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Biosynthesis of the major surface protease GP63 of Leishmania chagasi
Molecular & Biochemical Parasitology 121 (2002) 119 /128 www.parasitology-online.com
Biosynthesis of the major surface protease GP63 of Leishmania chagasi Chaoqun Yao a,b, Kevin G. Leidal a, Andrew Brittingham a,1, Deirdre E. Tarr b,2, John E. Donelson c, Mary E. Wilson a,b,d,* a
Department of Internal Medicine, University of Iowa, SW34-GH, 200 Hawkins Dr., Iowa City, IA 52242, USA b Department of Microbiology, University of Iowa, BSB Newton Road, Iowa City, IA 52242, USA c Department of Biochemistry, University of Iowa, BSB Newton Road, Iowa City, IA 52242, USA d The Veterans’ Affairs Medical Center, Iowa City, IA 52242, USA Received 2 January 2001; received in revised form 29 January 2002; accepted 1 February 2002
Abstract The protozoan Leishmania chagasi expresses a surface metalloprotease, GP63, whose abundance increases 14-fold as parasites grow from logarithmic to stationary phase. L. chagasi GP63 is encoded by three classes of MSP genes that are differentially expressed during parasite growth. Using metabolic labeling and immunoprecipitation, we found L. chagasi GP63 first appeared as a 66-kDa band that was replaced by a 63-kDa protein. This pattern also occurred in transfected L. donovani harboring detectable products of only one MSP gene, suggesting a precursor /product relationship. The half-life of GP63 increased from 29 h in logarithmic phase to /72 h in stationary phase promastigotes. GP63 loss from the cell was complemented by the appearance of a 63-kDa GP63 in extracellular medium in both membrane-associated and -free forms. Calculations suggested that the long and lengthening T1/2 of cell-associated GP63 accounts in part for its progressive accumulation in the cell during promastigote growth. The current findings add yet another level of complexity to post-transcriptionally regulated expression of an abundant surface molecule in a trypanosomatid protozoan. # 2002 Published by Elsevier Science B.V. Keywords: Leishmania chagasi ; Major surface protease; Protein biosynthesis; Half-life; Pulse-chase; Surface biotinylation
1. Introduction The Leishmania sp. are digenetic protozoa that cycle between the gut of the sand fly vector and phagolysosome of a mammalian host macrophage. Several para-
Abbreviations: CRD, cross-reactive determinant; DHIFCS, dialyzed heat-inactivated fetal calf serum; GPI, glycosylphosphatidylinositol; HBSS, Hanks’ Balanced Salts Solution; MSP, major surface protease; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate buffered saline; PI-PLC, phosphatidyl-inositol specific phospholipase C; T1/2, half-life; UTR, untranslated regions; VSG, variant surface glycoprotein. * Corresponding author. Tel.: 1-319-356-3169; fax: 1-319-3847208. E-mail address: [email protected] (M.E. Wilson). 1 Present address: Department of Microbiology, Osteopathic Medical Center, Des Moines University, 3200 Grand Avenue, Des Moines, IA 50312, USA. 2 Present address: Department of Public Health, Johns Hopkins University, Baltimore, MD 21205, USA.
site surface molecules facilitate survival in these diverse host environments. One such molecule is GP63, an abundant protease found on the surface membrane of all Leishmania sp. studied to date [1 /8]. When the parasite is first inoculated into mammalian tissue by a sand fly vector, GP63 facilitates survival of the extracellular promastigote stage in the presence of host complement, likely by converting complement component C3b to iC3b [9,10]. GP63 also promotes attachment of promastigotes to macrophage receptors such as CR3, the receptor for iC3b [9,11 /13], although GP63 protease activity is not absolutely required for CR3 attachment [9,14]. Upon achieving an intracellular location, promastigotes convert to obligate intracellular amastigote forms. There is evidence that GP63 promotes amastigote survival within macrophage phagolysosomes [15,16]. Thus, GP63 participates in the interaction of the parasite with its host at several points during its life cycle.
0166-6851/02/$ - see front matter # 2002 Published by Elsevier Science B.V. PII: S 0 1 6 6 - 6 8 5 1 ( 0 2 ) 0 0 0 3 0 - 0
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The amount of GP63 protein expressed by Leishmania sp. is modulated in different parasite growth and life cycle stages [5,17 /19]. Leishmania chagasi GP63 abundance is lowest in logarithmically growing promastigotes when parasites are least virulent for a murine host, and increases progressively by 14-fold as promastigotes develop in vitro to their highly infectious state in stationary phase of growth. This variation is associated with the expression of mRNAs from three distinct classes of major surface protease (MSP ) genes encoding GP63 [19,20]. Transcripts from MSPL genes are expressed solely during logarithmic growth when GP63 protein is lowest, whereas MSPS transcripts are expressed predominantly during stationary phase when GP63 protein levels are highest [19]. This switch between MSP class transcripts is determined largely by posttranscriptional events such as mRNA half-life (T1/2), rather than by the transcriptional rate [21,22]. However, considering transcripts of all three MSP classes together, the total abundance of MSP RNA does not increase during the transition from log to stationary phase. The purpose of this work, therefore, was to determine mechanisms regulating the increase in abundance of GP63 protein during the transition from logarithmic to stationary growth. We found that a great deal of this increase (five-fold) was due to a lengthening of the T1/2 of cell-associated GP63 protein. Much of the GP63 loss occurred through shedding from the parasite surface, suggesting changes in membrane characteristics could contribute to the rate of GP63 shedding. These findings add yet another level of complexity to the posttranscriptional regulation of GP63 expression in this trypanosomatid protozoan.
2. Materials and methods 2.1. Parasites and antibodies A Brazilian strain of L. chagasi (MHOM/BR/00/ 1669) was maintained in hamsters by serial intracardiac injection of amastigotes. Promastigotes were grown at 26 8C in hemoflagellate-modified minimal essential medium (HOMEM) containing 10% dialyzed heatinactivated fetal calf serum (DHIFCS) [23]. Cultures were seeded at 1/106 promastigotes ml1 and used at different points in growth from logarithmic (2 /4 days) to stationary (6 /10 days) phase as previously defined [24]. Antibodies used include polyclonal rabbit and sheep antisera against purified L. chagasi GP63 [13], polyclonal monospecific rabbit antibody to the crossreactive determinant (CRD) on glycosylphosphatidylinositol (GPI) membrane anchors treated with phosphatidyl-inositol specific phospholipase C (PI-PLC (kindly provided by Dr James Bangs, University of Wisconsin), polyclonal rabbit antisera against a cytosolic protein
P36 in L. amazonensis [25] (kindly provided by Dr K.-P. Chang of the Chicago Medical School and Dr Brad McGwire of Northwestern University), and monoclonal antibody to b-tubulin E7 (Development Studies of Hybridoma Bank, University of Iowa). Serum from patients with visceral leishmaniasis was provided by Dr Selma Jeronimo, Natal, Brazil. Peroxidase-conjugated goat anti -rabbit, anti -mouse, anti -human, or rabbit anti -sheep IgG were used as secondary antibodies (Cappel, Organon Teknika, Corp., Durham, NC). 2.2. Polyacrylamide gels and protein blots Total promastigote protein samples were prepared as described [20] and separated on 7.5% polyacrylamide gels [13]. To detect [35S], gels were soaked in 1 M Na salicylate, vacuum dried, and exposed to X-ray film (Kodak, Rochester, NY). Immunoblots were performed by electrophoretic transfer of proteins to Immobilon-P (Millipore, Bedford, MA) and blocking in 5% milk, 0.1% Tween-20, PBS. Primary antibodies were diluted 1:10,000 unless otherwise indicated. Secondary antibodies were diluted 1:20,000. Blots containing biotinylated proteins were incubated with 1:3000 peroxidase-labeled extravidin (Sigma Chemical Co.). Blots were developed by chemiluminescence (SuperSignal† , Pierce, Rockford, IL or ECLTM, Amersham Pharmacia Biotech, Arlington Heights, IL). For analysis of GPI anchors, amphipathic membrane molecules were separated from hydrophilic proteins by phase separation with Triton X-114 as described [5,26] and treated with either buffer or PI-PLC (Sigma Chemical Co.), for 30 min at 37 8C prior to a second phase separation. Samples were analyzed for GP63 or CRD on immunoblots. Secreted proteins were analyzed by washing promastigotes twice in Hanks’ Balanced Salts Solution (HBSS; GIBCO BRL, Rockville, MD) and resuspending in serum-, BSA-free medium at the starting concentration. After 24 /48 h cells were removed by centrifugation. Supernatants were filtered (0.22 mm) and concentrated with centriprep-10 and microcom-10 (Amicon, Beverly, MA). 2.3. Northern blots Total promastigote RNA was extracted by the method of Chomczynski [27]. Four micrograms of RNA were separated in lanes of 1.2% formaldehydecontaining agarose gels and transferred by capillary action to nylon filters (Roche, Germany). The blots were probed with [32P]-DNA corresponding to the conserved 5? untranslated regions (UTR) and coding region of MSP genes, or to the coding region of a-tubulin amplified by the polymerase chain reaction from L. chagasi genomic DNA. The blots were washed at high
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stringency (0.1 /SSC, 0.1% SDS, 65 8C) and exposed to X-ray film. 2.4. Metabolic labeling Nascent proteins were metabolically labeled with 50 mCi ml 1 of ProMix containing L-[35S]-methionine and 35 L-[ S]-cysteine (Amersham Pharmacia Biotech) in methionine-free RPMI (containing cysteine) with DHIFCS. The rate of GP63 synthesis was measured by detergent lysis of 4 /107 promastigotes between 15 min and 49 h after the addition of label, followed by immunoprecipitation with polyclonal serum to GP63. To measure GP63 T1/2, promastigotes were pulsed at their original cell density with [35S]-methionine for 1/2 h. Cultures were flooded with an excess of unlabeled methionine but maintained at the original (log or stationary) cell density. At times ranging from 0 to 72 h, a volume equivalent to 4/107 starting promastigotes was subjected to immunoprecipitation. To determine the rates of [35S]-methionine incorporation into total proteins, cells were pulsed with [35S]-methionine for 1 h and then lysed with 1.0% SDS. Proteins were precipitated with trichloroacetic acid and dried onto glass microfibre filters (Whatman, England) as described [28]. Label incorporation was measured with a scintillation counter. To measure the [35S]-methionine uptake, promastigotes were treated with 10 mg cycloheximide ml1 for 5 or 30 min in methionine-free RPMI. The cells were then pulsed with [35S]-methionine for 30 min without removing the inhibitor, and collected by microcentrifugation (12,000/g, 3 min) through an oil cushion (n -butyl phthalate /dinonyl phthalate /4:1) to separate them from free [35S]-methionine in the medium. After samples were taken from the medium, the whole tubes were snap frozen in ethanol/dry ice and pellet was cut off. Background counts were derived from pellets of control tubes with no cell. [35S]-methionine uptake rate was calculated as accounts of pellet (pellet/medium) 1, quantified on a scintillation counter. 2.5. Immunoprecipitation Aliquots of 4/107 promastigotes or spent culture medium from equivalent numbers of parasites were lysed by incubation for 4 /16 h in 1.0% Triton X-100, 20 mM EDTA, 20 mg antipain ml 1, and 20 mg leupeptin ml1 in TBS (10 mM Tris /HCl pH 7.2, 158 mM NaCl) at 4 8C. Results were identical using an alternate (RIPA) lysis buffer containing TBS and 1% Na deoxycholate, 1 mM phenylmethylsulfonyl fluoride, 0.1% SDS, 1 mM Na3VO4, 2 mM benzamidine, and 2 mM EDTA. Detergent-insoluble material was removed by microcentrifugation at 12,000 /g , 4 8C for 5 min. Samples were pre-cleared by incubation in rabbit or sheep pre-immune serum followed by PANSORBIN†
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(protein A) or OMNISORB† (protein G) Cells (Calbiochem Novabiochem, La Jolla, CA), respectively. Samples were heat denatured in 2% SDS, and incubated in 0.5% immune serum in 50 mM Tris /HCl pH 7.4, 190 mM NaCl, 6 mM EDTA, 2.5% Triton X-100 for 16 h at 4 8C. Antibody/protein complexes were captured with protein A or G. Cell-bound complexes were recovered by microcentrifugation, washed twice in 0.5% Triton X100 in TBS, washed twice in TBS, and denatured in reducing SDS-sample buffer [13]. 2.6. Surface biotinylation Five hundred micrograms NHS-Sulfo-Biotin ml 1 (Pierce) were incubated with 1.1 /108 promastigotes ml 1 in HBSS for 30 min at 26 8C. The reaction was stopped with 10 mM NH4Cl and cells were washed by centrifugation. Parallel cultures were metabolically labeled with [35S]-methionine as above. At time points between 0.5 and 93 h after removal from label, biotinylated or metabolically labeled cells were subjected to immunoprecipitation with anti-GP63 serum.
3. Results 3.1. GP63 proteins and RNAs are differentially expressed during promastigote growth from logarithmic to stationary phase in vitro L. chagasi promastigotes increase in infectivity as they develop from logarithmic to stationary phase in culture medium [24]. Coincident with this growth we observed a 14-fold increase in the amount of GP63 in day 7 (stationary phase) compared to day 3 (logarithmic phase) promastigotes documented on immunoblots (Fig. 1). The lower, less intense band in Fig. 1 was previously documented by our group to be a 59 kDa GP63 isoform that is probably encoded by MSPS2 [29]. This increase in GP63 protein was associated with a switch from a 2.7 to 3.0-kb MSP RNA species (Fig. 2A), which we previously found corresponds with a switch from expression of MSPL to MSPS transcripts, respectively [19]. Separation of amphipathic from hydrophilic molecules with Triton X-114 showed the major GP63 proteins separated in the detergent (amphipathic) phase (Fig. 2B). Proteins in both bands were susceptible to PI-PLC cleavage since the bands shifted from the amphipathic to the hydrophilic phase after enzyme treatment. The PI-PLC treated but not the untreated proteins were recognized by antibody to the CRD that is uniquely exposed after PI-PLC treatment (Fig. 2B, [30]). Together these results indicate that the predominant two isoforms are anchored to the L. chagasi promastigote with a GPI anchor similar to other trypanosomatid surface proteins [31,32].
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Fig. 1. Steady-state levels of GP63 during in vitro culture of promastigotes. (A) Promastigote proteins prepared on sequential days of culture were resolved by 7.5% SDS-PAGE gel electrophoresis and probed with E7 monoclonal antibody to b-tubulin (1:1000 dilution) to normalize for loading. The same filter was re-probed with rabbit polyclonal antiserum to purified GP63 to quantify its steady-state amount on different days of growth. (B) Protein quantitation of (A) by auto-background densitometry. The growth curve (cell density) represents the mean (SD) promastigote concentration in eight parallel cultures. The steady-state level of GP63 on each day was calculated by normalizing the GP63 to b-tubulin densitometry units, and setting the lowest (day 3) ratio at 1.0. Circles represent cell density; triangles indicate steady-state GP63 levels.
3.2. GP63 synthesis and loss
Fig. 2. (A) Total RNAs from L. chagasi promastigotes on different days of growth from logarithmic (days 2 /4) to stationary phase (days 6 /7) were probed with [32P]-labeled DNAs corresponding to either the MSP 5?-UTR and coding region to detect all MSP class RNAs (left panel), or the a-tubulin coding region (right panel). (B) GP63 was extracted from L. chagasi stationary promastigotes with Triton X-114 [26] and exposed to either PI-PLC (P) or buffer (C). Hydrophilic and amphipathic molecules were separated by Triton X-114 phase separation into aqueous (a) or detergent (d) phases, respectively, followed by immunoblot analysis. Blots were incubated with either polyclonal antiserum to GP63 (left panel) or antiserum to the CRD of cleaved GPI anchors (right panel [30]).
Metabolic labeling of GP63 with [35S]-methionine followed by immunoprecipitation with anti-GP63 serum allowed us to follow GP63 biosynthesis. In logarithmic phase L. chagasi promastigotes (day 3), a predominant immunoprecipitated GP63 appeared first as a 66-kDa protein followed by the additional appearance of a 63kDa GP63 protein after 30/60 min (left panel of Fig. 3A and B). The 63-kDa protein accumulated progressively during promastigote growth, resulting in high levels after about 2 days (Fig. 3B). Pulse-chase studies showed the loss of immunoprecipitated 66-kDa GP63 coincided with appearance of the lower Mr GP63 (Fig. 3A, right panel). This lower Mr GP63 had a T1/2 of 429/7 h in logarithmic promastigotes (day 3) quantified by densitometry in eight replicate experiments. These observations do not preclude the existence of additional GP63 isoforms that are not easily detected by metabolic labeling and immunoprecipitation. Since L. chagasi possesses more than 18 genes encoding GP63, we could not be certain whether the 66- and 63-kDa metabolically labeled proteins were the products of distinct genes with different rates of synthesis and degradation, or whether the above pattern represents processing of a precursor to a mature GP63 protein product. As such, we examined attenuated L. donovani parasites transfected with a single L. chagasi
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Fig. 3. The rates of GP63 synthesis and degradation. (A) (left panel) GP63 synthesis was quantified in logarithmic L. chagasi promastigotes incubated in [35S]-methionine for varying times before immunoprecipitating GP63. Right panel: A representative pulse-chase study was performed by incubating logarithmic phase promastigotes (1.1 107 ml 1) in [35S]-methionine for 1 h, after which an excess of unlabeled methionine was added (time 0). At the indicated time points, GP63 was immunoprecipitated from aliquots of cultures corresponding to 4 107 starting promastigotes. (B) GP63 synthesis was analyzed by metabolic labeling of logarithmic phase (1.1 107 ml 1) promastigotes in [35S]-methionine for 20 min /49 h and immunoprecipitating with anti-GP63 serum. Each lane contains GP63 proteins derived from 4 107 promastigotes.
MSPS or MSPL gene. Although L. donovani expresses GP63, the amount is low in attenuated promastigotes and L. donovani GP63 is poorly detected by polyclonal antiserum to L. chagasi GP63. Thus, only the transfected MSP gene product is detected in these cells [29]. We found that a higher Mr ‘precursor’ produced from a single MSP gene (MSPS1 , MSPS2 , or MSPL ) was chased into a lower Mr ‘mature’ form in transfectants (Fig. 4A; MSPL data not shown). Thus, a precursor / product relationship was observed in transfectants harboring only one MSP gene whose protein product could be detected by immunoprecipitation. To determine whether both the 66- and the 63-kDa GP63s are glycosylated, we utilized N -glycanase to remove N-linked oligosaccharides from L. chagasi proteins. Rather than collapsing into a single band, pulse-chase experiments showed both the 66- and the 63kDa proteins are susceptible to digestion with N glycanase (Fig. 4B, ‘/’ lanes). This suggests both forms of GP63 had undergone N -glycosylation, and that the difference between the bands is not merely due to N glycosylation. Incubation in 10 mg tunicamycin ml 1, which inhibits N -glycosylation, yielded parallel results (not shown). 3.3. Intracellular processing of GP63 We hypothesized that GP63 could mature from a 66to 63-kDa isoform through proteolytic cleavage of the
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Fig. 4. (A) The precursor /product relationship during processing of individual MSP genes. Attenuated L. donovani logarithmic promastigotes (2 107 cells ml 1) (L. don) or L. donovani transfected with the pX plasmid expressing individual MSPS genes (MSPS1 , MSPS2 ) were metabolically labeled and chased with an excess of unlabeled methionine. GP63 was immunoprecipitated from aliquots corresponding to 4 107 starting promastigotes and the pattern of protein maturation was detected on autoradiograms. Antiserum to L. chagasi GP63 did not recognize the homologous molecule in attenuated L. donovani alone (L. don) or transfected with pX (not shown). (B) L. chagasi logarithmic promastigotes underwent a 2 h pulse with [35S]methionine followed by a 0 /18 h chase with unlabeled methionine. Proteins were incubated in the absence () or presence () of N glycanase and GP63 was immunoprecipitated from aliquots of culture corresponding to 4 107 starting promastigotes. Autoradiograms of the reaction products are shown.
pro-enzyme, similar to the 3 kDa change in L. major GP63 after pro-peptide removal [33]. Extracellular protease inhibitors such as antipain, leupeptin, phenylmethylsulfonyl fluoride, or EDTA did not inhibit production of the 63 kDa GP63 (not shown), leading us to question whether both GP63s or only the 63-kDa form was expressed at the parasite surface. Parallel logarithmic phase promastigote cultures were treated by pulse-chase with [35S]-methionine or by surface biotinylation prior to lysis (Fig. 5A). Linking biotin to promastigote surface proteins labeled only the mature 63-kDa form of GP63, in contrast to parallel cultures metabolically labeled with [35S]-methionine in which both 66- and 63-kDa GP63 isoforms were labeled. If indeed the 66- and 63-kDa GP63s represent precursor and product, this result would suggest that generation of the ‘mature’ 63 kDa GP63 occurs intracellularly rather than on the promastigote surface. Alternatively, it is conceivable that the 66-kDa GP63 form is of very low abundance and poorly accessible for biotinylation. The 63-kDa GP63 protein slowly appeared in the extracellular medium (Fig. 5B). There was no associated change in Mr, suggesting proteolytic cleavage had not
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membrane-bound vesicles or large aggregated micelles containing GP63. To eliminate the possibility that GP63 was released due to cell lysis or death, an antibody against a well-characterized cytosolic protein P36 in L. amazonensis [25] was used to probe a western blot of L. chagasi cell pellets or concentrated extracellular medium. In contrast to GP63, which appeared in both cells and extracellular medium, P36 was found only in intact cells (Fig. 6A). A positive control cytoskeletal protein, b-tubulin, was also found exclusively in cell pellet. We conclude that the observed GP63 in the medium was not due to cell lysis. Taken together, these data suggest GP63 is lost from promastigotes by release in a soluble (possibly micellar) form, and also in membrane-bound vesicles. 3.4. GP63 has a longer T1/2 in stationary phase promastigotes than in logarithmically growing promastigotes Fig. 5. (A) GP63 processing occurs intracellularly. Logarithmic promastigote cultures were either metabolically labeled with [35S]methionine for 2 h followed by various times of chase in unlabeled methionine (left panel), or surface labeled with biotin at time 0 (right panel). 0.5 /48 h later, culture volumes equivalent to 4 107 starting promastigotes were subjected to GP63 immunoprecipitation. Panels contain an autoradiogram (left) or nitrocellulose developed with extravidin-peroxidase followed by enhanced chemiluminescence (right). (B) GP63 is slowly released into extracellular medium. L. chagasi logarithmic promastigotes were metabolically labeled and chased with unlabeled methionine. At different time point, parasites were recovered by centrifugation, growth medium was filtered to remove residual cells, and GP63 was immunoprecipitated. Lanes of autoradiograms contain GP63 from a volume of cells or growth medium equivalent to 4 107 starting promastigotes. (C) GP63 in the extracellular medium exists in both membrane-associated and -free forms. Logarithmic promastigote cultures were separated into supernatant (S1) and pellet (PM) by centrifugation (12,000 g ). S1 was filtered through 0.2 mm filter and further separated by centrifugation at 100,000 g into a membrane (P2) and supernatant (S2) fractions. P2 was solubilized in Triton X-100 and detergent-soluble (S3) versus detergent-insoluble (P3) fractions were separated by centrifugation. Proteins were analyzed by immunoblot probed with sheep antiserum to purified GP63.
occurred. In addition, GP63 release was not associated with the appearance of a CRD antibody-positive band in the supernatant that would have suggested PI-PLCmediated hydrolysis of the GPI anchor (data not shown). To further investigate whether 63-kDa GP63 in the extracellular medium is still membrane associated or free in the medium, filtered growth medium was centrifuged at 100,000 /g to pellet membranes. Equal amounts of 63-kDa GP63 were found in both supernatants and pellets of such centrifugation (Fig. 5C), suggesting both membrane-free and -associated forms of GP63 are in the medium. Furthermore, the pelletassociated GP63 was soluble in the detergent Triton X-100 (Fig. 5C), consistent with the presence of either
Our prior studies indicated there is equal transcription of the MSP genes in logarithmic versus stationary phase promastigotes [22]. Our current study indicates that GP63 is synthesized in logarithmic phase (day 3) promastigotes, and this GP63 has a T1/2 of about 429/ 7 h. Collectively these results do not explain 14-fold difference in GP63 abundance in logarithmic versus
Fig. 6. (A) GP63 release in extracellular medium. Logarithmic promastigotes (day 3) were suspended in serum- and BSA-free medium. After 24 and 48 h, filtered supernatants (S) and cell pellets (P) were blotted to nitrocellulose filters and probed with the rabbit sera against the cytoplasmic protein P36 [25], rabbit antisera against GP63, or control mAb E7 to b-tubulin. Each lane was loaded the equivalent amount of pellet or supernatant corresponding to 1 107 starting promastigotes. (B) The half-life of surface biotinylated GP63 is longer in stationary phase promastigotes than in logarithmic phase parasites. Promastigotes in day 4 (Log) or day 8 (Sta) were either metabolically labeled with [35S]-methionine (data not shown) or surface labeled with biotin for 30 min. From the end of labeling (time 0) to 93 h, samples were taken at various time points as indicated. Culture volumes equivalent 8 107 starting promastioges were subjected to GP63 immunoprecipitation. GP63 was detected with extravadin-peroxidase followed by enhanced chemiluminescence.
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stationary parasites. We therefore attempted to measure the rate of GP63 protein synthesis in logarithmic and stationary phase promastigotes. Unexpectedly, it initially appeared as though 12 times more GP63 were synthesized by day 2 (logarithmic phase) promastigotes than by day 8 (stationary phase) promastigotes (Table 1). This result is not compatible with the observed increase in steady-state GP63 levels in stationary promastigotes. To address this discrepancy, we measured the incorporation of [35S]-methionine into total proteins by logarithmic versus stationary phase promastigotes. Four repeat studies, each with triplicate conditions, showed that logarithmic parasites (day 3) incorporated three times more [35S]-methionine than stationary phase parasites (day 6 /9) (Table 1). We also determined the rate of [35S]-methionine uptake by promastigotes. In the latter experiment, cycloheximide was used to prevent [35S]-methionine incorporation into proteins. We previously showed that 10 mg cycloheximide ml1 inhibits L. chagasi protein synthesis by more than 95% [21]. Logarithmic phase promastigotes (day 3) took up 4.6-fold more [35S]-methionine than stationary phase promastigotes (day 7) in the presence of 10 mg cycloheximide ml 1 (Table 1). Furthermore, immunoprecipitation with pooled sera from the patients with visceral leishmaniasis showed multiple proteins were less abundant in stationary than in logarithmic promastigotes. Densitometric measurement of the abundant 80-, 49- and 23-kDa proteins showed ratios of 2.6, 1.9 and 2.3, respectively, comparing abundance in logarithmic versus stationary phase promastigotes. These data could reflect a decrease in amino acid transport into the cell. Whether there is also an overall decrease in protein synthesis cannot be discerned. The differences in [35S]met uptake prevent us from directly comparing the rates of GP63 translation in logarithmic versus stationary phase promastigotes. We can merely state that L.
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chagasi promastigotes synthesize some GP63 during logarithmic phase of growth, even though they have a relatively low amount of GP63 protein during this growth phase. Although we could not compare rates of translation, we were able to measure the T1/2 of GP63 protein in logarithmic versus stationary phase promastigotes. As shown in the representative experiment in Table 1, the T1/2 of GP63 in day 2 (logarithmic) promastigotes was 29.2 h and progressively increased to more than 72 h in day 5 promastigotes, at which time parasites were converting to stationary phase. The change in GP63 T1/2 does not reflect a ‘dilutional’ effect due to rapid growth of logarithmic promastigotes, since we controlled for this possibility in pulse-chase experiments by taking sample sizes corresponding to 4 /107 starting promastigotes. Each time point reflects the loss of GP63 from the same starting amount of labeled GP63. This result suggests that GP63 protein becomes more stable as promastigotes progress from logarithmic to stationary phase. Three replicate experiments displayed a similar pattern. Analyses of the coding regions do not predict structural differences between the GP63s detected in log versus stationary promastigotes. Therefore, we considered the possibility that modification of the promastigote membrane accounts for differences in GP63 loss from the cells. Logarithmic or stationary phase promastigotes were surface biotinylated. Starting at the end of the biotinylation period, we measured the loss of GP63 by immunoprecipitation. Two replicate experiments showed that log phase promastigotes lost their surface GP63 more rapidly than stationary phase promastigotes. The T1/2 of the surface GP63 was 34 and 77 h in log and stationary phase parasites, respectively (Fig. 6B). We calculated the predicted steady-state amount of GP63 from the T1/2 data, assuming that
Table 1 Changes in GP63 T1/2, measurable GP63 synthesis, [35S]-methionine uptake and incorporation during promastigote growth Day of growth
GP63 T1/2 (h)a
Measurable GP63 synthesisb
Relative [35S]-methionine incorporationc
[35S]-methionine uptake rated
2 3 4 5 6 /9
29.2 47.7 63.4 72.0 ND
12.1 8.7 5.9 1.7 1.3
ND 3.091.7 ND ND 1.0
ND 16.990.9 ND ND 3.790.3
ND, not done. a GP63 T1/2 was measured by pulse-chase and immunoprecipitation in the original cell density of sequential days of culture. The amount of protein was quantified by densitometry. Using linear regression, the time of 50% protein loss was calculated. b Parallel promastigote cultures were labeled for 1 h, and the protein was quantified by autoradiography and densitometry as described for GP63 T1/2 (a). The measurable GP63 synthesis was calculated as a ratio to the day 8 (lowest) densitometry value. The amount on day 6 /9 was the average of those on individual days (1.5, 1.2, 1.0, and 1.5, respectively). c 35 [ S]-methionine incorporation by promastigotes on different days of growth was normalized to 1.0 on day 6 /9 (triplicates, n 4). d Logarithmic (day 3) or stationary (day 7) promastigotes were treated with cycloheximide for 30 min, and then pulsed with [35S]-methionine for additional 30 min without removal of cycloheximide. [35S]-methionine uptake rate was calculated from triplicates. Treatment for 5 min with cycloheximide and a repeat study yielded similar results (not shown).
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Table 2 Predicted steady-state GP63 levels from protein half-life using linear regression Day
2
3
2 3 4 5 6 7 8 9
1.00 0.58 0.21 0.00 0.00 0.00 0.00 0.00
1.00 0.71 0.50 0.28 0.07 0.00 0.00
4
1.00 0.84 0.63 0.43 0.22 0.01
5
1.00 1.00 1.00 1.00 1.00
6
1.00 1.00 1.00 1.00
7
1.00 1.00 1.00
8
1.00 1.00
9
Total predicted
Amount measureda
1.00
1.00 1.58 1.92 2.34 2.91 3.49 4.22 5.01
1.9 1.0 5.5 7.6 12.5 14.7 11.5 12.1
Relative levels of GP63 protein were predicted according to the following linear regressions: day 2, f (x ) 0.9556670.015625x , r 2 0.9598; day 3, f (x ) 0.9293330.009x , r 2 0.7569; day 4, f (x ) 1.0499 /0.008676x , r 2 0.8198. Assumptions were: both logarithmic and stationary phase promastigotes have a steady translation rate of 1.00, and there was little degradation of proteins synthesized on day 5 and afterwards. a Western blot determination of the steady-state ratios of GP63 abundance on each day of growth compared to the lowest measured value (day 3). Values represent ratios of GP63 to b-tubulin in the same lanes of blots, reprobed with two different antibodies (see Fig. 1). Measurements were made by densitometry.
both logarithmic and stationary phase promastigotes have a steady translation rate set at 1.0 arbitrary units per day. As shown in Table 2, the difference in T1/2 alone could explain a five-fold increase in cell-associated GP63 abundance in stationary phase versus logarithmic phase promastigotes. Thus, even though we cannot reliably compare translation rates, the changing T1/2 of the membrane bound protein can account for a majority of the protein accumulation in stationary phase organisms.
4. Discussion GP63 of L. chagasi is encoded by a family of more than 18 tandem MSP genes. These genes fall into three classes (MSPL , MSPS , MSPC ) that have distinct sequences in their 3?UTRs and downstream intergenic regions. All MSP 5?UTRs and coding regions have a high degree of sequence identity, although there are regional differences in coding regions, particularly in the case of the MSPC gene. Transcripts of the three MSP class genes are differentially expressed, with MSPL RNAs predominating in logarithmic growth and MSPS RNAs predominating in stationary phase of promastigote growth. MSPC RNAs are constitutively expressed at low levels throughout growth [19]. Our prior studies of mRNA expression showed the MSP mRNAs are regulated post-transcriptionally. Expression of at least one of the three MSP classes (MSPL ) is determined by the stability of its mRNA [21,22]. During the current study we examined the biosynthesis and T1/2 of the MSP gene products. Metabolic labeling studies of wild-type L. chagasi showed that loss of a 66-kDa GP63 coincided with appearance of the predominant 63-kDa GP63 that was detectable by immunoprecipitation. A similar apparent precursor /product relationship was observed in trans-
fectants harboring only one detectable MSP gene product. GP63 of all Leishmania sp. is produced as a proenzyme that requires removal of the pro-peptide for generation of an active enzyme [4,17]. Our findings are consistent with production of a 66 kDa precursor containing the pro-peptide, and removal of the proregion to form a 63 kDa product. Pro-peptide removal may not require mature cellular GP63, since the transfected MSPS1 , MSPS2 or MSPL gene products were cleaved in a GP63-low host cell (attenuated L. donovani ). These findings are consistent with the published findings of Macdonald et al., in which pro-peptide removal from recombinant L. major GP63 synthesized in COS-7 cells underwent activation through an autocatalytic cis mechanism that apparently does not involve exogenous activated GP63 [33]. The authors hypothesized that this is consistent with the cysteine switch mechanism, in which latency is maintained through interactions between the active site and a Cys sulfhydryl group in the GP63 pro-region [33]. Biosynthesis of GP63 can be contrasted with that of the variant surface glycoprotein (VSG) of the related protozoan Trypanosoma brucei . VSG is synthesized in the endoplasmic reticulum where it undergoes addition of N-linked oligosaccharides, replacement of the Cterminal peptide with a GPI anchor [34 /36], and transport to a compartment at the cell surface containing an endogenous membrane-bound phospholipase C [37]. Despite being delivered to this compartment, VSG is quantitatively released from the native cell by the action of a Zn-metalloprotease, perhaps by a T. brucei version of GP63, rather than by PI-PLC cleavage. The net result is a released molecule with increased migration on SDS-PAGE and a kinetics of shedding that is much faster than that of Leishmania GP63 [38]. Similar to VSG, GP63 undergoes N -glycosylation and is linked to a GPI anchor prior to delivery to the surface membrane [39]. Unlike VSG, it appeared slowly in
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extracellular medium. The kinetics of GP63 loss inversely paralleled the kinetics of its appearance in extracellular medium, and the extracellular form of GP63 did not bind antibody to the CRD exposed after PI-PLC treatment. Thus, it seems that the shedding of GP63 occurs without PI-PLC mediated cleavage of the GPI anchor. Our prior studies established the fact that low levels of GP63 are present in logarithmic promastigotes that express predominantly MSPL class RNAs, whereas GP63 abundance increases in stationary phase parasites expressing MSPS RNAs [29]. To our surprise, there was abundant synthesis of GP63 by logarithmic phase promastigotes. It was not possible to directly compare the rates of GP63 translation in different growth phases due to discrepancies in uptake and incorporation of the metabolic label in log versus stationary phase parasites. Nonetheless, there was an increase in GP63 protein T1/2 as parasites developed in vitro from logarithmic to stationary phase. The change in T1/2 was observed either when GP63 was metabolically labeled, or when surface GP63 was labeled by biotinylation. These data are consistent with a change in GP63 shedding due to remodeling of the promastigote surface membrane during growth. We propose that the observed increase in the steadystate abundance of GP63 protein during growth of L. chagasi is due, at least in part, to the lengthening of the half-life of the membrane-bound protein. GP63 may be more rapidly ‘shed’ or ‘lost’ from the surface of logarithmic phase than of stationary phase cells, thus accounting for the lower half-life of cellular GP63 in logarithmic parasites. It is possible that additional contributions are made by differences in the rates of translation of MSPL versus MSPS genes, and/or the increased abundance in MSPS RNA in stationary phase. During prior work we documented that the regulation of MSPL RNA expression is related to growth-related shortening of the T1/2 of this transcript [21,40]. The current study documents the fact that the amount of GP63 protein expressed is also influenced by the kinetics of its loss from the cell.
5. Addendum Since submission of this manuscript, McGuire et al. have documented extracellular release of GP63 from Leishmania amazonensis and several other Leishmania sp. (J. Biol. Chem. 2002, in press).
Acknowledgements The authors are grateful to Dr James Bangs, University of Wisconsin, Dr Brad McGuire of North-
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western University and Dr K.-P. Chang, Chicago Medical School for their helpful suggestions and provision of antibodies used in this work. This work was supported by NIH grant AI32135 (MEW and JED), DK/AI52550 (MEW), AI40591 (JED), and a Veterans’ Affairs Merit Review grant (MEW). CY and AB were supported by NIH Training Grants T32 AI07343 and T32 AI07511, respectively.
References [1] Webb JR, Button LL, McMaster WR. Heterogeneity of the genes encoding the major surface glycoprotein of Leishmania donovani . Mol Biochem Parasitol 1991;48:173 /84. [2] Button LL, Russell DG, Klein HL, Medina-Acosta E, Karess RE, McMaster WR. Genes encoding the major surface glycoprotein in Leishmania are tandemly linked at a single chromosomal locus and are constitutively transcribed. Mol Biochem Parasitol 1989;32:271 /84. [3] Medina-Acosta E, Karess RE, Russell DG. Structurally distinct genes from the surface protease of Leishmania mexicana are developmentally regulated. Mol Biochem Parasitol 1993;57:31 / 46. [4] Button LL, McMaster WR. Molecular cloning of the major surface antigen of Leishmania . J Exp Med 1988;167:724 /9. [5] Wilson ME, Hardin KK, Donelson JE. Expression of the major surface glycoprotein of Leishmania donovani chagasi in virulent and attenuated promastigotes. J Immunol 1989;143:678 /84. [6] Steinkraus HB, Greer JM, Stephenson DC, Langer PJ. Sequence heterogeneity and polymorphic gene arrangements of the Leishmania guyanensis gp63 genes. Mol Biochem Parasitol 1993;62:173 /86. [7] Bordier C. The promastigote surface protease of Leishmania . Parasitol Today 1987;3:151 /3. [8] Fong D, Chang K-P. Surface antigenic change during differentiation of a parasitic protozoan, Leishmania mexicana : identification by monoclonal antibodies. Proc Natl Acad Sci USA 1982;79:7366 /70. [9] Brittingham A, Morrison CJ, McMaster WR, McGwire BS, Chang KP, Mosser DM. Role of the Leishmania surface protease gp63 in complement fixation, cell adhesion, and resistance to complement-mediated lysis. J Immunol 1995;155:3102 /11. [10] Chaudhuri G, Chang K-P. Acid protease activity of a major surface membrane glycoprotein (gp63) from Leishmania mexicana promastigotes. Mol Biochem Parasitol 1988;27:43 /52. [11] Russell DG, Wilhelm H. The involvement of the major surface glycoprotein (gp63) of Leishmania promastigotes in attachment to macrophages. J Immunol 1986;136:2613 /20. [12] Chang CS, Chang K-P. Monoclonal antibody affinity purification of a leishmania membrane glycoprotein and its inhibition of leishmania-macrophage binding. Proc Natl Acad Sci USA 1986;83:100 /4. [13] Wilson ME, Hardin KK. The major concanavalin A-binding surface glycoprotein of Leishmania donovani chagasi promastigotes is involved in attachment to human macrophages. J Immunol 1988;141:265 /72. [14] Wilson ME, Hardin KK. The major Leishmania donovani chagasi surface glycoprotein in tunicamycin-resistant promastigotes. J Immunol 1990;144:4825 /34. [15] Chaudhuri G, Chaudhuri M, Pan A, Chang K-P. Surface acid proteinase (gp63) of Leishmania mexicana : a metalloenzyme capable of protecting liposome-encapsulated proteins from pha-
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[27] Chomczynski P, Sacchi N. Single step method of RNA isolation by acid guanidinium thiocyanate /phenol /chloroform extraction. Anal Biochem 1987;162:156 /9. [28] Coligan JE, Kruisbeek AM, Margulies DH, Shevach EM, Strober W. Current protocols in immunology. New York: John Wiley and Sons, 1991 /2000. [29] Roberts SC, Wilson ME, Donelson JE. Developmentally regulated expression of a novel 59-kDa product of the major surface protease (Msp or gp63) gene family of Leishmania chagasi . J Biol Chem 1995;270:8884 /92. [30] Krakow JL, Hereld D, Bangs JD, Hart GW, Englund PT. Identification of a glycolipid precursor of the Trypanosoma brucei variant surface glycoprotein. J Biol Chem 1986;261:12 147 /53. [31] Etges R, Bouvier J, Bordier C. The major surface protein of Leishmania promastigotes is anchored in the membrane by a myristic acid-labeled phospholipid. EMBO J 1986;5:597 /601. [32] Ferguson MAJ, Brimacombe JS, Cottaz S, Field RA, Gu¨ther LS, Homans SW, et al. Glycosyl /phosphatidylinositol molecules of the parasite and the host. Parasitology 1994;108(Suppl.):S45 /54. [33] Macdonald MH, Morrison CJ, McMaster WR. Analysis of the active site and activation mechanism of the Leishmania surface metalloproteinase GP63. Biochim Biophys Acta 1995;1253:197 / 9. [34] Bangs JD, Brouch EM, Ransom DM, Roggy JL. A soluble secretory reporter system on Trypanosoma brucei . J Biol Chem 1996;271:18 387 /93. [35] Bangs JD, Doering TL, Englund PT, Hart GW. Biosynthesis of a variant surface glycoprotein of Trypanosoma brucei . J Biol Chem 1988;263:17 697 /705. [36] Bangs JD, Hereld D, Krakow JL, Hart GW, Englund PT. Rapid processing of the carboxyl terminus of a trypanosome variant surface glycoprotein. Proc Natl Acad Sci USA 1985;82:3207 /11. [37] Bangs JD, Andrews NW, Hart GW, Englund PT. Posttranslational modification and intracellular transport of a trypanosome variant surface glycoprotein. J Cell Biol 1986;103:255 /63. [38] Bangs JD, Ransom DM, McDowell MA, Brouch EM. Expression of bloodstream variant surface glycoproteins in procyclic stage Trypanosoma brucei : role of GPI anchors in secretion. EMBO J 1997;16:4285 /94. [39] McGwire BS, Chang K-P. Posttranslational regulation of a Leishmania HEXXH metalloprotease (gp63). J Biol Chem 1996;271:7903 /9. [40] Brittingham A, Miller MA, Donelson JE, Wilson ME. Regulation of GP63 mRNA stability in promastigotes of virulent and attenuated Leishmania chagasi . Mol Biochem Parasitol 2001;112:51 /9.
Biosynthesis of the major surface protease GP63 of Leishmania chagasi Chaoqun Yao a,b, Kevin G. Leidal a, Andrew Brittingham a,1, Deirdre E. Tarr b,2, John E. Donelson c, Mary E. Wilson a,b,d,* a
Department of Internal Medicine, University of Iowa, SW34-GH, 200 Hawkins Dr., Iowa City, IA 52242, USA b Department of Microbiology, University of Iowa, BSB Newton Road, Iowa City, IA 52242, USA c Department of Biochemistry, University of Iowa, BSB Newton Road, Iowa City, IA 52242, USA d The Veterans’ Affairs Medical Center, Iowa City, IA 52242, USA Received 2 January 2001; received in revised form 29 January 2002; accepted 1 February 2002
Abstract The protozoan Leishmania chagasi expresses a surface metalloprotease, GP63, whose abundance increases 14-fold as parasites grow from logarithmic to stationary phase. L. chagasi GP63 is encoded by three classes of MSP genes that are differentially expressed during parasite growth. Using metabolic labeling and immunoprecipitation, we found L. chagasi GP63 first appeared as a 66-kDa band that was replaced by a 63-kDa protein. This pattern also occurred in transfected L. donovani harboring detectable products of only one MSP gene, suggesting a precursor /product relationship. The half-life of GP63 increased from 29 h in logarithmic phase to /72 h in stationary phase promastigotes. GP63 loss from the cell was complemented by the appearance of a 63-kDa GP63 in extracellular medium in both membrane-associated and -free forms. Calculations suggested that the long and lengthening T1/2 of cell-associated GP63 accounts in part for its progressive accumulation in the cell during promastigote growth. The current findings add yet another level of complexity to post-transcriptionally regulated expression of an abundant surface molecule in a trypanosomatid protozoan. # 2002 Published by Elsevier Science B.V. Keywords: Leishmania chagasi ; Major surface protease; Protein biosynthesis; Half-life; Pulse-chase; Surface biotinylation
1. Introduction The Leishmania sp. are digenetic protozoa that cycle between the gut of the sand fly vector and phagolysosome of a mammalian host macrophage. Several para-
Abbreviations: CRD, cross-reactive determinant; DHIFCS, dialyzed heat-inactivated fetal calf serum; GPI, glycosylphosphatidylinositol; HBSS, Hanks’ Balanced Salts Solution; MSP, major surface protease; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate buffered saline; PI-PLC, phosphatidyl-inositol specific phospholipase C; T1/2, half-life; UTR, untranslated regions; VSG, variant surface glycoprotein. * Corresponding author. Tel.: 1-319-356-3169; fax: 1-319-3847208. E-mail address: [email protected] (M.E. Wilson). 1 Present address: Department of Microbiology, Osteopathic Medical Center, Des Moines University, 3200 Grand Avenue, Des Moines, IA 50312, USA. 2 Present address: Department of Public Health, Johns Hopkins University, Baltimore, MD 21205, USA.
site surface molecules facilitate survival in these diverse host environments. One such molecule is GP63, an abundant protease found on the surface membrane of all Leishmania sp. studied to date [1 /8]. When the parasite is first inoculated into mammalian tissue by a sand fly vector, GP63 facilitates survival of the extracellular promastigote stage in the presence of host complement, likely by converting complement component C3b to iC3b [9,10]. GP63 also promotes attachment of promastigotes to macrophage receptors such as CR3, the receptor for iC3b [9,11 /13], although GP63 protease activity is not absolutely required for CR3 attachment [9,14]. Upon achieving an intracellular location, promastigotes convert to obligate intracellular amastigote forms. There is evidence that GP63 promotes amastigote survival within macrophage phagolysosomes [15,16]. Thus, GP63 participates in the interaction of the parasite with its host at several points during its life cycle.
0166-6851/02/$ - see front matter # 2002 Published by Elsevier Science B.V. PII: S 0 1 6 6 - 6 8 5 1 ( 0 2 ) 0 0 0 3 0 - 0
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The amount of GP63 protein expressed by Leishmania sp. is modulated in different parasite growth and life cycle stages [5,17 /19]. Leishmania chagasi GP63 abundance is lowest in logarithmically growing promastigotes when parasites are least virulent for a murine host, and increases progressively by 14-fold as promastigotes develop in vitro to their highly infectious state in stationary phase of growth. This variation is associated with the expression of mRNAs from three distinct classes of major surface protease (MSP ) genes encoding GP63 [19,20]. Transcripts from MSPL genes are expressed solely during logarithmic growth when GP63 protein is lowest, whereas MSPS transcripts are expressed predominantly during stationary phase when GP63 protein levels are highest [19]. This switch between MSP class transcripts is determined largely by posttranscriptional events such as mRNA half-life (T1/2), rather than by the transcriptional rate [21,22]. However, considering transcripts of all three MSP classes together, the total abundance of MSP RNA does not increase during the transition from log to stationary phase. The purpose of this work, therefore, was to determine mechanisms regulating the increase in abundance of GP63 protein during the transition from logarithmic to stationary growth. We found that a great deal of this increase (five-fold) was due to a lengthening of the T1/2 of cell-associated GP63 protein. Much of the GP63 loss occurred through shedding from the parasite surface, suggesting changes in membrane characteristics could contribute to the rate of GP63 shedding. These findings add yet another level of complexity to the posttranscriptional regulation of GP63 expression in this trypanosomatid protozoan.
2. Materials and methods 2.1. Parasites and antibodies A Brazilian strain of L. chagasi (MHOM/BR/00/ 1669) was maintained in hamsters by serial intracardiac injection of amastigotes. Promastigotes were grown at 26 8C in hemoflagellate-modified minimal essential medium (HOMEM) containing 10% dialyzed heatinactivated fetal calf serum (DHIFCS) [23]. Cultures were seeded at 1/106 promastigotes ml1 and used at different points in growth from logarithmic (2 /4 days) to stationary (6 /10 days) phase as previously defined [24]. Antibodies used include polyclonal rabbit and sheep antisera against purified L. chagasi GP63 [13], polyclonal monospecific rabbit antibody to the crossreactive determinant (CRD) on glycosylphosphatidylinositol (GPI) membrane anchors treated with phosphatidyl-inositol specific phospholipase C (PI-PLC (kindly provided by Dr James Bangs, University of Wisconsin), polyclonal rabbit antisera against a cytosolic protein
P36 in L. amazonensis [25] (kindly provided by Dr K.-P. Chang of the Chicago Medical School and Dr Brad McGwire of Northwestern University), and monoclonal antibody to b-tubulin E7 (Development Studies of Hybridoma Bank, University of Iowa). Serum from patients with visceral leishmaniasis was provided by Dr Selma Jeronimo, Natal, Brazil. Peroxidase-conjugated goat anti -rabbit, anti -mouse, anti -human, or rabbit anti -sheep IgG were used as secondary antibodies (Cappel, Organon Teknika, Corp., Durham, NC). 2.2. Polyacrylamide gels and protein blots Total promastigote protein samples were prepared as described [20] and separated on 7.5% polyacrylamide gels [13]. To detect [35S], gels were soaked in 1 M Na salicylate, vacuum dried, and exposed to X-ray film (Kodak, Rochester, NY). Immunoblots were performed by electrophoretic transfer of proteins to Immobilon-P (Millipore, Bedford, MA) and blocking in 5% milk, 0.1% Tween-20, PBS. Primary antibodies were diluted 1:10,000 unless otherwise indicated. Secondary antibodies were diluted 1:20,000. Blots containing biotinylated proteins were incubated with 1:3000 peroxidase-labeled extravidin (Sigma Chemical Co.). Blots were developed by chemiluminescence (SuperSignal† , Pierce, Rockford, IL or ECLTM, Amersham Pharmacia Biotech, Arlington Heights, IL). For analysis of GPI anchors, amphipathic membrane molecules were separated from hydrophilic proteins by phase separation with Triton X-114 as described [5,26] and treated with either buffer or PI-PLC (Sigma Chemical Co.), for 30 min at 37 8C prior to a second phase separation. Samples were analyzed for GP63 or CRD on immunoblots. Secreted proteins were analyzed by washing promastigotes twice in Hanks’ Balanced Salts Solution (HBSS; GIBCO BRL, Rockville, MD) and resuspending in serum-, BSA-free medium at the starting concentration. After 24 /48 h cells were removed by centrifugation. Supernatants were filtered (0.22 mm) and concentrated with centriprep-10 and microcom-10 (Amicon, Beverly, MA). 2.3. Northern blots Total promastigote RNA was extracted by the method of Chomczynski [27]. Four micrograms of RNA were separated in lanes of 1.2% formaldehydecontaining agarose gels and transferred by capillary action to nylon filters (Roche, Germany). The blots were probed with [32P]-DNA corresponding to the conserved 5? untranslated regions (UTR) and coding region of MSP genes, or to the coding region of a-tubulin amplified by the polymerase chain reaction from L. chagasi genomic DNA. The blots were washed at high
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stringency (0.1 /SSC, 0.1% SDS, 65 8C) and exposed to X-ray film. 2.4. Metabolic labeling Nascent proteins were metabolically labeled with 50 mCi ml 1 of ProMix containing L-[35S]-methionine and 35 L-[ S]-cysteine (Amersham Pharmacia Biotech) in methionine-free RPMI (containing cysteine) with DHIFCS. The rate of GP63 synthesis was measured by detergent lysis of 4 /107 promastigotes between 15 min and 49 h after the addition of label, followed by immunoprecipitation with polyclonal serum to GP63. To measure GP63 T1/2, promastigotes were pulsed at their original cell density with [35S]-methionine for 1/2 h. Cultures were flooded with an excess of unlabeled methionine but maintained at the original (log or stationary) cell density. At times ranging from 0 to 72 h, a volume equivalent to 4/107 starting promastigotes was subjected to immunoprecipitation. To determine the rates of [35S]-methionine incorporation into total proteins, cells were pulsed with [35S]-methionine for 1 h and then lysed with 1.0% SDS. Proteins were precipitated with trichloroacetic acid and dried onto glass microfibre filters (Whatman, England) as described [28]. Label incorporation was measured with a scintillation counter. To measure the [35S]-methionine uptake, promastigotes were treated with 10 mg cycloheximide ml1 for 5 or 30 min in methionine-free RPMI. The cells were then pulsed with [35S]-methionine for 30 min without removing the inhibitor, and collected by microcentrifugation (12,000/g, 3 min) through an oil cushion (n -butyl phthalate /dinonyl phthalate /4:1) to separate them from free [35S]-methionine in the medium. After samples were taken from the medium, the whole tubes were snap frozen in ethanol/dry ice and pellet was cut off. Background counts were derived from pellets of control tubes with no cell. [35S]-methionine uptake rate was calculated as accounts of pellet (pellet/medium) 1, quantified on a scintillation counter. 2.5. Immunoprecipitation Aliquots of 4/107 promastigotes or spent culture medium from equivalent numbers of parasites were lysed by incubation for 4 /16 h in 1.0% Triton X-100, 20 mM EDTA, 20 mg antipain ml 1, and 20 mg leupeptin ml1 in TBS (10 mM Tris /HCl pH 7.2, 158 mM NaCl) at 4 8C. Results were identical using an alternate (RIPA) lysis buffer containing TBS and 1% Na deoxycholate, 1 mM phenylmethylsulfonyl fluoride, 0.1% SDS, 1 mM Na3VO4, 2 mM benzamidine, and 2 mM EDTA. Detergent-insoluble material was removed by microcentrifugation at 12,000 /g , 4 8C for 5 min. Samples were pre-cleared by incubation in rabbit or sheep pre-immune serum followed by PANSORBIN†
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(protein A) or OMNISORB† (protein G) Cells (Calbiochem Novabiochem, La Jolla, CA), respectively. Samples were heat denatured in 2% SDS, and incubated in 0.5% immune serum in 50 mM Tris /HCl pH 7.4, 190 mM NaCl, 6 mM EDTA, 2.5% Triton X-100 for 16 h at 4 8C. Antibody/protein complexes were captured with protein A or G. Cell-bound complexes were recovered by microcentrifugation, washed twice in 0.5% Triton X100 in TBS, washed twice in TBS, and denatured in reducing SDS-sample buffer [13]. 2.6. Surface biotinylation Five hundred micrograms NHS-Sulfo-Biotin ml 1 (Pierce) were incubated with 1.1 /108 promastigotes ml 1 in HBSS for 30 min at 26 8C. The reaction was stopped with 10 mM NH4Cl and cells were washed by centrifugation. Parallel cultures were metabolically labeled with [35S]-methionine as above. At time points between 0.5 and 93 h after removal from label, biotinylated or metabolically labeled cells were subjected to immunoprecipitation with anti-GP63 serum.
3. Results 3.1. GP63 proteins and RNAs are differentially expressed during promastigote growth from logarithmic to stationary phase in vitro L. chagasi promastigotes increase in infectivity as they develop from logarithmic to stationary phase in culture medium [24]. Coincident with this growth we observed a 14-fold increase in the amount of GP63 in day 7 (stationary phase) compared to day 3 (logarithmic phase) promastigotes documented on immunoblots (Fig. 1). The lower, less intense band in Fig. 1 was previously documented by our group to be a 59 kDa GP63 isoform that is probably encoded by MSPS2 [29]. This increase in GP63 protein was associated with a switch from a 2.7 to 3.0-kb MSP RNA species (Fig. 2A), which we previously found corresponds with a switch from expression of MSPL to MSPS transcripts, respectively [19]. Separation of amphipathic from hydrophilic molecules with Triton X-114 showed the major GP63 proteins separated in the detergent (amphipathic) phase (Fig. 2B). Proteins in both bands were susceptible to PI-PLC cleavage since the bands shifted from the amphipathic to the hydrophilic phase after enzyme treatment. The PI-PLC treated but not the untreated proteins were recognized by antibody to the CRD that is uniquely exposed after PI-PLC treatment (Fig. 2B, [30]). Together these results indicate that the predominant two isoforms are anchored to the L. chagasi promastigote with a GPI anchor similar to other trypanosomatid surface proteins [31,32].
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Fig. 1. Steady-state levels of GP63 during in vitro culture of promastigotes. (A) Promastigote proteins prepared on sequential days of culture were resolved by 7.5% SDS-PAGE gel electrophoresis and probed with E7 monoclonal antibody to b-tubulin (1:1000 dilution) to normalize for loading. The same filter was re-probed with rabbit polyclonal antiserum to purified GP63 to quantify its steady-state amount on different days of growth. (B) Protein quantitation of (A) by auto-background densitometry. The growth curve (cell density) represents the mean (SD) promastigote concentration in eight parallel cultures. The steady-state level of GP63 on each day was calculated by normalizing the GP63 to b-tubulin densitometry units, and setting the lowest (day 3) ratio at 1.0. Circles represent cell density; triangles indicate steady-state GP63 levels.
3.2. GP63 synthesis and loss
Fig. 2. (A) Total RNAs from L. chagasi promastigotes on different days of growth from logarithmic (days 2 /4) to stationary phase (days 6 /7) were probed with [32P]-labeled DNAs corresponding to either the MSP 5?-UTR and coding region to detect all MSP class RNAs (left panel), or the a-tubulin coding region (right panel). (B) GP63 was extracted from L. chagasi stationary promastigotes with Triton X-114 [26] and exposed to either PI-PLC (P) or buffer (C). Hydrophilic and amphipathic molecules were separated by Triton X-114 phase separation into aqueous (a) or detergent (d) phases, respectively, followed by immunoblot analysis. Blots were incubated with either polyclonal antiserum to GP63 (left panel) or antiserum to the CRD of cleaved GPI anchors (right panel [30]).
Metabolic labeling of GP63 with [35S]-methionine followed by immunoprecipitation with anti-GP63 serum allowed us to follow GP63 biosynthesis. In logarithmic phase L. chagasi promastigotes (day 3), a predominant immunoprecipitated GP63 appeared first as a 66-kDa protein followed by the additional appearance of a 63kDa GP63 protein after 30/60 min (left panel of Fig. 3A and B). The 63-kDa protein accumulated progressively during promastigote growth, resulting in high levels after about 2 days (Fig. 3B). Pulse-chase studies showed the loss of immunoprecipitated 66-kDa GP63 coincided with appearance of the lower Mr GP63 (Fig. 3A, right panel). This lower Mr GP63 had a T1/2 of 429/7 h in logarithmic promastigotes (day 3) quantified by densitometry in eight replicate experiments. These observations do not preclude the existence of additional GP63 isoforms that are not easily detected by metabolic labeling and immunoprecipitation. Since L. chagasi possesses more than 18 genes encoding GP63, we could not be certain whether the 66- and 63-kDa metabolically labeled proteins were the products of distinct genes with different rates of synthesis and degradation, or whether the above pattern represents processing of a precursor to a mature GP63 protein product. As such, we examined attenuated L. donovani parasites transfected with a single L. chagasi
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Fig. 3. The rates of GP63 synthesis and degradation. (A) (left panel) GP63 synthesis was quantified in logarithmic L. chagasi promastigotes incubated in [35S]-methionine for varying times before immunoprecipitating GP63. Right panel: A representative pulse-chase study was performed by incubating logarithmic phase promastigotes (1.1 107 ml 1) in [35S]-methionine for 1 h, after which an excess of unlabeled methionine was added (time 0). At the indicated time points, GP63 was immunoprecipitated from aliquots of cultures corresponding to 4 107 starting promastigotes. (B) GP63 synthesis was analyzed by metabolic labeling of logarithmic phase (1.1 107 ml 1) promastigotes in [35S]-methionine for 20 min /49 h and immunoprecipitating with anti-GP63 serum. Each lane contains GP63 proteins derived from 4 107 promastigotes.
MSPS or MSPL gene. Although L. donovani expresses GP63, the amount is low in attenuated promastigotes and L. donovani GP63 is poorly detected by polyclonal antiserum to L. chagasi GP63. Thus, only the transfected MSP gene product is detected in these cells [29]. We found that a higher Mr ‘precursor’ produced from a single MSP gene (MSPS1 , MSPS2 , or MSPL ) was chased into a lower Mr ‘mature’ form in transfectants (Fig. 4A; MSPL data not shown). Thus, a precursor / product relationship was observed in transfectants harboring only one MSP gene whose protein product could be detected by immunoprecipitation. To determine whether both the 66- and the 63-kDa GP63s are glycosylated, we utilized N -glycanase to remove N-linked oligosaccharides from L. chagasi proteins. Rather than collapsing into a single band, pulse-chase experiments showed both the 66- and the 63kDa proteins are susceptible to digestion with N glycanase (Fig. 4B, ‘/’ lanes). This suggests both forms of GP63 had undergone N -glycosylation, and that the difference between the bands is not merely due to N glycosylation. Incubation in 10 mg tunicamycin ml 1, which inhibits N -glycosylation, yielded parallel results (not shown). 3.3. Intracellular processing of GP63 We hypothesized that GP63 could mature from a 66to 63-kDa isoform through proteolytic cleavage of the
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Fig. 4. (A) The precursor /product relationship during processing of individual MSP genes. Attenuated L. donovani logarithmic promastigotes (2 107 cells ml 1) (L. don) or L. donovani transfected with the pX plasmid expressing individual MSPS genes (MSPS1 , MSPS2 ) were metabolically labeled and chased with an excess of unlabeled methionine. GP63 was immunoprecipitated from aliquots corresponding to 4 107 starting promastigotes and the pattern of protein maturation was detected on autoradiograms. Antiserum to L. chagasi GP63 did not recognize the homologous molecule in attenuated L. donovani alone (L. don) or transfected with pX (not shown). (B) L. chagasi logarithmic promastigotes underwent a 2 h pulse with [35S]methionine followed by a 0 /18 h chase with unlabeled methionine. Proteins were incubated in the absence () or presence () of N glycanase and GP63 was immunoprecipitated from aliquots of culture corresponding to 4 107 starting promastigotes. Autoradiograms of the reaction products are shown.
pro-enzyme, similar to the 3 kDa change in L. major GP63 after pro-peptide removal [33]. Extracellular protease inhibitors such as antipain, leupeptin, phenylmethylsulfonyl fluoride, or EDTA did not inhibit production of the 63 kDa GP63 (not shown), leading us to question whether both GP63s or only the 63-kDa form was expressed at the parasite surface. Parallel logarithmic phase promastigote cultures were treated by pulse-chase with [35S]-methionine or by surface biotinylation prior to lysis (Fig. 5A). Linking biotin to promastigote surface proteins labeled only the mature 63-kDa form of GP63, in contrast to parallel cultures metabolically labeled with [35S]-methionine in which both 66- and 63-kDa GP63 isoforms were labeled. If indeed the 66- and 63-kDa GP63s represent precursor and product, this result would suggest that generation of the ‘mature’ 63 kDa GP63 occurs intracellularly rather than on the promastigote surface. Alternatively, it is conceivable that the 66-kDa GP63 form is of very low abundance and poorly accessible for biotinylation. The 63-kDa GP63 protein slowly appeared in the extracellular medium (Fig. 5B). There was no associated change in Mr, suggesting proteolytic cleavage had not
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membrane-bound vesicles or large aggregated micelles containing GP63. To eliminate the possibility that GP63 was released due to cell lysis or death, an antibody against a well-characterized cytosolic protein P36 in L. amazonensis [25] was used to probe a western blot of L. chagasi cell pellets or concentrated extracellular medium. In contrast to GP63, which appeared in both cells and extracellular medium, P36 was found only in intact cells (Fig. 6A). A positive control cytoskeletal protein, b-tubulin, was also found exclusively in cell pellet. We conclude that the observed GP63 in the medium was not due to cell lysis. Taken together, these data suggest GP63 is lost from promastigotes by release in a soluble (possibly micellar) form, and also in membrane-bound vesicles. 3.4. GP63 has a longer T1/2 in stationary phase promastigotes than in logarithmically growing promastigotes Fig. 5. (A) GP63 processing occurs intracellularly. Logarithmic promastigote cultures were either metabolically labeled with [35S]methionine for 2 h followed by various times of chase in unlabeled methionine (left panel), or surface labeled with biotin at time 0 (right panel). 0.5 /48 h later, culture volumes equivalent to 4 107 starting promastigotes were subjected to GP63 immunoprecipitation. Panels contain an autoradiogram (left) or nitrocellulose developed with extravidin-peroxidase followed by enhanced chemiluminescence (right). (B) GP63 is slowly released into extracellular medium. L. chagasi logarithmic promastigotes were metabolically labeled and chased with unlabeled methionine. At different time point, parasites were recovered by centrifugation, growth medium was filtered to remove residual cells, and GP63 was immunoprecipitated. Lanes of autoradiograms contain GP63 from a volume of cells or growth medium equivalent to 4 107 starting promastigotes. (C) GP63 in the extracellular medium exists in both membrane-associated and -free forms. Logarithmic promastigote cultures were separated into supernatant (S1) and pellet (PM) by centrifugation (12,000 g ). S1 was filtered through 0.2 mm filter and further separated by centrifugation at 100,000 g into a membrane (P2) and supernatant (S2) fractions. P2 was solubilized in Triton X-100 and detergent-soluble (S3) versus detergent-insoluble (P3) fractions were separated by centrifugation. Proteins were analyzed by immunoblot probed with sheep antiserum to purified GP63.
occurred. In addition, GP63 release was not associated with the appearance of a CRD antibody-positive band in the supernatant that would have suggested PI-PLCmediated hydrolysis of the GPI anchor (data not shown). To further investigate whether 63-kDa GP63 in the extracellular medium is still membrane associated or free in the medium, filtered growth medium was centrifuged at 100,000 /g to pellet membranes. Equal amounts of 63-kDa GP63 were found in both supernatants and pellets of such centrifugation (Fig. 5C), suggesting both membrane-free and -associated forms of GP63 are in the medium. Furthermore, the pelletassociated GP63 was soluble in the detergent Triton X-100 (Fig. 5C), consistent with the presence of either
Our prior studies indicated there is equal transcription of the MSP genes in logarithmic versus stationary phase promastigotes [22]. Our current study indicates that GP63 is synthesized in logarithmic phase (day 3) promastigotes, and this GP63 has a T1/2 of about 429/ 7 h. Collectively these results do not explain 14-fold difference in GP63 abundance in logarithmic versus
Fig. 6. (A) GP63 release in extracellular medium. Logarithmic promastigotes (day 3) were suspended in serum- and BSA-free medium. After 24 and 48 h, filtered supernatants (S) and cell pellets (P) were blotted to nitrocellulose filters and probed with the rabbit sera against the cytoplasmic protein P36 [25], rabbit antisera against GP63, or control mAb E7 to b-tubulin. Each lane was loaded the equivalent amount of pellet or supernatant corresponding to 1 107 starting promastigotes. (B) The half-life of surface biotinylated GP63 is longer in stationary phase promastigotes than in logarithmic phase parasites. Promastigotes in day 4 (Log) or day 8 (Sta) were either metabolically labeled with [35S]-methionine (data not shown) or surface labeled with biotin for 30 min. From the end of labeling (time 0) to 93 h, samples were taken at various time points as indicated. Culture volumes equivalent 8 107 starting promastioges were subjected to GP63 immunoprecipitation. GP63 was detected with extravadin-peroxidase followed by enhanced chemiluminescence.
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stationary parasites. We therefore attempted to measure the rate of GP63 protein synthesis in logarithmic and stationary phase promastigotes. Unexpectedly, it initially appeared as though 12 times more GP63 were synthesized by day 2 (logarithmic phase) promastigotes than by day 8 (stationary phase) promastigotes (Table 1). This result is not compatible with the observed increase in steady-state GP63 levels in stationary promastigotes. To address this discrepancy, we measured the incorporation of [35S]-methionine into total proteins by logarithmic versus stationary phase promastigotes. Four repeat studies, each with triplicate conditions, showed that logarithmic parasites (day 3) incorporated three times more [35S]-methionine than stationary phase parasites (day 6 /9) (Table 1). We also determined the rate of [35S]-methionine uptake by promastigotes. In the latter experiment, cycloheximide was used to prevent [35S]-methionine incorporation into proteins. We previously showed that 10 mg cycloheximide ml1 inhibits L. chagasi protein synthesis by more than 95% [21]. Logarithmic phase promastigotes (day 3) took up 4.6-fold more [35S]-methionine than stationary phase promastigotes (day 7) in the presence of 10 mg cycloheximide ml 1 (Table 1). Furthermore, immunoprecipitation with pooled sera from the patients with visceral leishmaniasis showed multiple proteins were less abundant in stationary than in logarithmic promastigotes. Densitometric measurement of the abundant 80-, 49- and 23-kDa proteins showed ratios of 2.6, 1.9 and 2.3, respectively, comparing abundance in logarithmic versus stationary phase promastigotes. These data could reflect a decrease in amino acid transport into the cell. Whether there is also an overall decrease in protein synthesis cannot be discerned. The differences in [35S]met uptake prevent us from directly comparing the rates of GP63 translation in logarithmic versus stationary phase promastigotes. We can merely state that L.
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chagasi promastigotes synthesize some GP63 during logarithmic phase of growth, even though they have a relatively low amount of GP63 protein during this growth phase. Although we could not compare rates of translation, we were able to measure the T1/2 of GP63 protein in logarithmic versus stationary phase promastigotes. As shown in the representative experiment in Table 1, the T1/2 of GP63 in day 2 (logarithmic) promastigotes was 29.2 h and progressively increased to more than 72 h in day 5 promastigotes, at which time parasites were converting to stationary phase. The change in GP63 T1/2 does not reflect a ‘dilutional’ effect due to rapid growth of logarithmic promastigotes, since we controlled for this possibility in pulse-chase experiments by taking sample sizes corresponding to 4 /107 starting promastigotes. Each time point reflects the loss of GP63 from the same starting amount of labeled GP63. This result suggests that GP63 protein becomes more stable as promastigotes progress from logarithmic to stationary phase. Three replicate experiments displayed a similar pattern. Analyses of the coding regions do not predict structural differences between the GP63s detected in log versus stationary promastigotes. Therefore, we considered the possibility that modification of the promastigote membrane accounts for differences in GP63 loss from the cells. Logarithmic or stationary phase promastigotes were surface biotinylated. Starting at the end of the biotinylation period, we measured the loss of GP63 by immunoprecipitation. Two replicate experiments showed that log phase promastigotes lost their surface GP63 more rapidly than stationary phase promastigotes. The T1/2 of the surface GP63 was 34 and 77 h in log and stationary phase parasites, respectively (Fig. 6B). We calculated the predicted steady-state amount of GP63 from the T1/2 data, assuming that
Table 1 Changes in GP63 T1/2, measurable GP63 synthesis, [35S]-methionine uptake and incorporation during promastigote growth Day of growth
GP63 T1/2 (h)a
Measurable GP63 synthesisb
Relative [35S]-methionine incorporationc
[35S]-methionine uptake rated
2 3 4 5 6 /9
29.2 47.7 63.4 72.0 ND
12.1 8.7 5.9 1.7 1.3
ND 3.091.7 ND ND 1.0
ND 16.990.9 ND ND 3.790.3
ND, not done. a GP63 T1/2 was measured by pulse-chase and immunoprecipitation in the original cell density of sequential days of culture. The amount of protein was quantified by densitometry. Using linear regression, the time of 50% protein loss was calculated. b Parallel promastigote cultures were labeled for 1 h, and the protein was quantified by autoradiography and densitometry as described for GP63 T1/2 (a). The measurable GP63 synthesis was calculated as a ratio to the day 8 (lowest) densitometry value. The amount on day 6 /9 was the average of those on individual days (1.5, 1.2, 1.0, and 1.5, respectively). c 35 [ S]-methionine incorporation by promastigotes on different days of growth was normalized to 1.0 on day 6 /9 (triplicates, n 4). d Logarithmic (day 3) or stationary (day 7) promastigotes were treated with cycloheximide for 30 min, and then pulsed with [35S]-methionine for additional 30 min without removal of cycloheximide. [35S]-methionine uptake rate was calculated from triplicates. Treatment for 5 min with cycloheximide and a repeat study yielded similar results (not shown).
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Table 2 Predicted steady-state GP63 levels from protein half-life using linear regression Day
2
3
2 3 4 5 6 7 8 9
1.00 0.58 0.21 0.00 0.00 0.00 0.00 0.00
1.00 0.71 0.50 0.28 0.07 0.00 0.00
4
1.00 0.84 0.63 0.43 0.22 0.01
5
1.00 1.00 1.00 1.00 1.00
6
1.00 1.00 1.00 1.00
7
1.00 1.00 1.00
8
1.00 1.00
9
Total predicted
Amount measureda
1.00
1.00 1.58 1.92 2.34 2.91 3.49 4.22 5.01
1.9 1.0 5.5 7.6 12.5 14.7 11.5 12.1
Relative levels of GP63 protein were predicted according to the following linear regressions: day 2, f (x ) 0.9556670.015625x , r 2 0.9598; day 3, f (x ) 0.9293330.009x , r 2 0.7569; day 4, f (x ) 1.0499 /0.008676x , r 2 0.8198. Assumptions were: both logarithmic and stationary phase promastigotes have a steady translation rate of 1.00, and there was little degradation of proteins synthesized on day 5 and afterwards. a Western blot determination of the steady-state ratios of GP63 abundance on each day of growth compared to the lowest measured value (day 3). Values represent ratios of GP63 to b-tubulin in the same lanes of blots, reprobed with two different antibodies (see Fig. 1). Measurements were made by densitometry.
both logarithmic and stationary phase promastigotes have a steady translation rate set at 1.0 arbitrary units per day. As shown in Table 2, the difference in T1/2 alone could explain a five-fold increase in cell-associated GP63 abundance in stationary phase versus logarithmic phase promastigotes. Thus, even though we cannot reliably compare translation rates, the changing T1/2 of the membrane bound protein can account for a majority of the protein accumulation in stationary phase organisms.
4. Discussion GP63 of L. chagasi is encoded by a family of more than 18 tandem MSP genes. These genes fall into three classes (MSPL , MSPS , MSPC ) that have distinct sequences in their 3?UTRs and downstream intergenic regions. All MSP 5?UTRs and coding regions have a high degree of sequence identity, although there are regional differences in coding regions, particularly in the case of the MSPC gene. Transcripts of the three MSP class genes are differentially expressed, with MSPL RNAs predominating in logarithmic growth and MSPS RNAs predominating in stationary phase of promastigote growth. MSPC RNAs are constitutively expressed at low levels throughout growth [19]. Our prior studies of mRNA expression showed the MSP mRNAs are regulated post-transcriptionally. Expression of at least one of the three MSP classes (MSPL ) is determined by the stability of its mRNA [21,22]. During the current study we examined the biosynthesis and T1/2 of the MSP gene products. Metabolic labeling studies of wild-type L. chagasi showed that loss of a 66-kDa GP63 coincided with appearance of the predominant 63-kDa GP63 that was detectable by immunoprecipitation. A similar apparent precursor /product relationship was observed in trans-
fectants harboring only one detectable MSP gene product. GP63 of all Leishmania sp. is produced as a proenzyme that requires removal of the pro-peptide for generation of an active enzyme [4,17]. Our findings are consistent with production of a 66 kDa precursor containing the pro-peptide, and removal of the proregion to form a 63 kDa product. Pro-peptide removal may not require mature cellular GP63, since the transfected MSPS1 , MSPS2 or MSPL gene products were cleaved in a GP63-low host cell (attenuated L. donovani ). These findings are consistent with the published findings of Macdonald et al., in which pro-peptide removal from recombinant L. major GP63 synthesized in COS-7 cells underwent activation through an autocatalytic cis mechanism that apparently does not involve exogenous activated GP63 [33]. The authors hypothesized that this is consistent with the cysteine switch mechanism, in which latency is maintained through interactions between the active site and a Cys sulfhydryl group in the GP63 pro-region [33]. Biosynthesis of GP63 can be contrasted with that of the variant surface glycoprotein (VSG) of the related protozoan Trypanosoma brucei . VSG is synthesized in the endoplasmic reticulum where it undergoes addition of N-linked oligosaccharides, replacement of the Cterminal peptide with a GPI anchor [34 /36], and transport to a compartment at the cell surface containing an endogenous membrane-bound phospholipase C [37]. Despite being delivered to this compartment, VSG is quantitatively released from the native cell by the action of a Zn-metalloprotease, perhaps by a T. brucei version of GP63, rather than by PI-PLC cleavage. The net result is a released molecule with increased migration on SDS-PAGE and a kinetics of shedding that is much faster than that of Leishmania GP63 [38]. Similar to VSG, GP63 undergoes N -glycosylation and is linked to a GPI anchor prior to delivery to the surface membrane [39]. Unlike VSG, it appeared slowly in
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extracellular medium. The kinetics of GP63 loss inversely paralleled the kinetics of its appearance in extracellular medium, and the extracellular form of GP63 did not bind antibody to the CRD exposed after PI-PLC treatment. Thus, it seems that the shedding of GP63 occurs without PI-PLC mediated cleavage of the GPI anchor. Our prior studies established the fact that low levels of GP63 are present in logarithmic promastigotes that express predominantly MSPL class RNAs, whereas GP63 abundance increases in stationary phase parasites expressing MSPS RNAs [29]. To our surprise, there was abundant synthesis of GP63 by logarithmic phase promastigotes. It was not possible to directly compare the rates of GP63 translation in different growth phases due to discrepancies in uptake and incorporation of the metabolic label in log versus stationary phase parasites. Nonetheless, there was an increase in GP63 protein T1/2 as parasites developed in vitro from logarithmic to stationary phase. The change in T1/2 was observed either when GP63 was metabolically labeled, or when surface GP63 was labeled by biotinylation. These data are consistent with a change in GP63 shedding due to remodeling of the promastigote surface membrane during growth. We propose that the observed increase in the steadystate abundance of GP63 protein during growth of L. chagasi is due, at least in part, to the lengthening of the half-life of the membrane-bound protein. GP63 may be more rapidly ‘shed’ or ‘lost’ from the surface of logarithmic phase than of stationary phase cells, thus accounting for the lower half-life of cellular GP63 in logarithmic parasites. It is possible that additional contributions are made by differences in the rates of translation of MSPL versus MSPS genes, and/or the increased abundance in MSPS RNA in stationary phase. During prior work we documented that the regulation of MSPL RNA expression is related to growth-related shortening of the T1/2 of this transcript [21,40]. The current study documents the fact that the amount of GP63 protein expressed is also influenced by the kinetics of its loss from the cell.
5. Addendum Since submission of this manuscript, McGuire et al. have documented extracellular release of GP63 from Leishmania amazonensis and several other Leishmania sp. (J. Biol. Chem. 2002, in press).
Acknowledgements The authors are grateful to Dr James Bangs, University of Wisconsin, Dr Brad McGuire of North-
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western University and Dr K.-P. Chang, Chicago Medical School for their helpful suggestions and provision of antibodies used in this work. This work was supported by NIH grant AI32135 (MEW and JED), DK/AI52550 (MEW), AI40591 (JED), and a Veterans’ Affairs Merit Review grant (MEW). CY and AB were supported by NIH Training Grants T32 AI07343 and T32 AI07511, respectively.
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