contains minute quantities of saturated lysoGPCho species and instead synthesizes mostly polyunsaturated and highly unsaturated lysoGPCho by TbPLA1 .
T. brucei spends much of its life in the mammalian systemic circulation from which it acquires its nutrients, including saturated and unsaturated lysoGPCho [18,22]. Unsaturated lysoGPCho in mammalian plasma accounts for roughly only 1–4% of total plasma phospholipids  and, although it is formed by an undefined mechanism, it is derived from hepatic secretions . It was originally thought that plasma lysoGPCho acquired by the BSF of T. brucei was detoxified by the cell's robust PLA1 activity [18,19,25]. This is most probably not the case, however, since TbPLA1 is cytosolic and metabolizes endogenous GPCho . With a long term goal to elucidate the biological significance of TbPLA1 in T. brucei, in the present study, we investigate endogenous lysoGPCho synthesis in the BSF of T. brucei and relate this activity to its insect-stage equivalent. We also characterize further TbPLA1-mediated lysoGPCho synthesis in vitro in order to further understand the properties of this novel eukaryotic enzyme.
Analytical reagents were bought from Sigma unless stated otherwise. Synthetic phospholipids were purchased from Avanti Polar Lipids. Chemicals used for bulk buffer production were from BDH unless stated otherwise. All solvents were of HPLC grade and purchased from BDH.
Generation and analysis of BSF TbPLA1 mutants
Deletion constructs for BSF WT (wild-type) transformations were synthesized by a series of amplification and cloning steps similar to the methods used to obtain PCF TbPLA1 mutants . Briefly, the 5′-UTR (untranslated region) and 3′-UTR sequences of TbPLA1 were amplified, linked together and inserted into a cloning vector. Puromycin (puroR) and hygromycin (hygR) drug resistance genes were then ligated individually between the UTR sequences, which generated the deletion constructs 5′-puroR-3′ and 5′-hygR-3′. The overexpression vector pLEW82-PLA1-HA was constructed by cloning TbPLA1 into pLEW82 using the same primers used to construct pLEW100-TbPLA1 .
The BSF in vitro culture cell line (strain 427, MITat 1.2) used throughout the present study was from the long-term cultures of ‘single marker’ cells from Wirtz et al.  that express a TetR (tetracycline repressor) protein and T7 RNA polymerase, and was maintained under neomycin drug pressure at a final concentration of 2.5 μg/ml. BSF cultures were maintained below a cell density of 2×106/ml in HMI-9 medium at pH 7.5, supplemented with 10% (v/v) heat-inactivated foetal bovine serum (PAA Labs) and 10% (v/v) Serum Plus (JRH Biosciences) as lipid sources. BSF transformation with DNA constructs was achieved through electroporation as described previously , except that the cells were left to recover in the incubator for 6–24 h in 24 ml of HMI-9 before distribution and drug selection in a 12-well plate. Transformed cells containing the puroR and/or hygR genes were cultured in the presence of these drugs at final concentrations of 0.1 μg/ml and 4 μg/ml respectively. Drug resistant parasites appeared between 5 and 7 days, at which time 50 μl of cells was used to start a new culture. Clonal populations of cells were obtained through limiting dilution.
BSF cell lines were analysed by Southern and Northern blotting, performed exactly as described previously , as was the nano-ESI–MS/MS (electrospray ionization tandem MS) analysis. Western blot analysis was performed on 107 cell equivalents/lane that were obtained by resuspending cultured cells in boiling SDS/PAGE sample buffer at a concentration of 106 cells/μl.
Infectivity of genetically modified parasites was examined in mice (n=5 mice for each cell line). Adult mice (BALB/c) were infected intraperitoneally with a single injection of either 5×105 WT T. brucei bloodstream trypomastigotes or 5×105 genetically modified TbPLA1 null mutants. Blood parasitaemia was moni-tored at 24, 48 and 72 h.
Total lipids from 108 mid-log phase BSF trypanosomes were extracted and analysed as described previously . Briefly, the resultant lower phase lipid extract from a Bligh and Dyer extraction  was dried under nitrogen and re-dissolved in 20 μl of chloroform/methanol (1:2, v/v). Internal standards (0.5 nmol/standard), lysoGPCho17:0/− and lysoGPCho24:0/−, were added prior to lipid extraction. Aliquots (7 μl) were analysed by nanoflow capillary ESI–MS/MS in positive-ion mode for GPCho species with a Micromass Quattro Ultima triple quadrupole mass spectrometer with capillary/cone voltages and collision gas as previously described . Each spectrum encompasses at least 50 repetitive scans of 4 s duration.
‘Surface dilution’ kinetic scheme
For clarity, the concept of ‘surface dilution’ kinetics is briefly explained here. Lipolytic enzymes act preferentially on substrates that are aggregated at lipid/water interfaces, and this makes kinetic analysis with lipids more complicated than with simple water-soluble substrates. Interface-dependent enzymes work in an environment where bulk interactions occur three-dimensionally in solution, and surface interactions occur in two-dimensions, and any kinetic model for these types of enzymes must consider both types of actions in their proposed mechanism. Deems et al.  proposed the ‘surface dilution’ kinetic model after showing that both the surface concentration of lipid and the bulk concentration of lipid in a reaction play important roles in elucidating kinetic parameters of enzymes that require interfacial activation.
The ‘surface dilution’ kinetic model's interfacial activation principles are expressed in the following manner:
A soluble enzyme (E) initially associates with a lipid/Triton X-100 mixed micelle aggregate (A) to form an enzyme–aggregate complex (EA). Once the enzyme associates with the aggregate it proceeds to bind to a substrate lipid molecule (B) in its catalytic site thus forming another complex (EAB). Products of the reaction (Q) are formed during catalysis and the EA complex is regenerated. The previous two steps are confined to the surface, and so the reaction is a function of the surface concentration of the substrate and not its bulk concentration, which is a deviation from classic Michaelis–Menton kinetics. Owing to the surface being composed of both lipid and detergent, substrate surface concentration is expressed as a unitless mole fraction, Xs=[lipid]/[lipid+detergent], and it is thus possible to vary the substrate surface concentration simply by adding or subtracting detergent, hence the name ‘surface dilution’ kinetics.
The initial EA complex can be formed in two ways and ‘surface dilution’ kinetics can be applied to determine in what form EA exists. It is possible that the enzyme binds non-specifically to the micelle surface, the ‘surface binding’ model, in which case term A is expressed as the molar concentration of both the lipid and detergent that make up the micelle . Conversely, it is feasible that the enzyme could bind specifically to a lipid substrate in the micelle with one binding site, forming EA, before binding specifically at another site on another lipid molecule for catalysis, which has been termed the ‘dual phospholipid binding’ model [29,30]. The data analysis performed in the present study used equations derived previously and made the same assumptions necessary to ascertain kinetic parameters .
Bis-BODIPY® (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene) FL-C11-PC kinetic assay
Bis-BODIPY® FL-C11-PC (Molecular Probes) was used as a GPCho fluorescent substrate at a 1:100 molar ratio with GPCho16:0/16:0, and Triton X-100/lipid micelles were made with this mixture. In its diacyl state, the proximity of the BODIPY® fluorophores on adjacent phospholipid acyl chains causes an intramolecular self-quenching of fluorescence. Hydrolysis and release of an acyl chain by a phospholipase A alleviates quenching and an increase in fluorescence results. The flourophores are situated at the ends of two 11-carbon chains, thus mimicking a GPCho with an approximate length near to that of GPCho16:0/16:0.
The assay was developed for use in a 96-well plate. Phospholipid and Triton X-100 varied according to experimental conditions, and the micelles were made as an 8× working solution, which was then diluted accordingly for use in an 80 μl reaction/well. Each well initially contained 40 μl of buffer (50 mM Tris/HCl, pH 7.0) containing the desired amount of micelle substrate. The reaction was started by the addition of 40 μl of reaction buffer (50 mM Tris/HCl, pH 7.0, and 100 mM NaCl) containing 400 pg (11.4 pmol) of recombinant TbPLA1, obtained as described previously . The reaction was left to proceed at 22°C for 30 min, before quenching with 170 μl of 100% methanol. Liberated acyl chains containing a fluorophore were excited at 485 nm and detected through a 520-nm-centred bandpass filter using an FLX 800 Microplate Reader (Bio-Tek Instruments). All experiments were performed at least three times in triplicate and data points varied by less than one S.D. Velocities obtained were initial velocities. When the assay was used to test activity in the presence of metals and inhibitors, the variable conditions are stated in the legends to the respective Tables. Statistical significance was measured by applying a Student's paired t test with a minimum 95% confidence interval.
Preparation of lipid/detergent mixed micelles
Manufacturer stock vials of phospholipid were dissolved at 2.5–10 mg/ml with chloroform:methanol (1:2, v/v) and stored in the dark at −80°C until use. The desired amount of the fluorescent substrate mix was transferred into 1.5 ml Apex screw-cap microtubes (Alpha) and dried under nitrogen, while being protected from light. The lipids were resuspended in micelle buffer (50 mM Tris/HCl, pH 7.0, and the appropriate quantity of Triton X-100 to make the desired mole fraction) heated to 40°C. This solution was then vigorously vortexed for 1 min and placed in a 40°C sonicator (Ultrawave Limited) until the solution cleared, with intermittent vortexing. The fluorescent mixed micelles were prepared fresh.
A Superdex 200 10/300 GL column (Tricorn) with an approximate bed volume of 24 ml was connected to an ÄKTA Purifier pump and equilibrated in a detergent-free buffer (50 mM Tris/HCl, pH 7.5, and 50 mM NaCl) over one and a half column volumes with a flow rate of 0.5 ml/min at 22°C. The column was calibrated with a mixture of molecular mass markers (Bio-Rad 151-1901). The markers were prepared as instructed by the manufacturer and 150 μl of the standard solution was diluted with buffer to 250 μl, loaded onto the column, separated by size-exclusion chromatography and detected by UV absorbance at 280 nm. The markers and their respective molecular masses are as follows: thyroglobulin, 670 kDa; γ-globulin, 158 kDa; ovalbumin, 44 kDa; myoglobin, 17 kDa; and vitamin B12, 1.35 kDa. A calibration curve was prepared by measuring the elution volumes of the standards, and then calculating their corresponding partition coefficient (Kav) values, and plotting their Kav values versus the logarithm of their molecular mass. Kav values were calculated from the equation:
where Ve is the peak elution volume, Vo is the void volume and Vt is the total bed volume.
Purified His6·tagged recombinant TbPLA1 (125 μl at 10 mg/ml dialysed against buffer) and a 13.7 kDa RNase A internal standard (125 μl at 3 mg/ml dissolved in buffer) were combined and loaded onto the gel-filtration column. The peak elution volumes and times were used to calculate their Kav values, and the molecular mass of the recombinant TbPLA1 was determined from the calibration curve. After the last sample had eluted, the column was immediately re-equilibrated in a buffer containing Triton X-100 (50 mM Tris/HCl, pH7.5, 50 mM NaCl and 0.05% Triton X-100), then a new calibration run was performed and another sample containing recombinant TbPLA1 spiked with RNase A was loaded and analysed as described.
RESULTS AND DISCUSSION
Life cycle stage expression of TbPLA1
To initially examine the potential of PCF and BSF cells to synthesize lysoGPCho, an analysis of the expression of TbPLA1 in both these stages of the parasite life cycle was measured by Northern blotting. TbPLA1 mRNA levels are nearly equal in PCF and BSF cells (Figure 1A). However, Western blot analysis of these cells offered a different perspective, since TbPLA1 levels in BSF cells were 3.6-fold greater than in PCF cells (Figure 1B). These findings justified an examination of the in vivo activity of TbPLA1 in BSF cells so that lysoGPCho synthesis in PCF and BSF cells could be compared.
TbPLA1 expression profile
Construction and integrity of TbPLA1 mutants
Before further comparative biochemical and enzymic studies could be performed, experiments, which led to the generation of a BSF double knockout of TbPLA1, were warranted. Deletion constructs (5′-puroR-3′, 5′-hygR-3′) targeted to the 5′ and 3′ flanking regions of the TbPLA1 ORF (open reading frame) were generated and the genes for puroR and hygR were cloned between these flanking regions. These constructs were used to stably transform BSF parental cells that are referred to here as WT cells, these cells are commonly used in reverse genetic studies in T. brucei, and are transgenic clones that constitutively express T7 RNA polymerase and the TetR protein . TbPLA1 is a single copy gene that does not appear to be essential for PCF cell viability in culture . This seems also to be the case for BSF cells: two successive and successful rounds of gene replacement by UTR targeted homologous recombination using the 5′-puroR-3′ and 5′-hygR-3′ deletion constructs were sufficient to replace the two alleles of TbPLA1, with no apparent resulting phenotype. Evidence for the generation of three BSF TbPLA1 null mutant cell lines (Δpla1) is shown by Southern blot analysis (Figure 2A).
Validation of the generation of TbPLA1 mutants
To create an additional analytical tool, a genetic transformation was also carried out that attempted to generate a cell line (Δpla1-rescPLA1-HA) that overexpressed a rescue copy of TbPLA1 in a Δpla1 background. To achieve this, a transformation was carried out using the T. brucei tetracycline-inducible overexpression vector pLEW82 , engineered to contain a recombinant form of TbPLA1 (Figure 2A). Upon tetracycline induction, trypanosomes transformed with pLEW82-PLA1-HA produce an ectopic copy of TbPLA1 with a C-terminal HA (haemagglutinin) tag (PLA1–HA). The resultant tetracycline-induced Δpla1-rescPLA1-HA rescue clones produced PLA1–HA mRNA transcripts >6-fold over the level of TbPLA1 mRNA in both the WT and their respective uninduced counterpart cells (Figure 2B).
The TbPLA1 rescue cells were examined for their ability to translate the overexpressed TbPLA1 mRNA. A Western blot probed with anti-TbPLA1 antibody revealed that expression of PLA1–HA was observed to be much less abundant than TbPLA1 mRNA in WT cells despite the high level of PLA1–HA mRNA overexpression (Figure 2C). It has been observed previously that lysoGPCho synthesis in T. brucei is regulated partly at the level of translation of TbPLA1 mRNA to control enzyme levels , therefore an up-regulation of TbPLA1 mRNA processing as a response to its overexpression in BSF Δpla1-rescPLA1-HA mutant cells may account for a decrease in TbPLA1 expression. The existence of regulatory-like processes to control TbPLA1 concentration could be significant in the cell, considering the potential harm a membrane-degrading enzyme, such as TbPLA1, could foster if expressed at uncontrollable levels.
The growth rate of cultured BSF TbPLA1 mutants was assessed 2 months after the creation of the Δpla1-rescPLA1-HA rescue cell line. TbPLA1 mutants are not compromised in their ability to divide and establish long-term cultures, and the population growth rate between the WT and TbPLA1 mutants (under drug pressure) was not altered significantly (Figure 2D). Although TbPLA1 is not essential in culture, it remained to be investigated whether or not, under more physiologically relevant conditions, the loss of TbPLA1 would not be detrimental to the proper functioning of the cell. Thus, the virulence of BSF Δpla1 cells was compared with BSF WT cells in their capacity to establish an infection in an animal model system. Analysing the daily blood parasitaemia in inoculated mice revealed that the virulence of Δpla1 trypanosomes was not noticeably different from the WT: high parasitaemias of approx. 1.6×109 trypanosomes/ml of blood, which was nearly equal to that of the WT control, were reached within 72 h with an initial inoculation of 5×105 trypanosomes.
LysoGPCho analysis of TbPLA1 mutants
Phospholipids are vital components of membranes and the phospholipases that modify them are recognized as important factors in mediating membrane dynamics. In PCF trypanosomes, it was shown that TbPLA1 is a major lipolytic enzyme used by T. brucei to metabolize GPCho . To show this, an efficient qualitative and quantitative method was employed to separate and characterize the major T. brucei phospholipid classes and their molecular species from total lipid extracts using nano-ESI–MS/MS). The same method was used in the present study to analyse the choline–phospholipid content of BSF cellular lipids by scanning for the collision-induced specific fragment ion m/z 184 (Figure 3A). The [M+H]+ (mass plus hydrogen positive ion) GPCho molecular species composition of BSF trypomastigotes was examined and compared with that determined previously from PCF cells (compare Figure 3A with Figure 5A of ). The pool of GPCho in both the PCF and BSF WT lipid extracts of T. brucei is comprised of the same molecular species of diacyl, alkylacyl and alkenylacyl lipids. However, BSF T. brucei appear to contain more lipids in the series GPCho38:y.
TbPLA1-mediated lysoGPCho synthesis in BSF T. brucei
To examine lysoGPCho species, however, the dominating diacyl and alkylacyl/alkenylacyl GPCho species were excluded from a subsequent analysis by scanning for m/z 184 precursors in the mass range m/z 400–700. Peak analysis of the resultant short range spectra from BSF WT lipids revealed the following set of [M+H]+ lipid metabolites: lysoGPCho−/22:6, lysoGPCho−/22:5, lysoGPCho−/22:4, lysoGPCho−/18:2, lysoGPCho−/18:1, lysoGPCho−/18:0, lysoGPCho−/20:2, lysoGPCho−/20:3 and lysoGPCho−/20:4. The peaks representing these metabolites and their [M+H]+ value are shown (Figure 3B, panel 1).
Qualitative comparison of the BSF lysoGPCho spectrum peaks with those from PCF analysis indicated that their abundance was increased significantly in the BSF spectra. To further investigate this result the lysoGPCho series metabolites were quantified by comparison with non-natural lysoGPCho17:0/− and lysoGPCho24:0/− added to each sample prior to lipid extraction (Figure 3B, inset to panel 1). From these quantitative ESI–MS/MS experiments it was perceived that BSF WT cells possessed 3-fold the number of moles of total lysoGPCho than in PCF WT cells (Table 1). However, this lysoGPCho increase was not distributed evenly among the major lysoGPCho series. There was a 3.9-fold increase in the lysoGPCho−/22:y series whereas only a 1.3-fold increase was observed in the lysoGPCho−/20:y series. It is, however, important to recall that an observed increase or decrease in a lysoGPCho metabolite doesn't necessarily equate to its increased or decreased rate of synthesis; the change could be accounted for by an increase or decrease in the rate at which these intermediates are metabolized further.
|LysoGPCho series* (pmol/108 cells)|
|LysoGPCho series* (pmol/108 cells)|
Integration of m/z [M+H]+ signals to obtain peak areas was performed by assembling individual lysoGPCho into a series of peaks whereby one series comprises both the major and minor lysoGPCho molecules, and their isotopes, containing the same FA chain length, but with various degrees of unsaturation, represented by ‘y’.
The synthesis of lysoGPCho molecules in BSF T. brucei are mediated by TbPLA1 since Δpla1 cells show a very significant drop in their levels (Figure 3B, panel 2). The number of moles of lysoGPCho in BSF Δpla1 null mutants was recorded to be approx. 20% of those metabolite levels in WT cells (Table 1 and Figure 3B, inset to panel 2). The individual lysoGPCho−/18:y, lysoGPCho−/20:y and lysoGPCho−/22:y series levels decreased by 77%, 60% and 84% in the BSF TbPLA1 null mutants respectively. This decline in the ability to synthesize lysoGPCho was confirmed to be the direct result of the loss of TbPLA1, since tetracycline-induced Δpla1-rescPLA1-HA cells rescued lysoGPCho synthesis (Figure 3B, panels 3 and 4). This recovery is only modest due to the small amount of PLA1–HA synthesized from overexpressed PLA1–HA mRNA (Figure 2C). The individual lysoGPCho−/18:y, lysoGPCho−/20:y and lysoGPCho−/22:y series levels were restored to 41%, 73% and 46% of normal BSF levels of these metabolites in these cells respectively (Table 1 and Figure 3B, panels 3 and 4 and their respective inset panels).
TbPLA1 null mutants still possess a small amount of lysoGPCho. There are several explanations for this observation. It is possible that one or more alternative phospholipases is responsible for a low level of synthesis in vivo. However, lysates of PCF Δpla1 cells could no longer produce any lysoGPCho in an in vitro assay , and this is also true for BSF Δpla1 cells (results not shown). Much of the lysoGPCho peak area in Δpla1 cells could be contributed by background levels of fragment ions formed mechanically from diacyl-GPCho during the sample preparation and/or the ionization process. Also, BSF T. brucei is known to efficiently uptake exogenous lysoGPCho from both culture and the host's bloodstream [18,20]; therefore, some of the unsaturated lysoGPCho species present in Δpla1 mutants could be from low-density-lipoprotein- or albumin-linked unsaturated lysoGPCho from host plasma, the abundance of which are estimated to be 4.2 and 25.9 nmol/ml of human plasma respectively .
Kinetic analysis of lysoGPCho synthesis
TbPLA1 is a soluble enzyme that, both in vivo and in vitro, acts on insoluble lamellar GPCho substrates. In order to achieve lysoGPCho synthesis TbPLA1 therefore must: 1) adsorb to membrane surfaces, i.e. undergo interfacial activation, and 2) cleave the sn-1 ester of its substrate. The mechanism by which the latter is accomplished is through the action of a Ser-His-Asp catalytic triad . The following experiments give insight into the mechanism of interfacial activation and binding properties of TbPLA1.
Analytically, the actions of soluble lipolytic enzymes can be manifested by employing ‘surface dilution’ kinetics [28–30], and this can be applied to give insight into the mechanism by which an interfacial enzyme initially binds to an aggregate  (see the Experimental section for an explanation of this kinetic scheme). Surface dilution kinetics for recombinant TbPLA1 were carried out with the use of mixed micelles composed of GPCho16:0/16:0, Bis-BODIPY® FL-C11-PC and Triton X-100. Surface dilution was accomplished by adding the non-ionic detergent Triton X-100 in increments while keeping the substrate concentration constant. TbPLA1 activity at various substrate surface concentrations (0.036, 0.018 and 0.009 mole fractions) was then measured as a function of various substrate bulk concentrations.
First, an experiment was carried out to examine the possibility that the enzyme binds non-specifically to the micelle surface, known as the surface-binding model. In this instance the aggregate molecules are expressed as the molar concentration of both the lipid and detergent that make up the aggregate. Comparison of TbPLA1 activity at various surface concentrations shows that as the surface concentration of lipid decreases there is a decrease in the apparent Vmax, this is the surface dilution phenomenon (Figure 4A). The true Vmax is the Vmax at an infinite mole fraction of phospholipid substrate. The linear relationship between the decrease in activity as the surface concentration of substrate decreases also shows that TbPLA1 does not have a strong affinity for individual Triton X-100 molecules, since they are in great excess over the substrate at all mole fractions.
TbPLA1 activity in relation to the surface-binding model
Theoretical predictions for the surface-binding model state that in a double-reciprocal plot for Figure 4(A) the lines should converge at a point where its y-axis ordinate represents 1/Vmax (Figure 4B). The presented linear regression lines converge adequately, but kinetic constants were more easily obtained using the 1/v intercepts from Figure 4(B) in a replot against the reciprocal of the mole fractions from which they were derived (Figure 4C). The data from this plot produce a straight line where the intercept of the 1/v axis represents the true 1/Vmax, and the 1/[mole fraction] intercept is the −1/Km, where Km is the interfacial Michaelis constant for the mixed micelle. The Vmax and Km towards GPCho16:0/16:0 were calculated to be 487 μmol/min per mg and 0.22 respectively. The surface dilution kinetics equations also predict that if the kinetic model being employed is correct, then a replot of the slopes of the lines in Figure 4(B) versus the reciprocal of the mole fractions should be linear and pass through the origin, as seen in our replot (Figure 4D). The data presented seem to fit the surface-binding model.
It is also feasible that the enzyme could bind specifically to a lipid substrate in the micelle with one binding domain, forming EA, prior to specifically binding to another lipid molecule for catalysis; this case has been termed the dual phospholipid-binding model [29,30]. Analysis can be carried out according to this model using the same data used to test the first model . Accordingly, a plot of PLA1 activity as a function of the molar concentration of GPCho16:0/16:0 shows again that the enzyme activity is dependent on surface concentration of substrate (Figure 5A). The double reciprocal plot of the data in Figure 5(A) shows that the linear regression lines of the data do not converge as predicted in the dual phospholipid-binding model (Figure 5B). A replot of the 1/v intercepts from the three data sets used in Figure 5(B) versus the reciprocal of the mole fraction produces the same result as in Figure 5(C) and the Km and Vmax values are thus identical. A plot of the slopes in Figure 5(B) versus the reciprocal of the mole fraction produces a straight line that does not pass through the origin, which suggests that the data do not fit the dual phospholipid-binding model.
TbPLA1 activity in relation to the dual phospholipid-binding model
Kinetic analysis thus appears to indicate that TbPLA1 activation in vitro is mediated by non-specific binding to the mixed micelle interface, prior to binding and catalysing a phospholipid substrate. The use of Triton X-100–GPCho16:0/16:0–Bis-BODIPY® FL-C11-PC mixed micelles in a 96-well plate assay to perform kinetics greatly facilitated the experimental procedure and improved upon some drawbacks experienced in previously used assay methods, which were more laborious, lacked sensitivity  and/or used unnatural thiol ester substrate analogues, which are poor substrates compared with oxyester lipids [29,34].
Evidence for EA formation
The kinetic studies provided fundamental insight into the mechanism of activation of recombinant TbPLA1, but making a conclusion that interfacial activation is substrate independent could not be done until it had been verified independently. If the first kinetic model is accurate, TbPLA1 should be able to bind to an aggregate whose surface is devoid of phospholipid substrate and composed solely of non-ionic diluters. To test this prediction, a Superdex 200 size-exclusion column equilibrated in the absence or presence of lipid-free Triton X-100 micelles was loaded with a solution containing purified recombinant TbPLA1. The sample was spiked with RNase A as an internal standard and the elution absorbance was monitored at UV280 nm. In the absence of an interface, the majority of TbPLA1 was shown by peak area analysis to elute in monomer form (38.7 kDa; 96%), whereas a small proportion of enzyme eluted in the void volume and as a ‘dimer’ (75.9 kDa) (Figure 6).
Adsorption of TbPLA1 on to detergent micelles
In contrast, the affinity of recombinant TbPLA1 for a pure Triton X-100 micelle surface is demonstrated by a shift in its elution time from 29.7 to 25.8 min, representing an increase in apparent molecular mass from 38.7 kDa to 134 kDa (Figure 6). This increase of 95.3 kDa corresponds very well to an increase in molecular mass if a monomer of the enzyme were bound to exactly one micelle of Triton X-100, which has a molecular mass of approx. 90 kDa .
These results provide evidence for enzyme–aggregate formation and are a testament to the non-specific adsorption mechanism employed by this enzyme during interfacial activation onto mixed micelles. This association to the micelle surface is not dependent on surface charge, since Triton X-100 is non-ionic, and it is also not due to specific binding to Triton X-100 molecules (Figure 4A). The non-specific nature of binding to Triton X-100 mixed micelles for TbPLA1 contrasts with the nature of binding for cytosolic PA–PLA1 partially purified from bovine testis, which requires anionic phosphoglycerides to bind to surfaces . No other PLA1s have been examined for their binding properties.
Metal ion effects on lysoGPCho formation
To understand more precisely the fundamental basis behind lysoGPCho formation by TbPLA1, its biochemical properties and activity were examined further in vitro. Besides a requisite of an interface for activation, some phospholipases require co-factors for optimal activity, whereas others do not. For example, cytosolic PLA2 activity is calcium-dependent, whereas another PLA2 group, iPLA2, is calcium-independent . Conversely, the heavier metals can be potent inhibitors of lipase activity [17,38]. In the present study, both the metal ion co-factor and inhibitory profile for TbPLA1 was assessed.
TbPLA1 apparently has no absolute metal ion requirements for activity, since dialysis or incubation of the enzyme with EDTA prior to commencing the reaction did not abolish lysoGPCho synthesis (Table 2). This result is consistent with a previous finding that PLA1 activity in lysates of T. brucei is divalent cation independent . Also, magnesium and manganese did not enhance or alter TbPLA1 activity, as has been observed previously for mammalian PLA1 . On the other hand, a number of heavy metals inhibited TbPLA1 activity at various concentrations, a phenomenon that has been observed before for PLA1 activity in soluble fractions of T. brucei incubated with 5 mM metal ions . The most potent metals are cadmium and copper, whose inhibitory effects can be observed at concentrations as low as 2 μM (Table 2). Incubation with different levels of iron produced partial to total inhibition, whereas moderate inhibition was detected in the presence of relatively high concentrations of nickel and zinc. The inhibitory effects to TbPLA1in vitro upon pre-incubation with heavy metals are mediated by an undefined mechanism.
|% Relative activity|
|Additive||2 μM||20 μM||200 μM||2 mM|
|% Relative activity|
|Additive||2 μM||20 μM||200 μM||2 mM|
Enzyme only control, set at 100% (26 μmol/min per mg).
The maximum concentration of EDTA added was 10 mM, resulting in 91% relative activity.
Effects of serine inhibitors on TbPLA1 activity
Knowing that Ser131 is the active-site residue for TbPLA1 , the next line of experiments sought to inhibit enzyme activity with the known active-site serine modifiers iPr2P-F (di-isopropyl fluorophosphate), PMSF and E-600 (diethyl-p-nitrophenyl phosphate). It was anticipated that iPr2P-F would have little effect on PLA1 activity originating from T. brucei . Palmitoyl and arachidonyl trifluoromethyl ketone analogues (PACOCF3 and AACOCF3 respectively) were also examined for potential effects of product inhibition, as observed for cytosolic PLA2 enzymes when in the presence of these compounds . However, preincubation with moderate levels of all of these individual compounds with TbPLA1 resulted in no inhibition, and only relatively little inhibition of PLA1 activity was observed with a very high concentration of inhibitor (Table 3). These inhibitors covalently and permanently modify accessible serine residues, and, upon successful modification, a complete loss of activity would be anticipated. The fact that TbPLA1 is insensitive to working concentrations of these serine-modifying reagents suggests that the serine active-site residue of TbPLA1 is inaccessible. Many prokaryotic and eukaryotic lipases, which structurally have single and multiple domains respectively, contain active-sites buried within the enzyme that are sheltered by an α-helical lid structure, rendering the catalytic triad un-solvated [43,44]. The active-site of a lipase is able to cleave the substrate only after interfacial activation, and the α-helical lid structure plays a role in this enzyme activation . In light of the failure of serine-modifying reagents to inhibit TbPLA1, these results suggest that the active-site of TbPLA1 may also be covered by a similar lid structure when the enzyme is not adsorbed to membrane surfaces, and that interfacial activation of TbPLA1 engenders a conformational change to expose these otherwise inaccessible active-site elements that are needed for phospholipid hydrolysis, in a structural change that may be similar to lipase activation. Structural studies are underway to clarify this issue.
|% Relative activity|
|Serine modifier||10 mM||1 mM||0.1 mM|
|% Relative activity|
|Serine modifier||10 mM||1 mM||0.1 mM|
Enzyme only control set at 100% (38 μmol/min per mg).
TbPLA1 is constitutively expressed in both PCF and BSF trypanosomes. A reverse genetics approach was employed to study the effects of TbPLA1 deletion in BSF parasites. Ablation of TbPLA1 resulted in a very drastic reduction of all the molecular species of lysoGPCho metabolites that were present in WT cells. Interestingly, despite the adoption and adaptation of TbPLA1 in the PCF life cycle stage , lysoGPCho levels in the BSF stage are 3-fold greater than in the PCF stage. TbPLA1 does not appear to be essential for cell viability in culture or for virulence in a mammalian host. Evidence that the enzyme is insensitive to serine-modifying reagents suggests that the catalytic triad active-site of TbPLA1 is buried inside the enzyme and is sheltered by a lid domain, a property shared with other lipases. Furthermore, in a GPCho–Triton X-100 mixed micelle system, this soluble, monomeric enzyme appears to adsorb to lipid–micelle interfaces non-specifically, instead of requiring lipid substrates to achieve interfacial activation. On balance, the results suggest that this binding and activation mechanism could help to induce a favourable conformational change, which is thought to be needed to expose active-site elements.
This work was supported by Wellcome Trust Senior Fellowship Grant 067441 and a Wellcome Trust Prize Studentship (to G. S. R.).
mass plus hydrogen positive ion
electrospray ionization tandem MS
open reading frame
Trypanosoma brucei phospholipase A1
Present address: Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, CA 90095, U.S.A.