Elevated expression of cyclooxygenase-2 is a negative prognostic factor for overall survival in intrahepatic cholangiocarcinoma

The production of prostaglandins is regulated by cyclooxygenases (COXs), which also have a role in tumour development and progression in various human malignancies, including cholangiocarcinoma. Limited information is available of the correlation of COX-2 protein expression and prognosis in intrahepatic cholangiocarcinoma (ICC). The aim of the present study was to determine the clinical significance of COX-2 expression in ICC. In addition the correlation of COX-2 expression and apoptosis/proliferation was analysed. COX-2 expression was determined immunohistochemically in 62 resected ICCs. Proliferation was assessed using Ki67-immunohistochemistry, and apoptosis was measured with the TdT-mediated dUTP nick-end-labelling technique. COX-2 was identified as an independent prognostic factor (P = 0.028) in resected ICC by survival analysis. High levels of COX-2 expression were found to be associated both with reduced apoptosis and increased proliferation of tumour cells. This study demonstrates the independent prognostic value of the COX-2 expression in resected ICC, thus, offering a potential additional adjuvant therapeutic approach with COX-2 inhibitors.
Introduction
Cholangiocarcinoma (CCC) is composed of cells resembling those of bile ducts. CCC arises either from the extrahepatic duct including the hilar bifurcation or from the intrahepatic ducts. Intrahepatic cholangiocarcinoma (ICC) is less frequent than extrahepatic bile duct carcinoma but second among primary malignant liver tumours after hepatocellular carcinoma. In Western countries, ICC accounts for about 10% of the primary liver malignancies with increasing incidence in the last two decades [24]. The identification of patients with most aggressive ICC might help to optimise surgical treatment and avoid unnecessary surgical therapy. Therefore, it is desirable to identify molecular markers that individually predict tumour behaviour and may facilitate an individualized therapy.
Among the potential prognostic parameters, cyclooxygenase (COX)-2 is of particular interest, as it may also offer the option of treatment with nonsteroidal anti-inflammatory drugs (NSAIDs) [15]. COXs regulate the synthesis of prostaglandins and are, thus, the major target of NSAIDs. Its two isoforms (COX-1 and COX-2) have different expression patterns, with COX-1 being expressed in a broad variety of tissues. COX-2 has been shown to participate in tumour development and progression [11, 21]. Both elevated COX-2 messenger RNA (mRNA) and protein levels were found to be raised in ICC [3, 9]. There exist only few studies dealing with the potential prognostic value of the COX-2 expression. A small study with only 24 cases of CCC could not prove a significant association with survival; however, this study did not discriminate between extra- and intrahepatic cholangiocarcinoma [12]. Another study on extrahepatic cholangiocarcinoma failed to demonstrate a correlation of the COX-2 expression and the patients’ outcome in CCC [16]. In the present study, COX-2 expression was determined by immunohistochemistry in one of the largest contemporary series of consecutive resected ICC. The findings were correlated with a broad range of clinicopathological features. Recent observations suggest that COX-2 overexpression may cause a prostaglandin E2-mediated inhibition of apoptosis in CCC. Moreover, COX-2 overexpression was shown to increase cell growth by the activation of E series of prostaglandins (EP receptors) [8, 31]. Thus, apoptosis and proliferation was determined in this series to monitor a putative imbalance between cell death and cell proliferation induced by COX-2 protein overexpression [26].
Materials and methods
Between August 1998 and August 2006, a total of 62 patients with a mean age of 58 ± 11.5 years were available for this study. The study comprised consecutive patients who underwent surgery for liver resection. Patients solely undergoing an explorative laparatomy without subsequent resection were excluded from the study. Patients with hilar cholangiocarcinoma, gallbladder carcinoma, or mixed hepato/-CCC and liver cirrhosis were also excluded from the study. The diagnosis of ICC was based on histology by the examination of the resected liver specimen. The tumours were classified according to the pathologic TNM (pTNM) system (sixth edition) [28]. Detailed clinical data was available including preoperative therapy, operative details, and pathological findings including surgical radicability, tumour staging, and clinical follow-up. One patient suffered from primary sclerosing cholangitis without cirrhosis; hepatolithiasis was not present in any case. Data were completed by August 2006, and minimum follow-up was every six months or until death. The median length of the follow-up was 12 months.
Immunohistochemistry
Immunohistochemistry was performed with an automated staining device (Dako Autostainer, Glostrup, Denmark).
Cyclooxygenase-2
In this study, a monoclonal rabbit anti-human COX-2 antibody (DCS, Hamburg, Germany) was used. Immunohistochemistry was performed on 5-μm-thick paraffin, and antigen retrieval was carried out with 0.01-M citrate buffer at pH 6.1 for 20 min in a hot water bath (95°C). The primary antibody was incubated for 30 min at 1:250 dilution. Antibody demonstration was achieved using the commercially available anti-mouse IgG detection kit (EnVision, DakoCytomation, Carpenteria, CA, USA) The replacement of the primary antibodies by mouse immunoglobin served as negative controls. Positive controls (colorectal carcinoma) were included in each staining series. In ICC, COX-2 was scored according to the amount of positive stained tumour cells. One complete tumour slide was examined for specific cytoplasmic COX-2 immunostaining. If none or less than 10% of the tumour cells showed specific COX-2 immunostaining regardless of staining intensity, the case was classified as negative. The cases with 11–50% of the positively stained tumour cells were classified as moderately positive and tumours with more than 50% stained tumour cells as strongly positive.
Ki67 immunostaining and TdT-mediated dUTP nick-end labelling
Ki67 immunohistochemistry was performed on 5-μm-thick paraffin sections. Dewaxed and rehydrated sections were incubated with hydrogen peroxide to block endogenous peroxidase. After the antigen retrieval in a hot water bath, the prediluted monoclonal anti-Ki67 antibody (Biogenex, San Ramon, USA) was incubated for 30 min; antibody demonstration was performed with the commercially available anti-mouse IgG detection kit (EnVision, DakoCytomation). The replacement of the primary antibodies by mouse immunoglobin served as negative controls.
The growth fraction was defined as the percentage of Ki67-positive, randomly chosen nuclei per 600 tumour cells. In situ DNA fragmentation was established using the terminal desoxyribonucleotide transferase TdT-mediated dUTP nick-end-labelling technique (TUNEL) in paraffin-embedded sections. We used ApoTag™ plus peroxidase in situ apoptosis detection kit (Intergen). The staining procedures were performed according the manufacturer’s recommendations. The percentage of the stained apoptotic tumour cells per 600 randomly chosen tumour cells was calculated. To avoid miscounting of the necrotic cells, corresponding H&E sections were examined.
Statistical analysis
COX-2 immunostaining was assessed by two of the authors (K.J.S., H.R.) in a blind-trial fashion without knowledge of the clinical outcome. In case of disagreement, slides were re-evaluated by both investigators until agreement was reached. All data were converted to a PC and statistically analysed using SPSS version 12 for Windows Statistical Package for Social Sciences, Chicago, IL, USA.
The kappa coefficient of the inter-observer agreement was calculated for the semiquantitative COX-2 immunostaining results; the interpretation of the kappa value was performed using the commonly cited scale of Landis and Koch [19]. The interobserver agreement of COX-2 immunostaining was substantial (kappa = 0.73). Relationships between ordinal parameters were investigated using the two-tailed χ2 analysis (or the Fisher’s exact test where patient numbers were small). The relationship between categorical data (e.g. COX-2) and numeric data (e.g. number on Ki-67 positive tumour cells) was determined using the ANOVA test. The overall survival (OS) curves were estimated using the Kaplan–Meier method, and any differences in the survival curves were compared by the log-rank test. For multivariate analysis, the COX regression model was used. Overall, 95% confidence intervals were used throughout.
Results
COX-2 immunohistochemistry
The COX-2 immunostaining was located in the cytoplasm of epithelial cells. The plasma cells exhibited a positive cytoplasmic immunostaining, serving as internal control. Noncancerous intrahepatic bile duct epithelial cells next to invasive cancer showed frequently a positive COX-2 immunoreactivity, whereas no COX-2 staining could be detected distant from the tumour (Fig. 1). Hepatocytes near and distant from ICC revealed a positive staining in all cases. In all, 12 (19.4%) tumours were classified as negative, 25 (40.3%) as moderately positive, and 25 (40.3%) as strongly positive. Epithelial tumour cells exhibited a broad range of COX-2 immunoreactivity with a complete lack of staining or a specific and strong granular cytoplasmic COX-2 immunostaining (Fig. 2). COX-2 staining was not associated with any of the investigated clinicopathological parameters (Table 1).
Fig. 1Immunostaining for COX-2 in noncancerous intrahepatic bile duct epithelial cells. Whereas portal bile ducts distant from the ICC consistently lacked COX-2 immunostaining (asterisk, left side), bile duct epithelial cells (asterisk, right side) adjacent to ICC (arrow) exhibited cytoplasmic immunoreactivity of varying intensity. Notice the COX-2 protein expression in normal hepatocytes. Original magnification, ×400Fig. 2Representative COX-2 immunostaining results in ICC. Missing COX-2 staining (left) in contrast to strong cytoplasmic COX-2 immunoreactivity in ICC tumour cells (right). Original magnification, 400×. Inset: positive control (colorectal carcinoma) with strong immunostainingTable 1COX-2 expression, demographic characteristics, and clinicopathological characteristics in 62 patients of the resected ICC AllCOX-2 negativeCOX-2 moderateCOX-2 strongP values n (%)6212 (19.4)25 (40.3)25 (40.3)Gender (male/female)7/59/1610/150.422Mean age at diagnosis (y±SD)58.8 ± 11.552 ± 12.160.4 ± 9.8 60.6 ± 11.80.067Grading0.612 G1 (n%)2 (3.2)0 (0)2 (100)0 (0) G2 (n%)41 (66.1)7 (17.1)15 (36.6)19 (46.3) G3 (n%)19 (30.6)5 (26.3)8 (42.1)6 (31.6)UICC stage grouping0.277 I166 (37.5)3 (18.8)7 (43.8) II30 (0)1 (33.3)2 (66.7) IIIA,B,C385 (13.2)19 (50)14 (36.8) IV51 (20)2 (40)2 (40)Staging0.390 T1 (n,%)217 (33.3)5 (23.8)9 (42.9) T2 (n,%)70 (0)4 (57.1)3 (42.9) T3 (n,%)284 (14.3)13 (46.4)11 (39.3) T4 (n,%)6 1 (16.7)3 (50)2 (33.3)Lymph vessel infiltration0.121  L0477 (14.7)18 (38.3)22 (46.8)  L1155 (33.3)7 (46.7)3 (20)Blood vessel infiltration0.916 V0398 (20.5)15 (38.5)16 (41) V1234 (17.4)10 (43.5)9 (39.1)Nodal statusa0.281 03710 (27)15 (40.5)12 (32.4) 1212 (9.5)10 (47.6)9 (42.9)Distant metastasis0.999 No5711 (19.3)23 (40.4)23 (40.4) Yes51 (20)2 (40)2 (40)Solitary tumourb327 (21.9)11 (34.4)14 (43.8)0.545Multifocal tumour295 (17.2)14 (48.3)10 (34.5)Tumour size 8.18.75 ± 4.3 8.3 ± 2.97.4 ± 3.60.506Resection status0.627 R0399 (32.1)15 (38.5)15 (38.5) R1/2c233 (13)10 (43.5)10 (43.5)P values were calculated using chi-square analysis and ANOVA for continuous variablesaData available for 58 patients; bdata available for 61 patients; c20 cases were classified R1, only 3 were R2
Clinical outcome by COX-2 expression
OS dependent on COX-2 expression was analysed using Kaplan–Meier survival (n = 62; p = 0.036; Fig. 3). Patients with a strong COX-2 expression exhibited a significantly decreased OS, whereas patients classified as COX-2 negative revealed the most favourable OS. The unfavourable prognostic effect of tumours with elevated COX-2 protein expression remained in the subgroup of patients with primarily R0-resected ICC (n = 39; p = 0.038).
Fig. 3Kaplan–Meier survival curves in resected intrahepatic cholangiocarcinoma in relation to different COX-2 expression. Tumours with elevated COX-2 levels exhibit a significantly decreased overall survival (p = 0.036, Log Rank test)
The parallel univariate survival analysis showed a significant association of the resection status (p < 0.001), multifocal tumour growth (p = 0.005), higher International Union Against Cancer (UICC) stage (p = 0.012), and vascular invasion (p = 0.011) with a reduced OS. Table 2 shows the mean OS for all patients in the various subgroups.
Table 2Mean overall survival in relation to different patient subgroupsSubgroupsMean overall survival in all ICC patients (months)All patients16.5R0-resected patients19.7R1-resected patients11.4R2-resected patients9.3COX-negative patients22.8Moderate COX-2 expression18.7Strong COX-2 expression11.4
To clarify the independent prognostic value of the COX-2 expression in the patients with resected ICC, a multivariate analysis of the relevant parameters was performed. The COX regression analysis revealed the R-classification to be the best prognostic factor for OS followed by the COX-2 expression (Table 3).
Table 3Multivariate COX regression analysis for overall survival in 62 patients with resected intrahepatic cholangiocarcinoma HR (95% CI) P valuespTstage I–II/III–IV1.63 (0.44–6.02)0.465UICC I–II, III–IV1.20 (0.62–2.35)0.589Multifocality, solitary/multifocal1.26 (0.41–3.89)0.689Resection status, R0/R1, R2a3.08 (1.27–7.44)0.012Vascular invasion, V0/V11.98 (0.88–4.41)0.095COX-2, negative/moderate/strong1.07 (1.07–3.49)0.028aTwenty cases were classified R1, only three were R2
COX-2 and apoptosis/proliferation
There were significant lower mean numbers of apoptotic tumour cells per 600 tumour nuclei in tumours with strong COX-2 expression in contrast to tumours with moderate or negative COX-2 immunoreactivity (Table 4). In contrast, the proliferative activity increased in tumours with high levels of COX-2, reflected by a higher mean value of proliferating tumour cells. However, due to a substantial overlap of values, statistical significance was not reached.
Table 4Apoptosis (TUNEL) and proliferative activity (Ki67) in relation to COX-2 expression All (mean value/SD)COX-2 negativeCOX-2 moderate/strongP valuesApoptosis (percentage of positive tumour cells)2.17 ± 2.403.84 ± 4.041.76 ± 1.730.028Growth fraction (percentage of Ki67 positive tumour cells)9.74 ± 7.378.55 ± 8.419.99 ± 7.340.486P values were calculated using ANOVA for continuous variables
Discussion
CCC is a devastating cancer of the hepatic biliary tract. The assessment of the individual prognosis of ICC patients may be helpful to optimise decision making for surgical treatment and avoid unnecessary surgical therapy. At present, surgical therapy remains the only curative treatment for ICC; thus, novel therapeutic strategies for this aggressive cancer are necessary. This study on a large series of 62 patients with resected ICC showed the independent prognostic value of immunohistochemically determined COX-2 protein expression, thus, offering a potential additional adjuvant therapeutic approach with COX-2 inhibitors. Elevated COX-2 expression was associated with a marked reduced OS in multivariate analysis. The main advantage of our study is the large number of patients (n = 62) in combination with a high homogeneity of this series composed of consecutively resected ICC. A previous study on extrahepatic cholangiocarcinoma failed to demonstrate a relation of COX-2 expression with clinical outcome [16]. This suggests (genetic) differences depending on the site of origin within the biliary tract. Mutations of the RAS and TP53 genes are the most common genetic abnormalities identified both in ICC and cancer of extrahepatic bile duct (BDC) [13, 14, 16, 20, 29]. However, recent studies disclosed genetic differences regarding p53 protein overexpression among carcinomas of the proximal (ICC) and distal bile ducts (BDC), indicating different patterns of the inactivation of tumour-suppressor genes [1]. Because cell line studies suggest COX-2 both as an effector and inhibitor of the tumour suppressor p53 or DNA-damaging agents [4, 7], different genetic patterns in ICC and BDC, with varying induction of COX-2 gene/protein expression, might explain the diverse clinical impact of COX-2 in ICC and BDC.
A small study on 24 patients with CCC failed to show a prognostic significance of the COX-2 expression [12]. However, the power of this study is limited due to two reasons: (1) The authors did not discriminate between extra- and intrahepatic cholangiocarcinoma, and (2) the statistical power of a small series with 24 patients is very low. However, Javle et al. [12] demonstrated in their study that the median survival time of patients with low COX-2 expression was more than twice as high than that of patients with high COX-2 expression. This finding is in accordance with our results identifying COX-2 overexpression as a predictor of reduced OS. Javle et al. found COX-2 to be expressed in all 24 ICC, whereas in our study, only 81% of ICC exhibited an either moderate or strong COX-2 expression. Kim et al. [16] found COX-2 to be overexpressed in only 52% of the examined extrahepatic cholangiocarcinomas. These discrepancies are likely to be caused by differences in antigen retrieval and varying scoring systems, emphasizing the relevance of standardized protocols and immunoscores.
Previous studies suggest COX-2 overexpression as an early carcinogenetic event in the human biliary tract and pointed out that COX-2 expression was highest in more differentiated CCC, whereas COX-2 expression decreased with the loss of differentiation [5]. Our study did not reveal a significantly increased COX-2 expression in tumours with higher differentiation. Nevertheless, a detailed analysis of our data rather supports this notion. The group of well and moderately differentiated tumours exhibited the highest amount of cancers classified as strongly COX-2 positive (44.2%) in contrast to ICC with poor tumour differentiation (31.6%). On the other hand, we found the lowest levels of COX-2 expression to be more frequent in poorly differentiated tumours (26.3%) in contrast to well or moderately differentiated ICC (16.5%). The study by Kim et al. [16] on 102 extrahepatic cholangiocarcinoma describes no significant differences in terms of tumour differentiation. In the light of these varying data, one should be careful not to draw premature conclusions about the differential expression COX-2 protein in ICC.
The clinical conditions associated with ICC include parasitic liver fluke infestations, hepatolithiasis, which is frequently observed in clonorchiasis [10], nonbiliary cirrhosis, and primary sclerosing cholangitis [30]. It would be interesting to compare the levels of COX-2 expression in ICC with or without these established associated clinical risk factors. However, our series is not suitable to answer this question because all cases with liver cirrhosis, regardless of its origin, were excluded from our study to maintain the high homogeneity of our cohort and rule out cirrhosis- related influence on patient survival. As a consequence, this series comprises only one patient with associated primary sclerosing cholangitis. Patients with liver fluke infestations and associated hepatolithiasis were not present in this series because our cohort is composed from patients living in western Europe.
Interestingly, we found the up-regulation of COX-2 protein expression not only in cancerous epithelial cells but also in noncancerous epithelial cells adjacent to invasive cancer. Noncancerous epithelial cells distant from the ICC lacked COX-2 immunostaining. This staining pattern is in accordance with the results of Hayashi et al. [9]. The elevated expression of COX-2 in bile duct epithelial cells adjacent to invasive cancer is not surprising, as inflammation often accompanies invasive cancer and inflammatory cytokines such as IL-1α [22], IL-1β [2], or TNF-α are believed to induce COX-2 gene at the transcriptional level [23]. We observed a constant positive specific immunostaining in normal hepatocytes regardless of the distance to the ICC. This finding is in agreement with previous studies documenting elevated COX-2 protein and mRNA expression in nontumourous liver [3, 17].
Against the background of NSAIDs, recent studies have evaluated selective COX- inhibitors for their effect on CCC cells cell growth and invasion in vitro and in nude mice [6, 18, 27, 32, 33]. The treatment with COX-2 inhibitors resulted in induced apoptosis and inhibited proliferation. The mechanisms by which COX-2 contributes to the poor prognosis in variety of human cancers have not been completely elucidated yet. COX-2 has been shown to contribute to both tumourigenesis and the malignant phenotype of tumour cells by different mechanisms, including (1) the inhibition of apoptosis by the activation of the PI3-Kinase/Akt pathway and increased production of PGE2, leading to increased expression of Bcl-2 and attenuation of nitric oxide signalling, (2) increased angiogenesis by increased PGE2 production with subsequent vascular endothelial growth factor production, (3) increased invasiveness by the overexpression of CD44, and (4) increased cell growth by the activation of EP receptors [8, 31]. The above mentioned effect of COX-2 overexpression on apoptosis and proliferation can be confirmed in our study. COX-2 over-expressing ICC revealed a significantly decreased apoptosis and a higher mean value of proliferating tumour cells, although the latter finding did not reach statistical significance. This finding suggests an impaired balance between cell loss and cell gain, resulting in a shift towards tumour net tumour growth due to increased apoptosis. The observed decrease of apoptosis in COX-2 overexpressing ICC might be due to the PGE2-mediated induction of the antiapoptotic protein Bcl-2 and increased NFκB transcriptional activity, which is a key antiapoptotic mediator [25, 26].
In conclusion, this study demonstrates the independent prognostic value of immunohistochemical COX-2 protein expression in resected ICC, thus, offering a potential additional adjuvant therapeutic approach with COX-2 inhibitors and facilitating an optimised therapeutic strategy. Our results suggest that COX-2 overexpression causes a shift towards increased tumour cell proliferation and decreased apoptosis contributing to the unfavourable clinical course.


Asian wasp envenomation and acute renal failure: a report of two cases

Acute renal failure is an unusual complication of wasp stings. We report two cases of renal failure after multiple wasp stings (Vespa affinis). Both patients had evidence of intravascular haemolysis, hepatic dysfunction, oligo-anuria and azotaemia and required dialysis. The first patient had severe hemolysis, rhabdomyolysis, pigment and venom nephropathy and died on the 8th day in hospital. The second patient, who recovered completely in 3 weeks time with steroid and antihistaminic therapy, had interstitial nephritis. Although acute renal failure after wasp stings is typically caused by acute tubular necrosis (ATN) in the setting of haemolysis or rhabdomyolysis, in some patients, acute renal failure may result from a direct nephrotoxic effect or acute interstitial nephritis from a hypersensitivity reaction.
INTRODUCTION
In the Pokhara Valley, Nepal, there are many unpublished cases of wasp poisoning which take a heavy death toll annually. Wasp stings usually cause local allergic reactions but can sometimes lead to intravascular haemolysis, rhabdomyolysis, thrombocytopenia, acute tubular necrosis, acute hepatic injury (1) and even myocardial infarction (2) in addition to various respiratory and neurological (3) manifestations.
Death from wasp envenomation is a rare event and results from acute renal failure (ARF) involving various mechanisms. Although ARF after wasp stings is typically caused by ATN in the setting of haemolysis or rhabdomyolysis, in some patients, renal failure may result from a direct nephrotoxicity of wasp venom or acute interstitial nephritis from a hypersensitivity reaction. We here we report two cases of acute renal failure after wasp stings (Vespa affinis).
CASE 1
A 55-year-old farmer who had been collecting fodder from a jungle was admitted with dyspnea, hoarse voice and myalgia within 8 hours of being attacked by several wasps. The patient was given intravenous saline, oxygen, salbutamol (β2-adrenergic receptor agonist), chlorpheniramine (antihistamine), cyproheptadine (antihistamine), prednisolone (corticosteroid) and ranitidine (histamine H2-receptor antagonist). He also received fluid, mannitol and furosemide. By the next morning he had haemoptysis and had produced 400 ml of dark urine. On examination, the patient was drowsy, pale, icteric and cyanosed and had approximately one hundred and fifty red and swollen sting marks all over the body. Systemic examination revealed polyphonic wheezes and crepitations at the base of the right lung. Investigations are shown in Table 1. The onset of the oliguric phase was at 12 hours postenvenomation. Chest radiograph showed right basal consolidation. He died on the 8th day following admission despite aggressive therapy with medication, blood transfusions, assisted ventilation, and 16 cycles of dialysis.
CASE 2
A 40-year-old forest guard was attacked by a swarm of wasps. He presented with approximately twenty-five sting marks in exposed areas of face, throat, hands and legs [Fig. 1]. The patient was treated in the primary health centre with rubbing of saliva and papaya slices over the sting marks and referred to teaching hospital almost 24 hours after being stung. He developed anuria and had not passed any urine the previous night. On examination, the patient had a rapid pulse, unrecordable blood pressure, icterus, an urticarial rash in exposed parts of the face, legs and hands, facial puffiness, and a swollen left ankle and right knee joint. The rest of the physical examination was unremarkable. Investigations are shown in Table 1. He was treated with oxygen, salbutamol, chlorpheniramine, cyproheptadine, prednisolone and ranitidine. His hepatic and renal function improved gradually with fluid challenge, furosemide, mannitol, bicarbonate infusion, dopamine, and 12 cycles of dialysis in 3 weeks’ time.
OBSERVATION
DISCUSSION
Wasp stings are well-known causes of toxic and hypersensitivity reactions. Direct toxicity is rare, but has been reported in cases when a very large amount of venom is injected. Immediate hypersensitivity reactions, such as bronchospasm in the first case and an urticarial rash in the second, are known to occur. In our report, both reactions responded to steroids and antihistaminics. The second patient had swollen joints indicating serum sickness-like reaction in a sensitized individual.
Wasp venom contains toxic melittin, apamine, phospholipases A1, hyaluronidase, acid phosphatase, histamine, and degranulating peptide mastoparan (4). These components have direct and indirect cytotoxic (hepatic, renal and myocyte membrane), hemolytic, neurotoxic and vasoactive properties, which can cause intravascular haemolysis and rhabdomyolysis (5, 6).
Wasp venom can cause ARF by several mechanisms, which include ATN, acute interstitial nephritis, pigment nephropathy resulting from rhabdomyolysis (myoglobinuria) or intravascular haemolysis (haemoglobinuria) and hypotension caused by an anaphylactic reaction (7, 8).
Previously rhabdomyolysis and renal ischemia were thought to be main causes of nephropathy. Sakhuja et al had postulated that direct toxic injury could be one of the possible mechanisms of ARF following wasp poisoning (9).
Many cases of rhabdomyolysis-associated ARF have been published, but those due to wasp stings are rare. The wasp venom has deleterious effect on renal tubules and glomeruli (albuminuria, haematuria and ARF), red blood cells (haemolysis, reticulocytosis, unconjugated hyperbilirubinaemia), muscles (rhabdomyolysis, elevated creatinine phosphokinase and lactate dehydrogenase, myoglobinuria) and liver (elevated transaminases, hypoalbuminaemia and prolonged prothrombin time) (10). Kularatne et al had described similar multi-organ failure with high mortality following wasp poisoning owing to direct toxic effect (11).
In the first case we presented, the patient had myalgia, indicating muscle injury as evidenced by elevated CPK, LDH and AST and myoglobinuria (728 ng/ml). He also had intravascular haemolysis and haemoglobinuria. Toxic pigments might have caused nephropathy resulting in ARF. The alternative mechanism of ARF postulated was direct nephrotoxicity by massive wasp venom.
Zhang R et al. (12) reported for the first time that acute tubulointerstitial nephritis could lead to ARF in wasp sting cases. In the second case we present, the patient had eosinophiluria, indicating interstitial nephritis. He recovered fully with the mentioned treatment. He did not report taking any medication which might have had nephrotoxic side-effects, and no other causes of ATN could be found. Ultrasound abdomen was unremarkable. Kidney biopsy revealed proximal peritubular necrosis and eosinophilic infiltration. Hence it can be hypothesized that the ATN was caused by a hypersensitivity reaction to the wasp venom.
CONCLUSION
Wasp stings pose a great environmental hazard in Nepal and early recognition of anaphylactic shock, hepatic or renal dysfunction, rhabdomyolysis or haemolysis and rapid transport to hospital are essential steps of management to avoid fatalities. ARF due to toxic or pigment nephropathy and tubulointerstitial nephritis should be considered in any oliguric and azotemic patient following wasp attack.

wasp envenomation
acute renal failure
rhabdomyolysis
interstitial nephritis
nepal


Kinetoplastid PPEF phosphatases: Dual acylated proteins expressed in the endomembrane system of Leishmania

Bioinformatic analyses have been used to identify potential downstream targets of the essential enzyme N-myristoyl transferase in the TriTryp species, Leishmania major, Trypanosoma brucei and Trypanosoma cruzi. These database searches predict ∼60 putative N-myristoylated proteins with high confidence, including both previously characterised and novel molecules. One of the latter is an N-myristoylated protein phosphatase which has high sequence similarity to the Protein Phosphatase with EF-Hand (PPEF) proteins identified in sensory cells of higher eukaryotes. In L. major and T. brucei, the PPEF-like phosphatases are encoded by single-copy genes and are constitutively expressed in all parasite life cycle stages. The N-terminus of LmPPEF is a substrate for N-myristoyl transferase and is also palmitoylated in vivo. The wild type protein has been localised to the endocytic system by immunofluorescence. The catalytic and fused C-terminal domains of the kinetoplastid and other eukaryotic PPEFs share high sequence similarity, but unlike their higher eukaryotic relatives, the C-terminal parasite EF-hand domains are degenerate and do not bind calcium.
1
Introduction
Protein phosphorylation and dephosphorylation are critical processes in a variety of cellular mechanisms for the detection, transmission, and integration of intra- and extra-cellular signals. In eukaryotes, the extensively studied PPP family of serine/threonine protein phosphatases function in cellular processes as diverse as regulation of the cell-cycle, RNA splicing and T cell activation [1]. PPPs have been divided into three subfamilies, commonly referred to as the ‘Classical’ PPP phosphatases; PP1, PP2A and PP2B [1]. Novel phosphatases that cannot be categorised into these subfamilies include the retinal degeneration C protein (RdgC) from Drosophila melanogaster
[2]. RdgC homologues are found in a number of eukaryotic species, including Homo sapiens, Mus musculus, Gallus gallus and Caenorhabditis elegans
[3,4] but have not been identified in fungi, yeast or plants to date. These novel phosphatases, distinguished by several putative EF-hand motifs within a fused C-terminal domain, have subsequently been termed PPEFs or Protein Phosphatases with EF-Hands [3].
Interestingly, PPEFs exhibit a much narrower tissue distribution than classical PPPs, being restricted to the central nervous system or primary sensory structures in all metazoans studied to date. Thus, Dm RdgC has been principally localised to photoreceptors and the mushroom bodies of the central brain [2,5] while C. elegans CePPEF is highly enriched in primary sensory neurones [6]. In situ hybridisation and immunostaining have also localised mammalian PPEF isoforms to sensory structures such as the inner ear, dorsal root ganglia, embryonic brainstem nuclei, photoreceptors and pinealocytes [3,4]. These findings suggest that the PPEFs have conserved functions in diverse sensory systems and may have a role in development in mammals. The substrates of the RdgC/PPEF phosphatases, however, remain elusive.
The domain architecture of RdgC/PPEF homologues is highly conserved, consisting of three fused domains (Fig. 1A). The N-terminal regulatory domain often contains myristoyl/palmitoyl acylation motifs and a downstream conserved IQ (isoleucine-glutamine) calmodulin binding domain. The catalytic domain contains several RdgC/PPEF specific sequences/motifs together with insertions of unknown function. The C-terminal domain contains at least two EF-hand motifs that have been shown to bind calcium in HsPPEF-1 and CePPEF [6,7] and one or more degenerate EF-hand like motifs.
Here, we describe the characterisation of PPEF-like genes in the diverse lower eukaryotes Leishmania and Trypanosoma, the sole members of this phosphatase family in these parasites. The kinetoplastid PPEFs were identified following a genome-wide search for N-myristoylated proteins, carried out to identify downstream targets of the essential gene N-myristoyl transferase (NMT) in Leishmania major, Trypanosoma brucei and Trypanosoma cruzi. We show that LmPPEF and TbPPEF are substrates for NMT in vivo and that LmPPEF (and probably TbPPEF) can also be palmitoylated in vivo. Using immunofluorescence, LmPPEF has been localised to the endocytic system of Leishmania parasites, with some accumulation at the flagellar pocket. This location requires downstream regions of the protein in addition to the unique acylated N-terminus. Unlike other members of the RdgC/PPEF family, the EF-hand domains within the C-terminus of LmPPEF are degenerate and do not bind calcium under the experimental conditions used here.
2
Materials and methods
2.1
PCR amplification and sub cloning
The 2862-bp LmPPEF open reading frame (ORF) was amplified from cosmid 1567.3 (gift from Al Ivens) using Pfu DNA polymerase (Promega) at 64 °C annealing temperature and the primers LmPPEFFor (5′-ATGGGGTGTGACTCATCCAAG-3′) and LmPPEFRev (5′-TTAGCGACTAGTGCCGAGGC-3′). The amplified LmPPEF ORF was cloned into pPCR-Script AMP SK(+) (Stratagene) to generate pLmPPEF.
236-bp and 1056-bp fragments from the 3′ end of the LmPPEF ORF (nucleotides 2154–2862 and 1806–2862, respectively) were amplified at 60 °C annealing temperature using primers LmPPEF-Cterm1For (5′-GACGATcatatgCGCATCTGGTAC-3′) and LmPPEF-Cterm1Rev (-5′-TGGCggatccTCTAGCCCTTA-3′) or primers LmPPEF-Cterm2For (5′-ATTAATTTcatatgCAGGTGGTGAGTCTA-3′) and LmPPEF-Cterm2Rev (5′-AATAggatccTTAGCGACTAGTGCC-3′). Cloning sites are shown in lower case. The PCR fragments were digested with NdeI/BamHI and cloned into pET-15b (Novagen) generating pLmPPEF-Cterm1 and pLmPPEF-Cterm2, respectively.
The 2775-bp TbPPEF ORF was amplified from T. brucei genomic DNA at 59 °C annealing temperature, using primers TbPPEFFor (5′-CTTACGTTTccatggGTTGCTC-3′) and TbPPEFRev (5′-CCTCCcTcgagatCTCTCACAAA-3′), digested with NcoI/XhoI and cloned into pET-33b, generating pTbPPEF. The recombinant TbPPEF protein expressed from this plasmid has an N-terminal myristoylation motif (MGCSTSK).
2.2
Parasite culture, membrane fractionation and nucleic acid analysis
L. major Friedlin parasites (MHOM/IL/80/Friedlin) were cultured, nucleic acids extracted and DNA/RNA blotting and hybridisation carried out as previously described [8]. For membrane fractionation, mid-log phase parasites (5 × 107) were lysed by sonication on ice in either PBS alone, PBS plus 1 mM CaCl2 or PBS plus 1 mM EGTA. Undisrupted cells were removed by two centrifugation steps (500 × g, 4 °C, 10 min). Cell lysates were separated into membrane and cytosolic fractions by ultra centrifugation (100,000 × g, 4 °C, 1 h). Following separation, membrane fractions were washed twice in PBS and proteins from both fractions analysed by SDS-PAGE and immunoblotting as described [8].
2.3
Antibody production and immunoblotting
Expression of N-terminally His-tagged recombinant LmPPEF-Cterm1 was induced by isopropyl-β-d-thiogalactopyranoside (IPTG) in Escherichia coli Rosetta (DE3) pLysS (Novagen). Cells were subsequently lysed in 6 M Gu-HCl prior to affinity chromatography using Talon Ni2+-nitrilotriacetic acid-agarose (Ni-NTA; BD Biosciences). Eluted protein was precipitated using 10% trichloroacetic acid, air dried and used for immunisation and generation of rabbit polyclonal antiserum (Eurogentech). Partial purification of LmPPEF-specific polyclonal antibodies was carried out using ammonium sulphate precipitation as described [9], followed by affinity purification against purified recombinant LmPPEF-Cterm1 as described [10].
Parasites were lysed in SDS-PAGE gel loading buffer, and immunoblotted as above with purified LmPPEF antiserum (abSK2031, 1:200 dilution), anti-NMT (abSK805, 1:2000 [8]), peroxidase anti-peroxidase (PAP) complex (P-2026, 1:2000, Sigma), or anti-GFP (ab290, 1:10,000, Abcam). Immune complexes were detected using an ECL kit (Amersham Biosciences).
2.4
L. major episomal expression constructs and parasite transfection
A 111-bp fragment from the 5′ end of the LmPPEF ORF (nucleotides1–111) was amplified from pLmPPEF at 58 °C annealing temperature using primers Lm37WT-GFPFor (5′-TAAAggatccATGGGGTGTGACTC-3′) and Lm37WT-GFPRev (5′-TTATAgatatcGCTACAAGTGCGTCG-3′). The fragment was digested with BamHI/EcoRV and cloned into pX-GFP [11], generating pLm37WT-GFP. Plasmids pLm37G2A-GFP, pLm37C3S-GFP and pLm37G/A,C/S-GFP were generated as above, using forward primers Lm37G2A-GFPFor (5′-TAAAggatccATGGCGTGTGACTC-3′), Lm37C3S-GFPFor (5′-TAAAggatccATGGGGTCTGACTC-3′) and Lm37G/A,C/S-GFPFor (5′-TAAAggatccATGGCGTCTGACTC-3′), respectively, and the reverse primer Lm37WT-GFPRev.
The 2862-bp LmPPEF ORF was amplified from pLmPPEF at 60 °C annealing temperature using primers LmPPEF-TAPFor (5′-ATTAATTTcatatgGGGTGTGACTCAT-3′) and LmPPEF-TAPRev (5′-ATAtctagaCTTGCGGCTAGTGCC-3′), digested with NdeI/XbaI and cloned into the TAP vector pGL893 (gift from Sebastion Besteiro), generating pLmPPEF-TAP. The recombinant LmPPEF-TAP protein expressed from this plasmid has an N-myristoylation motif (MGCDSSK). All constructs used in this study are shown in Fig. 1B.
Mid-log phase L. major were electroporated with 20–50 μg of either pLm37WT-GFP, pLm37G2A-GFP, pLm37C3S-GFP, pLm37G/A,C/S-GFP or pLmPPEF-TAP as described [11] and cultures subsequently grown in media supplemented with 1 mg/ml G418 (Life Technologies, Inc.).
2.5
Metabolic labelling and immunoprecipitation
Mid-log phase L. major promastigotes were metabolically labelled as previously described [11]. Cells were lysed for 1 h at 4 °C in lysis buffer (PBS containing 50 mM Tris, pH 7.5, 150 mM NaCl, 5 mM EDTA, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 100 μg/ml leupeptin, 500 μg/ml pepstatin, 198 μg/ml 1,10 phenanthroline and 25 μg/ml E64). The lysates were pre-cleared by incubation for 1 h at 4 °C with protein A-coupled Sepharose (Amersham Biosciences). Labelled proteins were then recovered from the supernatant by incubation with either anti-LmPPEF or anti-GFP antibodies overnight at 4 °C. After a second protein A-coupled Sepharose incubation, the beads were collected by centrifugation, washed twice in lysis buffer and proteins removed by boiling in SDS-PAGE gel loading buffer, prior to separation by SDS-PAGE. Detection of radiolabelling was improved using EN3HANCE™ Autoradiography Enhancer (Kodak). DTT was omitted from the loading buffer for separation of [9,10-3H] palmitate-labelled proteins.
2.6
Calcium mobility shift assay
This assay was carried out as described [12]. In brief, proteins were lysed in SDS-PAGE gel loading buffer and separated by SDS-PAGE using either 5 mM CaCl2 or 5 mM EGTA in both the stacking and resolving gels. Separated proteins were Coomassie-stained or analysed by immunoblotting.
2.7
N-Myristoylation co-expression assay
This assay was performed as described [8,13]. In brief, E. coli BL21(DE3) pLysS cells were co-transformed with pTbPPEF and either pNMT [8] or pTbNMT [14]. Expression of recombinant TbPPEF and NMT protein was induced by IPTG in the presence of [3H]-myristate (Amersham Biosciences) and, following SDS-PAGE, radiolabelled proteins were detected by autoradiography.
2.8
Fluorescence microscopy
For indirect immunofluorescence, L. major promastigotes were fixed and processed as previously described [15]. Cells were stained prior to fixation by incubation in 10 μM FM4-64 FX (Invitrogen) in serum-free medium at 26 °C for 10 min. For direct immunofluorescence, parasites were fixed in 4% (w/v) paraformaldehyde at room temperature for 15 min, washed in PBS and immobilised on poly-l-Lysine coated coverslips (4 × 106 cells per cover slip). Slides were mounted using Vectashield plus 4′,6′-diamidino-2-phenylindole (DAPI; Vecta Laboratories).
3
Results
3.1
The kinetoplastid N-myristoylomes
N-Myristoylation describes the co-translational addition of the C14:0 fatty acid myristate to the amino-terminal glycine residue of a subset of eukaryotic and viral proteins. Catalysed by the enzyme myristoyl-CoA:protein N-myristoyltransferase (NMT), N-myristoylation often plays a role in targeting proteins to membrane locations and can additionally be involved in mediating protein–protein interactions and stabilising protein structure. We have previously shown that NMT is essential for viability in kinetoplastid parasites [8] while myristate analogues (non-specific inhibitors of NMT) are lethal to L. major promastigotes and bloodstream T. brucei
[8,16,17]. A range of more specific NMT inhibitory compounds has recently been tested and several shown to inhibit T. brucei NMT activity in vitro, with toxicity to bloodstream T. brucei at low μM concentrations [18]. However, the cellular processes contributing to lethality in NMT-depleted cells are as yet unknown, although identification of the essential downstream targets of this enzyme may be crucial to the successful development of NMT as a potential drug target. Few kinetoplastid N-myristoylated proteins have been studied in depth to date—the Leishmania HASPB and SMP-1 proteins and the T. brucei ARL1 protein are exceptions [11,14,19].
We used several strategies, all based on the well characterised eukaryotic N-myristoylation consensus motif, to search the L. major, T. brucei and T. cruzi genome datasets for putative N-myristoylated proteins. The first publicly available prediction algorithm for these proteins, the PDOC00008 PROSITE myristoylation signature [20], is known to generate high numbers of false positive as well as false negative predictions. The more recent NMT Predictor program was developed using a set of ‘positive’ amino acid sequences for profile training, substantially reducing the number of false results generated and extending the N-myristoylation motif from 6 to 17 amino acids [21,22]. Further refinement has come with the Myristoylator program, based on a neural network model and incorporating an additional ‘negative’ training set of amino acids [23]. We firstly used the ‘relaxed’ version of PROSITE PDOC00008 derived from the NMT Predictor, G-{EDRKHPFYW}-x(2)-[STAGCNDEF]-{P} (described in [21]), to probe the kinetoplastid genome datasets (http://www.genedb.org/), thereby reducing the >8000 predicted ORFs to ∼250–300 potentially N-myristoylated proteins. The Myristoylator prediction algorithm was then applied to this refined data set, generating a final list of predicted N-myristoylated proteins within high confidence boundaries (scores: 0.85–1; Table 1; Tables S1–S3, supplementary data). For the three genomes analysed, 62 N-myristoylated proteins were predicted in L. major, 62 in T. brucei and 123 in T. cruzi, with the latter higher figure a consequence of the presence of two different haplotypes in the T. cruzi CL Brener genome sequence [24]. Predicted N-myristoylated proteins contribute 0.5–0.8% of the proteomes of other eukaryotes studied to date [22,25], and the kinetoplastid data fall well within this range (Table 1). Further analysis of the N-termini of the high confidence kinetoplastid proteins revealed a strong preference (>70% of the total) for serine at position 6 in all three species, with threonine used in a further ∼8% of proteins. In Saccharomyces cerevisiae, serine or threonine in these positions allows stabilisation of the peptide–NMT complex through hydrogen bonding with Asp417, Gly418 and His221 in the ScNMT crystal structure [21].
Of the putative N-myristoylated proteins identified with high confidence, 8 were specific to L. major, including the dual acylated HASPB [11] and the ADP-ribosylation factor LmARF3 [14], with a similar number of targets specific to T. brucei and T. cruzi (Tables S1–S3, supplementary data). Other previously characterised N-myristoylated proteins included the calpain-like proteins, T. brucei CAP 5.5 [26] and L. major SMP-1 [19], while a number of putative signal transduction pathway proteins were also identified, including protein kinases and phosphatases (Table 2). However, the largest group of kinetoplastid N-myristoylated proteins was that currently characterised as of “unknown function”. A majority of the proteins within this group are conserved between the three Tri-Tryp species.
3.2
Kinetoplastid PPEF-like protein phosphatases
Analyses of the kinetoplastid genomes have identified a range of kinases and protein phosphatases, some possessing novel motifs and insertions suggesting possible structural and functional differences from their mammalian homologues [24,27]. Of the phosphatases, only a few of the classical types have been characterised to date: protein phosphatase 2C (PP2C) from L. chagasi
[28], PP1 from T. cruzi
[29], PP1, PP2A and PP5 from T. brucei
[30]. The N-myristoylome analysis described above identified a conserved serine/threonine protein PPEF-like phosphatase (hereafter called PPEF) in all three kinetoplastid species: LmjF12.0660 in L. major, Tb927.1.4050 in T. brucei, Tc00.1047053506529.380/Tc00.1047053510889.80 in T. cruzi. These kinetoplastid PPEFs are of similar size and composition: 954 aa, pI 8.0, 109.0 kDa in L. major; 925 aa, pI 7.7, 105.7 kDa in T. brucei; 923 aa, pI 7.7, 104.8 kDa in T. cruzi.
The phylogenetic relationship between these three proteins, the RdgC/PP5 family and the classical phosphatase groups was investigated by multiple sequence alignment (using CLUSTAL W [31]) and generation of an unrooted tree (using the Tree View software, version 1.5.2 [32]). This analysis confirmed that the trypanosomatid PPEFs are more closely related to the RdgC/PPEF subfamily than to the classical PPP subfamilies, PP1, PP2A or PP2B, and are also separate from the PP5/PPT and PP7 subfamilies (Fig. 2A). Alignments revealed sequence similarity between the kinetoplastid PPEFs and other members of the RdgC/PPEF subfamily (Fig. S1, supplementary data), with the overall percentage of amino acid identity between representative proteins of this group shown in Fig. 2B. While the kinetoplastid proteins share 60–70% identity with each other, they are only ∼20% conserved when compared to their higher eukaryotic relatives. The similarity between these proteins is more apparent, however, when comparing their domain organisation, both at the primary sequence level (Fig. 1A; Fig. S2, supplementary data) and by homology modelling of the catalytic domain of LmPPEF with that of mammalian PP1 for which a high resolution structure is available [33] (Fig. S1, supplementary data). The central catalytic domains of the kinetoplastid and mammalian PPEFs, together with D. melanogaster RdgC, share significant sequence similarity (42%), including several RdgC/PP5 specific catalytic motifs [1]. Kinetoplastid PPEFs also share RdgC/PPEF specific mutations found in the conserved SAPNYC motif (common to all PPP phosphatases) that is found within the β12/β13 loop of the catalytic domain (Fig. S2, supplementary data). The first of these, a Pro to Ser substitution at position 3, is found in all members of the Rdg/PP5 family including the kinetoplastid proteins and at least two other protozoan phosphatases, PP1 from T. cruzi
[29] and PfPPJ, a novel protein phosphatase from Plasmodium falciparum
[34]. The second substitution within the SAPNYC motif, replacement of the Cys-6 residue by either Tyr, Asp or Asn, is restricted to the RdgC/PPEF sub-family. Overall, the level of conservation within the catalytic domain of LmPPEF, including those residues acting as metal ligands, suggest that this protein and its kinetoplastid orthologues are functional phosphatases (Fig. S1, supplementary data).
At their C-termini, the kinetoplastid PPEFs are more closely related to the C-termini of the RdgC/PPEFs than to any other calcium binding proteins or EF-hand domain-containing enzymes [35]. This observation supports the hypothesis that the catalytic domain of the ancestral form of RdgcC/PPEF fused with an EF-hand Ca2+-binding protein prior to the acquisition of the N-terminal domain [1]. At their N-termini, there is little similarity between the kinetoplastid PPEFs and the RdgC/PPEFs except for the presence of putative N-myristoylation and palmitoylation motifs, which are found in some but not all members of the RdgC/PPEF phosphatase family.
3.3
Genomic organisation and expression of the L. major and T. brucei PPEFs
The Artemis annotation tool [36] was used to examine the level of gene synteny in the regions flanking the PPEF genes in the L. major and T. brucei genomes. LmPPEF is found on chromosome 12 in L. major while TbPPEF is found on chromosome 1 in T. brucei (http://www.genedb.org/). The chromosomal regions surrounding these genes have been analysed previously as part of a wider study, revealing high conservation of gene order [37]. Thus the kinetoplastid PPEFs, including TcPPEF, can be classified as true orthologues.
To confirm the in silico gene analysis, genomic DNA blotting and hybridisation were used to show that both LmPPEF and TbPPEF are present as single copy genes in the L. major and T. brucei genomes, respectively (Fig. 3A and B). RNA expression from these genes in different parasite life cycle stages was initially demonstrated using RT-PCR (Fig. 3Ci and Di). RNA blotting and hybridisation were then used to confirm constitutive expression of single PPEF transcripts (of 4.3 and 4.8 kb, respectively) in insect and mammalian stages of L. major and T. brucei (Fig. 3Cii and Dii).
Expression of the LmPPEF protein was analysed by immunoblotting, using affinity-purified antibodies raised against the C-terminal 236 residues of the recombinant protein (expressed from construct LmPPEF-Cterm1, Fig. 1B). These antibodies recognised a single polypeptide band of 109 kDa on immunoblots of whole L. major parasite lysates, correlating with the size of the deduced open reading frame of gene LmjF12.0660 (Fig. 4A). Whole cell lysates from L. major procyclics, metacyclics and amastigotes were then analysed and a single 109 kDa protein detected in all three life cycle stages (Fig. 4B). However, no signal was detected in T. brucei procyclic or bloodstream form parasite lysates using the same anti-LmPPEF (data not shown). This suggests that this C-terminal antibody does not cross react with TbPPEF (despite the 51% amino acid conservation in this domain) and/or that TbPPEF is expressed at low levels during the parasite life cycle.
3.4
LmPPEF is not a calcium-binding protein
Given the degeneracy of EF-hand motifs in the C-terminal domains of the kinetoplastid PPEFs, and the absence of N-terminal IQ calmodulin binding motifs, we investigated whether LmPPEF can bind calcium, either as a recombinant or wild type protein, in a mobility shift assay. In this analysis, target and control proteins were separated by electrophoresis through denaturing gels in the presence of either Ca2+ or EGTA. Fig. 5A shows the positive (calreticulin) and negative (BSA) control proteins after separation: the migration of BSA was similar in both gel types whereas the migration of calreticulin was significantly altered on electrophoresis through Ca2+ as compared to EGTA, with >50% showing a mobility shift. To analyse the behaviour of LmPPEF in this assay, the C-terminal 351 residues, containing all three EF-hand like motifs, were expressed (from construct LmPPEF-Cterm 2) as a 40 kDa N-terminally His-tagged protein in L. major. Comparing the mobility of this protein and wild type LmPPEF in whole cell lysates, in the presence of Ca2+ or EGTA, revealed no significant differences in migration, suggesting that LmPPEF does not bind calcium (Fig. 5B).
Cell fractionation of total L. major proteins was also carried out, to analyse the relative distribution of LmPPEF between membrane and cytoplasm and to investigate whether this distribution was altered in the presence or absence of calcium. Wild type parasites were lysed in either PBS alone, PBS plus 1 mM CaCl2 or PBS plus 1 mM EGTA and separated by electrophoresis. Cell fractionation carried out in PBS alone revealed that LmPPEF is predominantly membrane-associated, with ∼20% of the protein detected in the soluble or cytoplasmic fraction (Fig. 5B). The presence or absence of calcium did not alter this membrane versus cytoplasmic distribution, again suggesting that the degenerate EF-hands in LmPPEF are unlikely to bind calcium. A similar conclusion emerged from 45Ca overlay experiments with both wild type and recombinant protein (data not shown).
3.5
The N-termini of recombinant and wild type kinetoplastid PPEFs are substrates for acylation in vivo
Given the presence of N-myristoylation motifs together with cysteine residues predicted to be palmitoylation sites at the N-termini of kinetoplastid PPEFs (Tables S1–S3, supplementary data), we firstly confirmed that these proteins could act as templates for NMT in an E. coli co-expression assay [8]. In these experiments, plasmids expressing NMT and TbPPEF as substrate were co-expressed in the presence of [3H]-myristoyl CoA and incorporation of radioactivity into myristoylated product detected by autoradiography following SDS-PAGE (Fig. 6A). Using NMTs from both L. major and T. brucei with TbPPEF, these results demonstrate radiolabelling of a ∼106 kDa product in the presence of each enzyme, indicative of the transfer of myristate to TbPPEF (as previously demonstrated for HASPA and TbARL1) [8,14]. While the amount of radiolabelled product appears greater with LmNMT rather than TbNMT in this experiment, this is likely due to an artefact of loading (as indicated by the higher amount of NMT-myristoyl CoA binary complex loaded on to the gel) rather than substrate specificity.
To demonstrate acylation in parasites in vivo, metabolic labelling experiments were carried out with L. major transgenic lines expressing the first 37 N-terminal residues of PPEF fused with GFP (Lm37WT-GFP, Fig. 1Bii) or a mutated version, in which the Gly in position 2, essential for N-myristoylation, is substituted with Ala (Lm37G2A-GFP, Fig. 1Bii). These parasites express equivalent amounts of the two fusion proteins, as indicated by immunoblotting with anti-GFP and anti-NMT (Fig. 6B). Following radiolabelling with [3H]-myristate or [3H]-palmitate, the fusion proteins were immunoprecipitated using anti-GFP and subjected to SDS-PAGE and autoradiography. This analysis detected a [3H]-myristate-labelled protein of 35 kDa, corresponding to the predicted molecular mass of the Lm37WT-GFP protein, which was absent from parasites expressing Lm37G2A-GFP lacking the residue required for N-myristoylation (Fig. 6C). Similarly, autoradiography of parasites labelled with [3H]-palmitate revealed a single radiolabelled protein of the same molecular mass, corresponding to [3H]-palmitate-labelled Lm37WT-GFP. This protein was again absent in parasites expressing Lm37G2A-GFP. These data correlate with similar experiments with the dual acylated HASPB protein, in which loss of the residue required for N-myristoylation also prevented palmitoylation, due to mislocalisation of the protein within the cell [11].
The experiments in Fig. 6A and C confirm that TbPPEF and LmPPEF can be acylated in vivo, both in a heterologous cell system and in live parasites. To confirm that endogenous LmPPEF is N-myristoylated in vivo, anti-LmPPEF was used in immunoprecipitations from parasite lysates following metabolic labelling with [3H]-myristate. As shown by autoradiography in Fig. 6D, a radiolabelled protein of ∼109 kDa, corresponding to the predicted molecular mass of LmPPEF, was detected in the lysate from wild type L. major parasites, while no proteins were detected following precipitation with protein A beads alone. Radiolabelled parasites expressing LmPPEF-TAP (Fig. 1Bi) were also subjected to immunoprecipitation with anti-LmPPEF, generating two detectable [3H]-myristate-labelled proteins in the lysate: the smaller corresponding to the 109 kDa native LmPPEF and the larger weaker ∼129 kDa band corresponding to the predicted size of the LmPPEF-TAP fusion protein.
3.6
LmPPEF is localised to the endomembrane system of L. major
Immunofluorescence microscopy with the affinity-purified LmPPEF antibody was used to investigate the location of LmPPEF in procyclic L. major (Fig. 7A). Overlay of the fluorescent signal with DAPI (for identification of the nucleus and kinetoplast) indicated a punctate distribution of signal within the cytosol, reminiscent of the endocytic system, together with some concentration in the region of the flagellar pocket. These observations are consistent with the predominantly membrane association of LmPPEF revealed by cellular fractionation (Fig. 5). Counter-staining with anti-LCB2, which recognises an ER-resident sub-unit of serine palmitoyltransferase [38] showed no overlap with PPEF (Fig. 7B) while labelling with the endocytic lipophilic tracer, FM4-64, demonstrated some co-localisation with the flagellar pocket and endosomes (Fig. 7C). PPEF staining was excluded from the nucleus, plasma membrane and flagellum.
We attempted to express and analyse GFP-fusions with either full-length wild type LmPPEF or the N-terminal 473 residues alone (lacking the C-terminal EF-hands). However, these constructs did not express after transfection into L. major, probably due to the large sizes of the recombinant proteins generated. As an alternative approach, given that the N-terminus of LmPPEF is a target for acylation (Fig. 6), the N-terminal GFP transgenic parasite lines (see Fig. 1B) were used to investigate the role of these modifications in intracellular localisation of the fusion proteins. Counter-staining with FM4-64 was also utilized to detect the flagellar pocket in these analyses. Transfected parasites expressing the first 37 residues of LmPPEF fused to GFP (Lm37WT-GFP) targeted the fusion protein predominantly to the flagellum and the flagellar pocket, with weak staining only in the cell body (Fig. 7D). Loss of the N-myristoylation site by mutation of Gly to Ala (Lm37G2A-GFP) resulted in homogeneous distribution of cytosolic fluorescence throughout the parasite (Fig. 7E), in a pattern almost identical to the localisation of GFP alone (data not shown). Separate mutation of the putative palmitoylation site alone, by substituting Cys-3 with Ser (Lm37C3S-GFP), resulted in concentrated signal in a region adjacent to the flagellar pocket most likely to be the Golgi (Fig. 7F). Expression of the double mutant Lm37G2A, C3S-GFP, lacking both acylation sites, caused the fusion protein to remain localised within the cytosol (Fig. 7G). These observations correlate with those previously observed using similar N-terminal mutations of the L. major HASPB protein [11], suggesting that the wild type fusion protein (Lm37WT-GFP) requires N-myristoylation in the cytosol to reach the Golgi region, where palmitoylation further modifies the protein for trafficking to other locations, predominantly the flagellum. Clearly, the localisations of these GFP fusion proteins are distinct from the endocytic location of wild type LmPPEF, indicating that other regions and/or signals within this large protein are important for subcellular localisation. More generally, these data suggest that N-terminal dual acylation might act as a primary signal in targeting proteins to the flagellum in Leishmania, as exemplified by HASPB, SMP-1 and PPEF [11,19 and this study].
4
Discussion
In this paper, as part of a wider study of the downstream targets of NMT, we describe the PPEF-like protein phosphatases that are encoded by single copy genes in three kinetoplastid species. These phosphatases were identified following in silico genome analysis that predicted a subset of kinetoplastid proteins as “high confidence” substrates for NMT. Within this subset were several proteins already confirmed as N-myristoylated in L. major or T. brucei, together with a number of species-specific molecules and a larger group of proteins found in all three species, many of no known function. We predict that all of these “high confidence” putative substrates are acylated in vivo but do not exclude other “medium confidence” proteins as candidates for NMT modification.
LmPPEF and TbPPEF, studied in detail here, are constitutively expressed, membrane-associated acylated proteins. LmPPEF is found in the endocytic system and the flagellar pocket. Conservation of key features and residues within the catalytic domains, which have been modelled against the mammalian PP1 catalytic region, suggest that these proteins are active enzymes (although this has not yet been demonstrated biochemically). Unlike these conserved central regions, however, divergence in the N- and C-terminal domains has led to loss of the IQ calmodulin binding motif and degeneration of the EF-hands, features that characterise the higher eukaryotic PPEFs [39,40]. Thus, there is no evidence that the kinetoplastid proteins are regulated by cytoplasmic Ca2+ levels and/or calmodulin, in contrast to the demonstrated interactions of both human PPEF and Drosophila RdgC with these regulators [39,40]. The analysis in Fig. 5 would support this conclusion.
Although PPEF-like proteins have been characterised in a number of species, the functions of these unusual phosphatases are not well understood. The Drosophila RdgC protein is implicated in dephosphorylation of rhodopsin, a G protein-coupled receptor (GPCR) that initiates vertebrate and invertebrate phototransduction. However, mutant mice with targeted disruptions in each of their two PPEF genes show no retinal degeneration and normal rhodopsin dephosphorylation kinetics, suggesting interspecies functional differences despite high similarities in protein sequence [41].
Completion of the Tri-Tryp genome projects has confirmed earlier predictions that these parasites have ‘stream-lined’ signal transduction mechanisms as compared to their higher eukaryotic multicellular counterparts [42]. The lack of several classes of signalling molecules, including serpentine receptors, heterotrimeric G proteins and most classes of catalytic receptors, contrasts with the presence of a large and diverse family of kinase and phosphatases, suggesting complex cellular interactions [43]. While the conservation and expression of PPEF genes in the Kinetoplastida strongly suggest a functional role for these unusual phosphatases, it is clear that this is unlikely to be similar to that demonstrated for the RdgC protein. We have perturbed TbPPEF expression by inducible RNAi in both bloodstream and procyclic stages of T. brucei, which results in a partial growth defect under normal culture conditions (data not shown). However, it cannot be discounted in these experiments that a more extreme phenotype is masked by sufficient residual expression to support enzyme activity. Functional analysis of conditional gene deletion mutants will be required, together with substrate identification, to delineate roles for the kinetoplastid PPEFs in parasite viability.

bioinformatics
n-myristoylation
protein phosphatases
palmitoylation
ppef, protein phosphatase with ef-hands
nmt, n-myristoyl transferase
bsf, bloodstream form
pcf, procyclic form