Identification of the Major Cysteine Protease of Giardia and Its Role in Encystation
2008; Elsevier BV; Volume: 283; Issue: 26 Linguagem: Inglês
10.1074/jbc.m802133200
ISSN1083-351X
AutoresKelly N. DuBois, Marla Abodeely, Judy A. Sakanari, Charles S. Craik, Malinda Lee, James H. McKerrow, Mohammed Sajid,
Tópico(s)Toxoplasma gondii Research Studies
ResumoGiardia lamblia is a protozoan parasite and the earliest branching clade of eukaryota. The Giardia life cycle alternates between an asexually replicating vegetative form and an infectious cyst form. Encystation and excystation are crucial processes for the survival and transmission of Giardia. Cysteine proteases in Giardia have been implicated in proteolytic processing events that enable the continuance of the life cycle throughout encystation and excystation. Using quantitative real-time PCR, the expression of twenty-seven clan CA cysteine protease genes in the Giardia genome was measured during both vegetative growth and encystation. Giardia cysteine protease 2 was the most highly expressed cysteine protease during both life cycle stages measured, with a dramatic expression increase during encystation. The mRNA transcript for Giardia cysteine protease 2 was 7-fold up-regulated during encystation and was greater than 3-fold higher than any other Giardia protease gene product. Recombinant Giardia cysteine protease 2 was expressed, purified, and biochemically characterized. The activity of the recombinant cysteine protease 2 protein was confirmed to be identical to the dominant cysteine protease activity found in G. lamblia lysates. Giardia cysteine protease 2 was co-localized with cyst wall protein in encystation-specific vesicles during encystation and processed cyst wall protein 2 to the size found in Giardia cyst walls. These data suggest that Giardia cysteine protease 2 is not only the major cysteine endoprotease expressed in Giardia, but is also central to the encystation process. Giardia lamblia is a protozoan parasite and the earliest branching clade of eukaryota. The Giardia life cycle alternates between an asexually replicating vegetative form and an infectious cyst form. Encystation and excystation are crucial processes for the survival and transmission of Giardia. Cysteine proteases in Giardia have been implicated in proteolytic processing events that enable the continuance of the life cycle throughout encystation and excystation. Using quantitative real-time PCR, the expression of twenty-seven clan CA cysteine protease genes in the Giardia genome was measured during both vegetative growth and encystation. Giardia cysteine protease 2 was the most highly expressed cysteine protease during both life cycle stages measured, with a dramatic expression increase during encystation. The mRNA transcript for Giardia cysteine protease 2 was 7-fold up-regulated during encystation and was greater than 3-fold higher than any other Giardia protease gene product. Recombinant Giardia cysteine protease 2 was expressed, purified, and biochemically characterized. The activity of the recombinant cysteine protease 2 protein was confirmed to be identical to the dominant cysteine protease activity found in G. lamblia lysates. Giardia cysteine protease 2 was co-localized with cyst wall protein in encystation-specific vesicles during encystation and processed cyst wall protein 2 to the size found in Giardia cyst walls. These data suggest that Giardia cysteine protease 2 is not only the major cysteine endoprotease expressed in Giardia, but is also central to the encystation process. Giardia lamblia is a protozoan parasite that inhabits the upper small intestine of many vertebrate hosts and is the most commonly isolated intestinal parasite world wide (1Adam R.D. Clin. Microbiol. Rev. 2001; 14: 447-475Crossref PubMed Scopus (876) Google Scholar). In addition to its medical importance, Giardia is of interest as a model cell system because it represents the most early branching clade of eukaryota (2Hedges S.B. Nat. Rev. Genet. 2002; 3: 838-849Crossref PubMed Scopus (579) Google Scholar, 3Sogin M.L. Gunderson J.H. Elwood H.J. Alonso R.A. Peattie D.A. Science. 1989; 243: 75-77Crossref PubMed Scopus (585) Google Scholar). Giardia has a simple two-stage life cycle consisting of a vegetative replicating trophozoite and an infectious cyst. Infection is initiated with cyst ingestion by a vertebrate host. After passage through the acidic host stomach, vegetative trophozoites emerge from the cyst by the process of excystation, asexually divide by binary fission, establish the duodenal infection, and give rise to the characteristic symptoms of giardiasis. Trophozoites can form infective cysts that are passed in the host feces and ingested by another host to propagate the life cycle (1Adam R.D. Clin. Microbiol. Rev. 2001; 14: 447-475Crossref PubMed Scopus (876) Google Scholar). The process of encystation is a coordinated secretion of cyst wall materials to the periphery of a cell to form the cyst wall (4Lujan H.D. Mowatt M.R. Nash T.E. Microbiol. Mol. Biol. Rev. 1997; 61: 294-304Crossref PubMed Scopus (129) Google Scholar, 5Hehl A.B. Marti M. Kohler P. Mol. Biol. Cell. 2000; 11: 1789-1800Crossref PubMed Scopus (84) Google Scholar). In response to environmental cues, trophozoites produce abundant cyst wall proteins that are packaged into encystation-specific vesicles (ESVs). 3The abbreviations used are: ESVs, encystation-specific vesicles; CWP2, cyst wall protein 2; ESCP, encystation-specific cysteine protease; GAPDH, glutaraldehyde phosphate dehydrogenase; GlCP2, G. lamblia cysteine protease 2; DTT, dithiothreitol; AMC, amino-methylcoumarin; Z, benzyloxycarbonyl; GFP, green fluorescent protein. 3The abbreviations used are: ESVs, encystation-specific vesicles; CWP2, cyst wall protein 2; ESCP, encystation-specific cysteine protease; GAPDH, glutaraldehyde phosphate dehydrogenase; GlCP2, G. lamblia cysteine protease 2; DTT, dithiothreitol; AMC, amino-methylcoumarin; Z, benzyloxycarbonyl; GFP, green fluorescent protein. These vesicles grow, mature, and eventually traffic to the plasma membrane of the trophozoite, where cyst wall precursor material is secreted to form the environmentally stable cyst wall (4Lujan H.D. Mowatt M.R. Nash T.E. Microbiol. Mol. Biol. Rev. 1997; 61: 294-304Crossref PubMed Scopus (129) Google Scholar, 6Reiner D.S. McCaffery M. Gillin F.D. Eur. J. Cell Biol. 1990; 53: 142-153PubMed Google Scholar, 7Lujan H.D. Mowatt M.R. Conrad J.T. Bowers B. Nash T.E. J. Biol. Chem. 1995; 270: 29307-29313Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). The expression of many proteins is up-regulated during the encystation process (4Lujan H.D. Mowatt M.R. Nash T.E. Microbiol. Mol. Biol. Rev. 1997; 61: 294-304Crossref PubMed Scopus (129) Google Scholar). Cysteine proteases have been found to be essential to the life cycles of several parasitic organisms, catalyzing diverse processes such as parasite immunevasion, tissue invasion, and encystment/excystment in addition to well established roles in protein processing and catabolism (8Sajid M. McKerrow J.H. Mol. Biochem. Parasitol. 2002; 120: 1-21Crossref PubMed Scopus (663) Google Scholar, 9McKerrow J.H. Int. J. Parasitol. 1999; 29: 833-837Crossref PubMed Scopus (134) Google Scholar). Indeed, in G. lamblia (10Ward W. Alvarado L. Rawlings N.D. Engel J.C. Franklin C. McKerrow J.H. Cell. 1997; 89: 437-444Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar) indispensable roles for cysteine proteases have been documented in the processes of encystation and excystation. Ward et al. (10Ward W. Alvarado L. Rawlings N.D. Engel J.C. Franklin C. McKerrow J.H. Cell. 1997; 89: 437-444Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar) validated a role for cysteine endoprotease activity in the excystation process by demonstrating that excystation was inhibited by the addition of small molecule cysteine protease inhibitors to the excystation media. Touz et al. (11Touz M.C. Nores M.J. Slavin I. Carmona C. Conrad J.T. Mowatt M.R. Nash T.E. Coronel C.E. Lujan H.D. J. Biol. Chem. 2002; 277: 8474-8481Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar) implicated a cysteine exoprotease in the process of encystation. Processing of cyst wall protein 2 (CWP2), one of the main cyst wall proteins that form the structure of the cyst wall, was also blocked by cysteine protease inhibitors. Whereas the role of these chemical knock-out experiments focused attention on clan CA cysteine proteases in Giardia, the recent completion of the Giardia genome indicated that there are twenty-seven candidate clan CA cysteine protease genes in Giardia. To address the question of which gene product(s) was responsible for key events in the life cycle, such as cyst wall processing, we analyzed the transcription levels of all twenty-seven genes and found that G. lamblia cysteine protease 2 (GlCP2) was in fact the major expressed cysteine protease gene in Giardia. We therefore cloned, heterologously expressed, and biochemically characterized this protease, and specifically evaluated its role in encystation. Cell Culture, Transfection, and Differentiation—WB isolated G. lamblia trophozoites (ATCC number 30957) were maintained in a modified TYI-S-33 medium supplemented with 10% fetal bovine serum (Omega Scientific, Inc.), penicillin-streptomycin (UCSF CCF), vitamins (Invitrogen), and Fungizone (UCSF CCF). The pGFP.pac vector (gift from Theodore Nash, National Institutes of Health; modified by Lei Li from the C. C. Wang laboratory, UCSF) was used to episomally express C-terminal GFP fusion proteins in Giardia. The transfection protocol used by Singer et al. (12Singer S.M. Yee J. Nash T.E. Mol. Biochem. Parasitol. 1998; 92: 59-69Crossref PubMed Scopus (107) Google Scholar) was followed with modifications: 1–2 × 106 trophozoites were electroporated with 50 μg of plasmid DNA (GenePulser XCell, Bio-Rad) at 0.45 kV, 950 μF. Transfectants were selected with puromycin dihydrochloride (Sigma) and increased in 5–20 μg/ml increments to a final concentration of 80–120 μg/ml. Trophozoites were induced to encyst as indicated by Abel et al. (13Abel E.S. Davids B.J. Robles L.D. Loflin C.E. Gillin F.D. Chakrabarti R. J. Biol. Chem. 2001; 276: 10320-10329Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). Transformation and Expression of GlCP2 in Pichia pastoris—The GlCP2 gene was re-synthesized to optimize for yeast codon usage (DNA 2.0). The rGlCP2 gene was amplified by PCR from the pJ31:7972 vector into which the full-length cDNA had previously been cloned and modified to include a polyhistidine tag using the forward primer GlCP2pPicF: CTCGAGAAAAGACATCATCATCATCATCATGAGTTGAATCATATTACTC and the reverse primer GlCP2pPicR: TCTAGATTACTCATCGAAAAATCCAGCATAGGCC. The 920-bp amplicon was subcloned in the XhoI/XbaI site of the P. pastoris expression vector pPicZαB (Invitrogen). The plasmid was linearized by digestion with SalI and introduced into P. pastoris by electroporation (GenePulser XCell, Bio-Rad) according to the manufacturer's specifications. Transformants were screened by growth on YPD+100 μg/ml zeocin (Invitrogen). Purification of rGlCP2—P. pastoris was grown under expression induction conditions in a BioFlo 110 Fermentor/Bioreactor (New Brunswick Scientific) for 3 days according to the manufacturer's specifications. Methanol was maintained at 0.5% (calculated by a methanol sensor) by addition of 100% methanol 2×/day. 0.2-μm filtered supernatant was lyophilized. 8 g of lyophilized material was resuspended in 40 ml of ddH2O + 1 mm Pefabloc (Sigma). Solution was filtered at 0.2 μm, dialyzed in 10,000 MWC dialysis tubing (Pierce) against 10 mm Tris-HCl, pH 8.0 at 4 °C, and fractionated by ion exchange chromatography with Fast Flow Q resin (GE Healthcare) followed by dialysis to desalt and a Mono Q anion exchange column (GE Healthcare). Purification of Cysteine Protease Activity from G. lamblia Lysates—Giardia cells were incubated in a 20 mm Tris-HCl, pH 7.2 and 0.2% Triton X-100 (Sigma-Aldrich) buffer at 4 °C with stirring for 2 h. Debris was pelleted, and supernatant was 0.2-μm filtered and subjected to anion exchange chromatography using a Mono Q column. Expression and Purification of CWP2—The open-reading frame for CWP2 was amplified from genomic DNA, and a C-terminal polyhistidine tag was added using the primers CWP2pMalF: TCTAGAATGGCTTGCCCTGCCACCGAGG and CWP2pMalR: GCGGCCGCTTTAATGATGATGATGATGATGCCTTCCCTGGATCCTTCTGCGGACAATAG and inserted into the NotI/XbaI site of the expression vector pCMVTnT (Promega). 1 μg of plasmid DNA was used as a template for in vitro transcription using the TnT Quick-coupled Transcription/Translation kit according to the manufacturer's specifications (Promega). [35S]rCWP2 was further purified on a nickel-nitrilotriacetic acid column (Qiagen). Protease Activity Assays—40 μm of the fluorogenic substrates Z-FR-AMC (N-carbobenzoxy-phenylalanyl-arginyl-7-amido-4-methylcoumarin) and Z-RR-AMC (N-carbobenzoxy-arginyl-arginyl-7-amido-4-methylcoumarin; excitation/emission of AMC: 360 nm/470 nm) (Bachem) were incubated with Giardia lysates or recombinant enzyme in Tris-HCl buffer (pH 7.2) or citrate/dibasic sodium phosphate buffers (pH 4.0–8.0) containing 4 mm DTT, 1 mm Pefabloc, and 10 mm EDTA. Subsequent protease activity was measured by monitoring the increase in relative fluorescence units (RFU) over time. rGlCP2 was incubated for 30 min with 50 μg of casein-resorufin (Molecular Probes) in 200 μl of citrate/dibasic sodium phosphate buffers. 960 μl of 5% (w/v) trichloroacetic acid was added, samples were incubated 10 min, and centrifuged. 400 μl of supernatant were added to 600 μl of 0.5 m Tris, pH 8.8. Hydrolysis was quantified by measuring fluorescence (excitation/emission: 574 nm/584 nm). Purified rCWP2 was incubated with enzyme in Tris-HCl buffer, pH 7.2, 4 mm DTT at 25 °C. The sample was fractionated by SDS-PAGE, dried, and visualized by phosphorimaging (Typhoon Trio, GE Healthcare). DCG04 (radio125I-iodinated or BODIPY-labeled), the clan CA cysteine protease inhibitor (14Greenbaum D. Medzihradszky K.F. Burlingame A. Bogyo M. Chem. Biol. 2000; 7: 569-581Abstract Full Text Full Text PDF PubMed Scopus (471) Google Scholar), was incubated with enzyme and 4 mm DTT for 30 min. Proteins were fractionated by SDS-PAGE, dried, and visualized by phosphorimaging (Typhoon Trio, GE Healthcare). To determine the Km of GlCP2, the fluorogenic peptide substrates Z-FR-AMC, Z-RR-AMC, and Z-VLK-AMC (Bachem) were incubated with enzyme at a range of concentrations, and the Vmax units/s was recorded. The non-linear regression and Km calculations were determined using Prism 4 software (Graphpad). rGlCP2 was fractionated on a Novex® 10% zymogram (gelatin) gel (Invitrogen) under native conditions as recommended by the manufacturer. The gel was stained with SimplyBlue™ Safestain (Invitrogen) and destained in water to visualize bands of protease activity. rGlCP2 was fractionated by SDS-PAGE under non-reducing conditions on a 15% Tris-HCl gel. The gel was washed 2× in 20 mm Tris-HCl, 0.2% Triton X-100. The gel was incubated in 20 mm Tris-HCl, 0.2% Triton X-100, 5 mm DTT, and 10 μm Z-FR-MNA (N-carbobenzoxy-phenylalanyl-arginyl-4-methoxy-β-naphthylamide) for 2 h at room temperature. Two volumes (compared with substrate) of 2 m coupling reagent (5-nitro-2-salicylaldehyde) was added to the reaction, and the reaction was incubated for an additional 4 h at room temperature. Fluorescence was visualized on a Typhoon Trio (GE Healthcare). Positional Scanning Synthetic Combinatorial Library (PS-SCL)—Protease activity was assayed at 25 °C in a buffer containing 20 mm Tris-HCl, pH 7.2, 5 mm DTT, 0.2% Triton X-100 (Sigma-Aldrich), and 1% Me2SO (from the substrates) or in buffer with NaOAc replacing Tris-HCl (pH 5.5) as referenced in the text. Assays were performed as previously described using 250 μm substrate in each assay (15Choe Y. Leonetti F. Greenbaum D.C. Lecaille F. Bogyo M. Bromme D. Ellman J.A. Craik C.S. J. Biol. Chem. 2006; 281: 12824-12832Abstract Full Text Full Text PDF PubMed Scopus (304) Google Scholar). RNA Methods—Total RNA from vegetative or encysting Giardia cells was isolated with TRIzol reagent (Invitrogen). 2 μg of RNA was treated with 1 unit of amplification grade DNase I (Sigma). cDNA was synthesized with Superscript III reverse transcriptase according to the manufacturer's specifications (Invitrogen). cDNA samples were stored at -80 °C until use. Control samples were prepared as above using nuclease-free ddH2O in place of RNA. Real-time PCR—PCR was performed in an Mx3005P™ QPCR system using MxPro™ QPCR software (Stratagene). Amplification was performed in a final volume of 25 μl, containing cDNA from the reverse-transcribed reaction, primer mixture (0.3 μm each of sense and antisense primers), and 12.5 μl of 2× SYBR Green Master Mix (Applied Biosystems). The final mRNA levels of the genes studied were normalized to GAPDH expression using the comparative CT method (Stratagene). The sequences of Giardia cysteine proteases were obtained from the Giardia genome project. For the GenBank™ protein accession numbers and primers see supplemental Table S1. Microscopy—A confocal microscope (LSM510 META; Carl Zeiss MicroImaging, Inc.) equipped with multiline (458, 477, 488, and 514 nm) Ar, Diode 405 nm, 543 nm HeNe, and 633 nm HeNe visible lasers with a “Plan-Apochromat” 63×/1.40 Oil DIC oil immersion lens (Carl Zeiss MicroImaging, Inc.) was used for fluorescence imaging. Cells were pulsed with oxygen at 37 °C for 1–3 h, fixed in 3% paraformaldehyde (Electron Microscopy Sciences) for 40 min, and mounted with ProLong Gold mounting media (+ or - DAPI) (Molecular Probes). LSM Image Browser software (Carl Zeiss MicroImaging, Inc.) was used for confocal image acquisition and analysis. Adobe Photoshop CS (Adobe Systems, Inc.) was used for subsequent processing. Antibodies and Reagents—Anti-Giardia cyst wall protein polyclonal was used at 1:100 (Waterborne, Inc.) Anti-GlCP2 peptide polyclonal (raised against the peptide SSKVHLATATSYKDYGLDI) was used at 1:500. Inhibitors: phenylmethylsulfonyl fluoride (Sigma), EDTA (Sigma), aprotinin (Sigma), pepstatin A (Calbiochem), leupeptin (Sigma), TLCK (1-chloro-3-tosylamido-7-amino-2-heptanone HCl) (Sigma), TPCK (1-chloro-3-tosylamido-4-phenyl-2-butanone) (Sigma), E64 (Sigma), CA074 (Sigma), lactacystin (Sigma), α-1 antitrypsin (Sigma), calpain inhibitor I (ALLN, Sigma), calpain inhibitor II (ALLM, Sigma). R. norvegicus cathepsin C was a gift from John Pederson (Unizyme, Denmark). Mass Spectrometry—Tryptic digest sample was analyzed by liquid chromatography-mass spectrometry/mass spectrometry. Analyses were performed with an LTQ ion trap (Thermo Scientific) and a QSTAR (Applied Biosystems). The data base search was conducted using Mascot (Matrix Science Inc) on the full NCBI protein data base. Mass accuracy for the QSTAR data: 100 ppm in MS; 0.2 Da in MS/MS. Mass accuracy for the LTQ data: 3 Da in MS; 0.8 Da in MS/MS. Two-dimensional Gel Electrophoresis—Protein samples were de-salted with Centricon spin columns (Millipore). Two-dimensional gel electrophoresis was performed according to the manufacturer's specifications using the Zoom IPGRunner system (Invitrogen). Gels were silver-stained with the Silver Stain Plus kit (Bio-Rad), and protein spots were excised and trypsin-digested. There Are Twenty-seven Clan CA Cysteine Proteases in the Genome of Giardia lamblia—Prior to the completion of the Giardia genome, only four cysteine proteases from Giardia had been identified (10Ward W. Alvarado L. Rawlings N.D. Engel J.C. Franklin C. McKerrow J.H. Cell. 1997; 89: 437-444Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 11Touz M.C. Nores M.J. Slavin I. Carmona C. Conrad J.T. Mowatt M.R. Nash T.E. Coronel C.E. Lujan H.D. J. Biol. Chem. 2002; 277: 8474-8481Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). Three of these genes encode cathepsin B-like cysteine proteases, the fourth a cathepsin C-like protease. Using these papain family enzymes as a query, the Giardia genome was mined for additional genes coding for cysteine proteases. In total, twenty-seven clan CA cysteine protease genes were located in the Giardia genome, and these could be classified by sequence homology into cathepsin B-like, cathepsin C-like, or cathepsin K/L-like categories (Fig. 1). Three of these (GenBank™ accession number EAA38990) are greater than 95% identical to each other and yet are found in three discrete regions of the genome. There is also a set of four genes greater than 95% identical and assigned to the same GenBank™ accession number (AAK92150). GlCP2 Is the Most Highly Expressed Cysteine Protease of the Twenty-seven in the Giardia Genome—RT-PCR was used to determine if each of these genes was expressed in the vegetative and encysting stages of the Giardia life cycle. It was found that twenty-five of these twenty-seven genes are expressed, while no expression could be seen for the genes with GenBank™ accession numbers EAA37191 and EAA39030 (data not shown). Quantitative RT-PCR was performed to determine the relative levels of gene expression among the expressed Giardia cysteine proteases in the vegetative and encysting life stages. Expression was normalized to the expression of the housekeeping gene glutaraldehyde phosphate dehydrogenase (GAPDH), which has been found to have stable expression during the Giardia life cycle (5Hehl A.B. Marti M. Kohler P. Mol. Biol. Cell. 2000; 11: 1789-1800Crossref PubMed Scopus (84) Google Scholar, 16Mowatt M.R. Lujan H.D. Cotten D.B. Bowers B. Yee J. Nash T.E. Stibbs H.H. Mol. Microbiol. 1995; 15: 955-963Crossref PubMed Scopus (136) Google Scholar). It is notable that the cathepsin B-like cysteine proteases are more highly expressed than the cathepsin C-like or K/L-like proteases (Fig. 1). Expression of all of the cysteine protease genes was increased marginally during encystation. GlCP2 was the most highly expressed transcript in both vegetative and encysting life stages by 1.6-fold and 3.5-fold, respectively, over the next most highly expressed transcript (Giardia cysteine protease 3, with 89% homology to GlCP2). This is consistent with GlCP2 being the only cysteine protease Ward et al. (10Ward W. Alvarado L. Rawlings N.D. Engel J.C. Franklin C. McKerrow J.H. Cell. 1997; 89: 437-444Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar) biochemically identified from the parasite by affinity purification and N-terminal sequencing. The expression of this gene was also increased by 7-fold during encystation (Fig. 1). GlCP2 Is Identified in Lysate Fractions Enriched for Cysteine Protease Activity—Biochemical characterization of total cysteine protease activity found in Giardia lysates was concurrently undertaken to complement the gene expression analysis. Giardia lysates were fractionated by ion exchange chromatography, and each fraction tested against an array of N-terminally blocked fluorescent peptide substrates (data not shown). Two main peaks of cysteine protease activity were resolved against the substrates Z-FR-AMC and Z-RR-AMC (Fig. 2A). The first peak eluted (Peak A) exhibited activity against both Z-RR-AMC and Z-FR-AMC, while the second peak (Peak B) had activity against Z-FR-AMC but far less against Z-RR-AMC. The activity-containing fractions from each peak were subsequently enriched with two additional rounds of ion exchange chromatography, concentrated, probed with a labeled irreversible cysteine protease active site inhibitor and resolved by one-dimensional SDS-PAGE or two-dimensional gel electrophoresis. The active site probe labeled two discrete protein bands in Peak A and only one protein band in Peak B (Fig. 2B). Protein bands from one-dimensional SDS-PAGE or spots from two-dimensional gel electrophoresis were subjected to tryptic digest and analyzed using liquid chromatography-mass spectrometry/mass spectrometry. The only cysteine protease identified from these methods was GlCP2, of which peptides were identified in both Peak A and Peak B (Table 1). This was consistent with the observation that GlCP2 was the major cysteine protease transcript expressed by G. lamblia.TABLE 1Amino acid sequences of peptide fragments of cysteine protease activity Peak A and Peak B Sequences were identified by liquid chromatography-mass spectrometry/mass spectrometry of cysteine protease activity Peak A and Peak B eluted from anion exchange chromatography of Giardia lysates.Peak APeak BCVAGLDKCVAGLDKTGTTTDECVPYKTGTTTDECVPYKVHLATATSYKDYGLDIPAMMKGINDCSIEEQAYAGFFDENSWGPDWGEDGYFRSGSTTLRGTCPTKCADGSSK Open table in a new tab Expression and Characterization of Recombinant GlCP2—To further analyze the activity and biological role of GlCP2, a resynthesized GlCP2 (rGlCP2) gene (resynthesized to optimize for yeast codon bias) of 34 kDa was expressed heterologously in P. pastoris (Fig. 3A). The polyhistidine-tagged rGlCP2 was purified by affinity and anion exchange chromatography and was found to autoactivate during the purification process to the mature form of 28 kDa (Fig. 3A). The full-length and mature forms of rGlCP2 had activity on a 10% gelatin zymogram native gel and migrated to an apparent mobility of 60-kDa marker (supplemental Fig. S1). To compare rGlCP2 activity to that predominantly seen in Giardia lysates, the activity profile of rGlCP2 by ion exchange chromatography was examined against Z-FR-AMC and Z-RR-AMC. The two activity peaks seen in Giardia lysates were reproduced with purified recombinant protein; the peaks of activity represent the pro and mature forms of the protease in Peak A and the mature protease alone in Peak B (Fig. 4A). This is consistent with the two protein bands labeled with the cysteine protease active site probe in Peak A, and the single band labeled in Peak B (Fig. 4B). To determine if full-length rGlCP2 has activity against a peptide substrate, protein from Peak A and Peak B was fractionated by SDS-PAGE under non-reducing conditions. In-gel activity was tested against the fluorogenic substrate Z-FR-MNA. Two bands of activity could be visualized in Peak A, while only one band of endoprotease activity was resolved in Peak B (Fig. 4B). A Western blot of these fractions using an antibody against GlCP2 also demonstrates that two bands in Peak A and one in Peak B are identified as GlCP2 (Fig. 4C). These data are consistent with the biochemical and mass spectrometry evidence that GlCP2 is responsible for the activity found in both peaks A and B. Purified peak B was utilized for further biochemical studies. An array of protease inhibitors was tested for their ability to inhibit rGlCP2 activity against Z-FR-AMC and Z-RR-AMC. Leupeptin and E64 were the most effective inhibitors of rGlCP2 (Table 2). The pH profile of rGlCP2 was elucidated using the peptide substrates Z-FR-AMC and Z-RR-AMC and the macromolecular substrate casein-resorufin. The pH optimum for rGlCP2 was found to be in the neutral range for each of these substrates (Fig. 3B). This is consistent with the localization of GlCP2 in non-acidified compartments and its absence in the acidified peripheral vacuoles (PVs) (data not shown). The Km and kcat/Km of Z-FR-AMC for GlCP2 were found to be 40 μm and 17.5 μm/s, respectively. The Km and kcat/Km of Z-RR-AMC for GlCP2 were found to be 9 μm and 72 μm/sec, respectively.TABLE 2Inhibition of rGlCP2 activity against the N-terminally blocked fluorogenic peptides substrate Z-FR-AMC and Z-RR-AMC Activity is expressed as percent activity relative to a control reaction.Inhibitor% ActivityZ-FR-AMCZ-RR-AMCPMSF 1 mm11393EDTA 10 mm4740Aprotinin 10 μg/ml9331Pepstatin A 1 μm100112Pepstatin A 10 μm118126Leupeptin 1 μm114Leupeptin 10 μm01TLCK 1 μm3826TLCK 10 μm179TPCK 1 μm5016TPCK 10 μm259E64 1 μm00E64 10 μm00CA074 1 μm7799CA074 10 μm3485Lactacystin 1 μm100101Lactacystin 10 μm119114α1antitrypsin 1 μm90130α1antitrypsin 10 μm74146ALLNaALLN: N-Acetyl-Leu-Leu-Nle-CHO 1 μm510ALLN 10 μm55ALLMbALLM: N-Acetyl-Leu-Leu-Met-CHO 1 μm613ALLM 10 μm55a ALLN: N-Acetyl-Leu-Leu-Nle-CHOb ALLM: N-Acetyl-Leu-Leu-Met-CHO Open table in a new tab To characterize the substrate specificity of rGlCP2, a positional scanning synthetic combinatorial library was used to determine the substrate preference of the substrate binding sites for P1-P4 (15Choe Y. Leonetti F. Greenbaum D.C. Lecaille F. Bogyo M. Bromme D. Ellman J.A. Craik C.S. J. Biol. Chem. 2006; 281: 12824-12832Abstract Full Text Full Text PDF PubMed Scopus (304) Google Scholar) (supplemental Fig. S2). rGlCP2 displays an amino acid preference at subsites P1 and P2 (P1: K>>R, Q, P; P2: L, M, V, F) while subsites P3 and P4 have relaxed specificity. These libraries were tested both at the optimal pH for the enzyme (7.2) and at pH 5.5, the conventional pH for this class of enzymes. The substrate specificity did not change over this pH range, though the level of enzyme activity was decreased by approximately 50% at the lower pH (data not shown). Based on the substrate specificity, an ideal substrate (Z-VLK-AMC) was used to measure the Km and kcat/Km, which were found to be 19 μm and 1,473 μm/s, respectively. GlCP2 Is Found in Giardia ESVs and Can Proteolytically Process CWP2 to the Predicted Size Found in the Cyst Wall—The localization of GlCP2 during encystation was determined by episomal expression of a GlCP2-GFP fusion in Giardia. Encysting cells were probed with an antibody against cyst wall protein to highlight the ESVs, and GlCP2-GFP was found to localize to ESVs (Fig. 5). Ward et al. (10Ward W. Alvarado L. Rawlings N.D. Engel J.C. Franklin C. McKerrow J.H. Cell. 1997; 89: 437-444Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar) previously implicated GlCP2 in Giardia excystation. However, whether this protease could also play a role in the encystation process was not addressed directly. Total Giardia lysates or purified rGlCP2 was incubated with recombinant CWP2 (rCWP2). In the presence of either Giardia lysates or rGlCP2, rCWP2 was processed from its original 39-kDa size to a 26-kDa fragment, the same size of the protein found in the cyst
Referência(s)