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Trypanosoma simiae and Trypanosoma congolense: surface glycoconjugates of procyclic forms—the same coats on different hangers?
Experimental Parasitology 100 (2002) 257–268 www.academicpress.com
Trypanosoma simiae and Trypanosoma congolense: surface glycoconjugates of procyclic forms—the same coats on different hangers? N. Mookherjee1 and T.W. Pearson* Department of Biochemistry and Microbiology, Petch Building, University of Victoria, P.O. Box 3055, Victoria, BC, Canada V8W 3P6 Received 20 November 2001; received in revised form 8 April 2002; accepted 15 April 2002
Abstract Organic solvent extraction, reverse-phase high performance liquid chromatography and enzyme-linked immunosorbent assay with surface binding monoclonal antibodies were used to isolate membrane molecules of procyclic culture forms of Trypanosoma simiae and Trypanosoma congolense. Gel electrophoresis of the purified molecules revealed two predominant molecular species from each parasite that were broadly similar yet showed different apparent molecular masses and staining characteristics. The molecules were shown to be glycosylphosphatidylinositol-lipid anchored glycoconjugates, rich in carbohydrates. Each moiety displayed surface-disposed carbohydrate epitopes that were recognized on the surface of both species of trypanosomes by monoclonal antibodies specific for procyclic parasites of the subgenus Nannomonas. The epitopes were previously shown to be displayed on the glutamic acid-alanine rich protein of T. congolense yet neither this protein, nor its encoding gene is present in T. simiae. The results indicate that although T. congolense and T. simiae share common carbohydrate surface epitopes, these are displayed on biochemically different molecules. We speculate that the surface disposed carbohydrate structures are involved in parasite–tsetse interactions since these species have the same developmental cycles in the insect vector. Index Descriptors and Abbreviations: African trypanosomes; procyclics, Trypanosoma simiae, Trypanosoma congolense, BSF, bloodstream forms; VSG, variant surface glycoprotein; GPI, glycosylphosphatidylinositol; GARP, glutamic acid alanine rich protein; mAb, monoclonal antibodies; PCF, procyclic culture forms; FBS, fetal bovine serum; KMP-11, kinetoplastid membrane protein-11; ELISA, enzyme-linked immunosorbent assay; SDS–PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; CBB, Coomassie brilliant blue; HPLC, high performance liquid chromatography; PVDF, polyvinylidene difluoride; MALDI-TOF MS, matrix-assisted laser desorption ionization time-of-flight mass spectrometry. ESI, electrospray ionization. Ó 2002 Elsevier Science (USA). All rights reserved.
1. Introduction Bloodstream forms (BSF) of African trypanosomes that are ingested by the tsetse fly differentiate into procyclic forms in the tsetse fly midgut (Vickerman et al., 1988). The transformation to procyclic forms results in the loss of BSF-specific variant surface glycoprotein *
Corresponding author. Fax: +250-721-8855. E-mail addresses: [email protected] (N. Mookherjee), [email protected] (T.W. Pearson). 1 Present address: Veterinary Infectious Disease Organisation (VIDO), 120 Veterinary Road, Saskatoon, Saskatchewan, Canada S7N 5E3; Fax: +306-966-7478.
(VSG) coat and expression of other stage-specific surface molecules, the procyclins (Mowatt et al., 1989; Richardson et al., 1988, 1987; Roditi et al., 1998). The procyclins have been extensively characterized in Trypanosoma brucei (Roditi et al., 1998) and have been shown to be essential for establishment of heavy infections in the tsetse fly (Nagamune et al., 2000; Ruepp et al., 1997). There are two major types of procyclins in T. brucei: the EP procyclins with extensive glutamic acid-proline repeats and the GPEET procyclins with glycine–proline–glutamic acid–glutamic acid–threonine repeats. Both types are glycosylphosphatidylinositol (GPI)-anchored proteins or glycoproteins and are encoded by multigene families. Both EP- and GPEET-
0014-4894/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII: S 0 0 1 4 - 4 8 9 4 ( 0 2 ) 0 0 0 2 3 - 1
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procyclins have several isoforms (Roditi and Clayton, 1999) that are expressed differentially depending on the trypanosome clone and on the culture conditions in vitro and in vivo at different times post infection (Vassella et al., 2000). It has been hypothesized that the procyclin coat plays a central role in the parasite life cycle, by its involvement in cell signaling which influences parasite differentiation and survival (Acosta-Serrano et al., 2001) or cell death (Pearson et al., 2000). Molecular interactions between procyclins and other tsetse molecules in the midgut have been hypothesized to aid in migration and homing of trypanosomes to different compartments in the fly vector (Roditi and Pearson, 1990). Another major function attributed to procyclins is protection of the parasite surface from tsetse proteases (Roditi and Pearson, 1990). In this regard, it has been recently shown that the N-terminal domains of procyclins are quantitatively cleaved in the tsetse midgut by tsetse proteases, whereas the C-terminal domains of procyclins are resistant (Acosta-Serrano et al., 2001). While procyclins have been characterized from T. brucei (subgenus Trypanozoon), the only procyclin analog characterized from the members of the subgenus Nannomonas is the glutamic acid-alanine rich protein (GARP) from T. congolense (Bayne et al., 1993; Beecroft et al., 1993). The genes encoding GARP are conserved among the T. congolense Savannah, Forest and Kilifi subgroups of the parasites (Asbeck et al., 2000) and are present in multiple copies (Rangarajan et al., 2000). Although the GARP gene was not detected in at least one clone of T. simiae (Garside and Gibson, 1995), surface directed monoclonal antibodies (mAbs) specific for T. congolense procyclic forms and which recognize an epitope on GARP and on other molecules (B€ utikofer et al., 2002) also bind to the surface of T. simiae (Beecroft et al., 1993). Since metacyclic trypanosomes of the subgenus Nannomonas (T. congolense, T. simiae, and T. godfreyi) develop in the proboscis of tsetse (Mulligan, 1970) it is tempting to suggest that this tissue tropism is influenced by the surface disposed, predominant carbohydrates on the parasites. Here we report a comparative analysis of the surface molecules of procyclic T. congolense and T. simiae, parasites that have a similar developmental cycle in the tsetse vector.
2. Materials and methods 2.1. Trypanosomes BSF of T. b. brucei 427.1 (Cross and Manning, 1973) and T. congolense K45/1 (derived from T. congolense STIB744) were obtained from Dr. R. Brun (Swiss Tropical Institute, Basel, Switzerland). BSF of T. simiae CP11 (Zweygarth and R€ otcher, 1987) were obtained from E. Zweygarth, Veterinary Laboratories, Kabete,
Kenya. Procyclic culture forms (PCF) of the above trypanosomes were derived from their corresponding BSF by transformation at 27 °C as previously described (Brun and Sch€ onberger, 1979). All PCF were maintained at 27 °C in a modified minimum essential medium containing 10–20% fetal bovine serum (FBS) (Fish et al., 1989). 2.2. Biosynthetic labeling of PCF with
35
[S]-methionine
Biosynthetic labeling of methionine-containing proteins in T. simiae and T. congolense PCF was performed as previously described (Pearson et al., 1987). 2.3. High resolution 2-D gel electrophoresis Multiple 2-D gel analysis using the ISO-DALT system (Anderson and Anderson, 1979) was performed as described by Pearson et al. (1987). For each parasite lysate 2 106 cpm were loaded onto the first dimension gel. The ampholines used were Pharmacia pH 3.0–10. Gradient gels of 5–15% acrylamide were used for the second dimension. The gels were dried and exposed to Kodak Biomax MR film (Eastman Kodak Company, Rochester, NY, USA) with intensifying screens for 6 days at )80 °C. The scanned autoradiographs were converted to JPEG files using Photoshop 5.5 graphics software (Adobe Systems, San Jose, CA). The autoradiograph profiles were analyzed using 2-D gel image analysis software, Melanie 3 (GeneBio, Geneva Bioinformatics SA, Geneva, Switzerland). 2.4. Southern blot analysis To determine whether the gene encoding GARP of T. congolense was present in T. simiae CP11, in-gel Southern blot analysis was performed with low-moderate stringency, using a PCR fragment corresponding to the T. congolense GARP coding region (Bayne et al., 1993) as the probe. The probe was obtained in the plasmid pGARP-PCR (Hehl et al., 1995) and was a gift from Dr. Isabel Roditi, Bern, Switzerland. The GARP probe was amplified from the plasmid by PCR using standard universal sequencing primers. Genomic DNA from PCF of T. simiae CP11, T. congolense K45/1 and T. b. brucei 427 was isolated from lysates of the parasites using standard methods (Sambrook et al., 1989). DNA (10 lg from each parasite) was digested in an Eppendorf microcentrifuge tube overnight at 37 °C with 100 U of the restriction enzyme Bam HI (New England Biolabs, Beverly, MA, USA) followed by a further 4 h digestion with another 100 U of enzyme at 37 °C. The digested DNA was ethanol precipitated, resuspended in 30 ll of distilled water and electrophoresed for 18 h at 10 V on a 0.6% agarose gel. The gel was denatured and neutralized using standard procedures (Sambrook et al., 1989) and
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dried using a BioRad model 483 slab dryer (BioRad Laboratories, Hercules, CA, USA). Prior to probing, the gel was rehydrated and treated with hybridization buffer (0.25 M Na2 HPO4 , pH 7.2/7% SDS) for 5 min at 55 °C (Bridge et al., 1998). The gel was probed overnight at 55 °C with 25 ng of the GARP PCR fragment endlabeled with 32 P dCTP (specific activity 3000 Ci/mM) and was washed with 0.25 M Na2 HPO4 , pH 7.2/7% SDS. Radioactivity in the gel was subsequently measured using a STORM 820 storage phosphor imaging system (Molecular Dynamics, Sunnyvale, CA, USA).
of procyclins from T. brucei spp. (Ferguson et al., 1993). The HPLC fractions were collected and screened by indirect ELISA (see below) with mAb TS 126, mAb TC 491, or mAb L98/L157 mixture as primary antibodies. Immunoreactive fractions were pooled, concentrated by vacuum evaporation (EYELA vapor mix S-10, Tokyo Rikakikai Company, Tokyo, Japan), and lyophilized. The lyophilized preparations were dissolved in 500 ll of 9% propan-1-ol in water (v/v) containing 0.1% Triton X100 and were stored frozen at )20 °C until further use.
2.5. Monoclonal antibodies and antisera
2.8. Enzyme-linked immunosorbent assay
Monoclonal antibody TC 491 (IgG3 ) is specific for procyclin-like molecules on the surface of T. congolense (Beecroft et al., 1993). The mAb binds most strongly to a T. congolense protease-resistant surface molecule (B€ utikofer et al., 2002) but also binds to epitopes of GARP (Beecroft et al., 1993). MAb TS 126 (IgM) was raised against living T. simiae PCF using immunization and screening procedures as previously described for T. congolense (Beecroft et al., 1993) except that the ClonaCell-HY system (StemCell Technologies, Vancouver, BC) was used for hybridoma growth. In this procedure the cell fusion mixture was diluted and plated in a semi-solid methylcellulose matrix containing HAT selective medium and B-cell growth factors, allowing single-step selection and cloning of hybridomas. MAbs L98 and L157 are both specific for kinetoplastid membrane protein-11 (KMP-11; Tolson et al., 1998) found in all kinetoplastid parasites (Stebeck et al., 1996). A rabbit antiserum specific for proaerolysin toxin of Aeromonas hydrophila was produced as previously described (Howard and Buckley, 1985).
Indirect enzyme-linked immunosorbent assay (ELISA) was performed essentially as previously described (Richardson et al., 1986). HPLC fractions (60 ll of each of the 2.9 ml fractions) from the octyl-Sepharose column (see above) were coated onto wells of ELISA plates by drying overnight at 37 °C. Ascites fluids containing mAb TS 126, mAb TC 491, or mAb L98/L157 mixture were used as primary antibodies at a dilution of 1:1000. A 1:2500 dilution of alkaline-phosphatase-conjugated goat anti-mouse IgG/IgM (Caltag, South San Francisco, CA, USA) was used as the secondary antibody. The substrate used was p-nitrophenyl phosphate (Sigma Chemical Company, Mississauga, ON, Canada). Absorbance at 405 nm was read after 20 minutes using an automated EIA plate reader (Model EL 310, Bio-Tek Instruments, Burlington, VT, USA).
2.6. Immunofluorescence Indirect immunofluorescence was performed on suspensions of live parasites (Pearson et al., 1981). Tissue culture supernatants containing mAb TS 126 or mAb TC 491 were used at a 1:4 dilution as the source of primary antibody. Alexa 488-labeled goat anti-mouse IgG (H + L) (Molecular probes, Eugene OR, USA) was used as secondary antibody. Immunofluorescence was observed using a Zeiss Standard microscope fitted with an epifluorescence attachment and an oil-immersion 63 X Neofluor objective. Photographs were taken using a digital camera. 2.7. Antigen purification Molecules were purified from T. simiae and T. congolense PCF by reverse-phase HPLC using a semi-preparative octyl-Sepharose column (1 cm 25 cm) and a procedure that was originally developed for purification
2.9. Polyacrylamide gel electrophoresis Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS–PAGE) was performed according to Laemmli (1970) using a Mini-Protean II minigel apparatus (BioRad Laboratories, Hercules, CA, USA). Unlabeled, biotin-labeled or radiolabeled HPLC-purified molecules from T. congolense and T. simiae PCF were separated using a 3% stacking gel and a 10% resolving gel at 100 V. The gels were subsequently stained with GelCode Blue (Pierce Chemical Company, Rockford, IL, USA), with silver stain (Merril et al., 1984) or StainsAll (Sigma Chemical Company, Mississauga, ON, Canada) (Green et al., 1973), or were used for immunoblotting, autoradiography, or fluorography. Rainbow high molecular weight colored markers (14.3–220 kDa, Amersham Pharmacia Biotech, Baie d’Urfe, Quebec, Canada) were run on each gel. For mass spectrometry analysis, gels were stained with colloidal Coomassie blue (see below). 2.10. In situ oxidation and labeling of glycoproteins SDS–PAGE-separated proteins were blotted onto PVDF membranes. Blots were washed extensively with
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PBS prior to oxidation of the oligosaccharide moieties of the glycoproteins with 10 mM sodium meta-periodate in 0.1 M sodium acetate buffer, pH 5.5, for 30 min at 0 °C in the dark. Blots were incubated with 15 mM glycerol for 5 min at 0 °C to quench the oxidation reaction and then were washed with PBS and incubated with 5 mM biotin hydrazide (EZ-Link, Pierce Chemical Company, Rockford, IL, USA) for 2 h at room temperature with agitation in the dark (O’Shannessy et al., 1987). Blots were washed extensively with PBS/ 0.1% Tween-20 (3 5 min, 1 15 min) and blocked with 5% skim milk powder (DIFCO, Becton Dickinson, Sparks, MD, USA) in PBS. Biotinylated molecules were detected using a 1/ 5000 dilution of streptavidin labeled with horseradish peroxidase (Cedarlane Laboratories Ltd., Hornby, ON, Canada). Blots were developed with SuperSignal Dura enhanced chemiluminescence substrate and exposed to Kodak Biomax MR film.
Signal Dura enhanced chemiluminescence substrate and exposed to Kodak Biomax MR film. 2.13. Amino acid microanalysis Amino acid microanalysis was performed on the pooled HPLC peaks from T. congolense and T. simiae PCF. Amino acid microanalysis was also performed on SDS–PAGE separated, HPLC-purified molecules from T. simiae PCF after blotting to PVDF membrane. In this case, bands were cut from unstained membrane after immunodetection of the bands in adjacent lanes. These PVDF bands were air dried and hydrolyzed in 6 N HCl at 165 °C for 45 min under argon gas. Amino acid microanalysis was performed using an Applied Biosystems model 420 derivatizeranalyzer by the University of Victoria Tripartite Microanalytical Center. 2.14. Protein microsequencing
2.11. Biosynthetic labeling of lipid anchors PCF (5 107 in 5 ml PCF medium) were added to 5 ml of fresh PCF medium, and radiolabeled with [13 H] ethan-1-ol-2-amine hydrochloride as described. Butanol extracts were prepared, lyophilized, and the dried material was dissolved in 50 ll of 9% (v/v) propan-1-ol (B€ utikofer et al., 1997). Radiolabeled proteins were separated by SDS–PAGE (10% resolving gels), the gels were soaked in Amplify (Amersham Pharmacia Biotech), dried, and exposed to Kodak Biomax MS film (Eastman Kodak Company, Rochester, NY, USA) with intensifying screens for 7 days at )70 °C. 2.12. Detection of proaerolysin-binding proteins Proaerolysin, the inactive precursor of the channelforming protein toxin aerolysin, binds specifically and with high affinity to several GPI anchors of cell surface proteins (Nelson et al., 1997). HPLC-purified molecules from T.simiae and T. congolense were separated by SDS–PAGE and electrophoretically transferred onto polyvinylidene difluoride membranes. The membranes were blocked using 5% skim milk powder in PBS/0.1% Tween 20 and were incubated with 50 lg of proaerolysin in 25 ml of blocking buffer for 1 h at RT. A 1/10,000 dilution of rabbit anti-aerolysin polyclonal antiserum was used as the primary antibody. Proaerolysin and antiserum specific for proaerolysin were obtained from Dr. J.T. Buckley, Dept. of Biochemistry, University of Victoria, BC, Canada. Anti-rabbit IgG/IgM labeled with horseradish peroxidase (Caltag, South San Francisco, CA, USA) was used as the secondary antibody at a 1/50,000 dilution. Blots were developed with Super-
PVDF membrane bands containing SDS–PAGE separated, HPLC-purified molecules from T. simiae PCF were placed directly in a gas-phase sequencer (model 470A, Applied Biosystems, Foster City, CA) and sequence analysis was performed by the University of Victoria Tripartite Microanalytical Center. 2.15. Peptide mass mapping by mass spectrometry For mass spectrometry, gels were stained with colloidal Coomassie brilliant blue G-250 by the method of Neuhoff et al. (1988) and stained gel bands were excised and the proteins reduced, alkylated, and trypsin-cleaved in-gel as previously described (Haddow et al., 2002). Peptide masses were determined using matrix-assisted laser desorption ionization time-of flight (MALDITOF) mass spectrometry as described previously (Haddow et al., 2002). The masses of the observed peptides were submitted to MS-Fit (Protein Prospector software package; http://prospector.ucsf.edu/) and Mascot (Matrix Science; http://www.matrixscience.com/) to perform the peptide mass fingerprint searches. 2.16. Peptide sequencing by mass spectrometry Nanospray ESI was used to introduce peptide ions into a PE-SCIEX Q-STRi quadrupole time-of-flight mass spectrometer (Applied Biosystems, Foster City, CA). as described (Haddow et al., 2002). Data were managed with Bioanalyst Software version 8.7 (PESCIEX, Boston, MA). Peptide fragmentation data searching was performed using the Mascot MS/MS Ions Search algorithm (Matrix Science; London, UK: http:// www.matrixscience.com/).
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3. Results 3.1. 2-D gel analysis of somes
35
S-methionine labeled trypano-
To assess the relatedness of the two members of the subgenus Nannomonas, methionine-containing proteins of T. simiae and T. congolense procyclic culture forms were compared by 2-D gel electrophoresis. The 2-D gel autoradiograph patterns of the 35 S-methionine labeled proteins of T.simiae and T. congolense PCF are shown in Fig. 1. The gel spots were identified by the Melanie 3 software and the profiles of T. simiae and T. congolense were superimposed. The T. simiae gel spots are indicated by ‘+’ and the T. congolense gel spots are outlined. Even though several major spot constellations were identical and were used as reference constellations by the Melanie 3 software, a gel automatch function was unable to superimpose the other protein spots. The composite image reveals that
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most of the labeled proteins migrated to different positions, indicating that the majority of the T. simiae and T. congolense labeled proteins are different from each other. 3.2. Southern blot analysis To determine whether the gene encoding GARP (originally identified in T. congolense) was also present in our T. simiae clone, hybridization of the T. congolense GARP probe to Bam HI-digested genomic DNA of PCF of T. simiae, T. congolense, and T. b.brucei, 427-01 was performed. The results are shown in Fig. 2. No hybridization was seen with DNA from T. simiae (lane 1) or from T. b.brucei (lane 3) even at lowmoderate stringency conditions, whereas a single band of approximately 700 bp was detected with genomic DNA of T.congolense (lane 2). The GARP gene was thus determined not to be present in T. simiae CP11 PCF.
Fig. 1. Superimposed, composite 2-D gel autoradiograms of 35 S-methionine-labeled T. simiae and T. congolense proteins. T. simiae CP11 gel spots are indicated by ‘+’. T. congolense K45/1 gel spots are represented by outlines. The overlapping spot constellations were recognized both visually, by overlapping the autoradiograms on a light box, and by Melanie 3 two-dimensional gel analysis software. The apparent molecular masses (in kiloDaltons) are indicated on the edge of the composite image.
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ELISA profiles of the fractions from a semi-preparative octyl-Sepharose reverse-phase HPLC column are shown in Fig. 4. This profile is representative of the results obtained in more than 10 experiments. Molecules detected with mAbs specific for the surface of the parasites eluted from both T. simiae (Fig. 4A) and T. congolense (Fig. 4B) between fractions #45 and #75 (range of 38% propanol to 56% propanol). It is clear that the HPLC-eluted molecules from both species cross-reacted with both of the mAbs. The kinetoplastid membrane protein-11 (KMP11) non-surface disposed membrane molecules eluted slightly before the major immunoreactive peak from T. simiae and at the beginning of the major antigen peak from T. congolense. It was thus apparent that surface reactive membrane molecules of both parasite species could be enriched by octyl-Sepharose reversed-phase chromatography. The surface mAb-immunoreactive HPLC fractions (minus those containing KMP-11) were pooled for further analysis. 3.5. SDS–PAGE analysis of HPLC-purified molecules
Fig. 2. Southern blot analysis of Bam-HI-digested genomic DNA of procyclic trypanosomes probed with GARP DNA from T. congolense K45/1. Lane 1: T. simiae CP11. Lane 2: T. congolense K45/1. Lane 3: T. brucei 427.01. The molecular size is shown in base pairs.
3.3. Immunofluorescence The anti-T. simiae mAb TS 126 and the anti-T. congolense mAb TC 491, both selected for reactivity with their respective PCF, were tested for cross-reactivity by surface immunofluorescence on living PCF. The results are shown in Fig. 3. Strong surface fluorescence on live PCF of both T. simiae and T. congolense was seen with mAb TS 126 (panels a and b) and with mAb TC 491 (panels c and d). No fluorescence was seen with a control mAb (anti-human transferrin) on either species of PCF (panels e and f). None of the three mAbs showed any fluorescence on T. brucei PCF (not shown). Both mAbs thus detected cross-reactive surface epitopes on trypanosomes of the subgenus Nannomonas. 3.4. HPLC separation of trypanosome molecules To prepare for biochemical characterization of the surface molecules detected by the cross-reactive, surface directed monoclonal antibodies, the relevant molecules of T. simiae and T. congolense PCF were isolated by organic solvent extraction and reversed-phase HPLC.
To analyze the molecules present in the HPLC-enriched immunoreactive material from T. simiae and T. congolense, SDS–PAGE gels were run and developed with three different stains. Representative results (selected from more than 10 different experiments) are shown in Fig. 5. Staining with GelCode Blue (Fig. 5A) showed a major broad band from 60 to 66 kDa for T. simiae (lane 1). This band was not present in the HPLCpurified material from T. congolense (lane 2). However, a major band at 50–52 kDa with a less prominent band centered at 44–46 kDa was seen with the molecules purified from T. congolense (lane 2). Stains-All (Fig. 5B) revealed a broad smear from 25 to 66 kDa with the highest intensity at 60–66 kDa for T. simiae (lane 1). With T. congolense (lane 2) the entire smear was shifted to a lower molecular mass with the peak intensity centered at 50 kDa. It was clear that the darkest intensities for both T. simiae and T. congolense corresponded to the major GelCode-Blue stained bands in Fig. 5A. For mass spectroscopic analysis, staining of SDS–PAGE gels was performed using highly sensitive colloidal Coomassie brilliant blue G-250. The staining pattern was similar to that obtained with Stains-All. Sections of these broad bands were cut and processed for mass spectroscopy (see below). Silver staining of the same material (Fig. 5C) revealed a complex pattern with both dark-staining bands and clear, unstained areas. The most prominent dark bands were a rough smear from 60 to 68 kDa in T. simiae (lane 1) and one centered at 46 kDa with T. congolense (lane 2). It was interesting that very preponderant clear (unstained) areas were revealed with silver staining, between 28 and 40 kDa with T. simiae (lane 1) and slightly lower with T. congolense (lane 2). Taken together, the staining results indicate that the
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Fig. 3. Indirect immunofluorescence analysis of anti-trypanosome monoclonal antibodies on living procyclic culture forms. Panels a, c, and e: mAbs TS 126, TC 491 and anti-HT respectively, on T. simiae CP 11 PCF. Panels b, d, and f: the same mAbs on T. congolense K45/1 PCF.
HPLC-enriched molecules from T. simiae are markedly distinct from those of T. congolense. 3.6. Biotin labeling of HPLC-purified PCF molecules To examine whether the HPLC-purified molecules from PCF of T. simiae and T. congolense were glycosylated, SDS–PAGE separated molecules on blots were oxidized with sodium meta-periodate and treated with biotin hydrazide. Biotin-labeled molecules were then detected with streptavidin-horseradish peroxidase. The results are shown in Fig. 6. Two major glycosylated bands were observed in the butanol extracts of the delipidated cell pellets from both parasites. With T. simiae, there was a broad, very dark smear at approximately 20–95 kDa (lane 1). With T. congolense, a less dark smear was seen from 20 to 95 kDa. This smear showed darker bands centered at approximately 30 kDa and 60 kDa (lane 2). The results clearly show that the HPLCenriched surface molecules from both T. simiae and T. congolense contain carbohydrates.
3.7. Biosynthetic labeling of lipid anchors To study whether the reverse-phase HPLC-purified molecules from T. simiae and T.congolense PCF were GPI-anchored, the parasites were biosynthetically labeled with ½1-3 H ethan-1-ol-2-amine hydrochloride. The cell counts after an overnight incubation were between 1:0–1:25 108 cells for both T. simiae and T. congolense. This was observed in several experiments and represents a doubling of cell numbers. Butanol extracts of the delipidated pellets contained 2:9 105 cpm (T. simiae) and 1:4 105 cpm (T. congolense). These labeled molecules were separated by SDS–PAGE and the gel was fluorographed. The results are shown in Fig. 7. A broad band centered at 66 kDa and another broad band between 25 and 40 kDa was seen with T. simiae (lane 1). A similar pattern was seen with T. congolense, although the bands were shifted slightly lower (lane 2). Both parasite species thus contained ethanolamine in their GPI anchors.
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3.8. Detection of GPI-anchored HPLC-purified molecules that bind to proaerolysin SDS–PAGE-separated HPLC-purified molecules from T. simiae and T. congolense PCF were analyzed for their ability to bind to proaerolysin, a molecule which has previously been demonstrated to bind to GPI anchor structures. The results are shown in Fig. 8. No proaerolysin-binding molecules were detected in the HPLC-purified fractions from T. simiae PCF (lane 1), whereas a molecule of approximately 60 kDa was detected in T. congolense PCF (lane 2), indicating that the anchor structures of the two trypanosome species are different. 3.9. Mass spectroscopic analysis of HPLC purifiedsurface molecules
Fig. 4. ELISA profile of T. simiae CP11 and T. congolense K45/1 PCF fractions eluted from a semi-preparative octyl-Sepharose HPLC column. A: T. simiae CP11. B: T. congolense K45/1. The primary mAbs were: TS 126, solid line. TC 491, dashed line. L98/L157 mixture gray line.
Although amino acid microanalysis confirmed that there was protein present in the HPLC-purified material from both T. simiae and T.congolense (data not shown), the samples could not be sequenced by gas-phase Nterminal sequencing, possibly due to interference by an abundance of carbohydrates. Therefore, in-gel tryptic digestion of the SDS–PAGE separated, HPLC-isolated protein bands was performed to obtain peptides for peptide mass mapping and for sequencing by MALDITOF and Q-TOF mass spectrometry, respectively. Although most of the protein bands gave good MALDITOF spectra, none yielded reliable protein identification after database searching with both MS Fit (Protein
Fig. 5. SDS–PAGE analysis of reverse-phase HPLC-purified molecules from PCF of T. simiae CP11 and T. congolense K45/1. (A) GelCode Blue stain. (B) Stains-All. (C) Silver stain. Lane 1: T. simiae CP11 PCF. Lane 2: T. congolense K45/1 PCF. The apparent molecular masses are shown in kiloDaltons.
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Fig. 6. Detection of biotin-labeled carbohydrates on blotted, SDS– PAGE separated reverse-phase HPLC-purified molecules from T. simiae CP11 and T. congolense K45/1. Lane 1: T. simiae CP11 PCF. Lane 2: T. congolense K45/1 PCF. Apparent molecular masses are shown in kiloDaltons.
Fig. 8. Detection of proaerolysin-binding molecules in SDS–PAGEseparated, HPLC-purified molecules from procyclic trypanosomes. Lane 1: T. simiae CP11 PCF. Lane 2: T. congolense K45/1 PCF. Apparent molecular masses are shown in kiloDaltons.
Prospector) and Mascot (Matrix Science) search algorithms. In contrast, peptide sequencing by Q-TOF mass spectrometry of the T. congolense major 50–52 kDa upper band and a fainter, lower 44–46 kDa band from this doublet (colloidal Coomassie blue-stained bands corresponding to those shown in Fig. 5, panel A, lane 2) Table 1 Peptide sequences obtained by Tandem nanospray MS/MS analysis of tryptic peptides from HPLC-purified molecules of T. simiae
Fig. 7. Fluorograph of SDS–PAGE separated, [1-3 H] ethan-1-ol-2amine hydrochloride labeled, reverse-phase purified molecules from T. simiae CP11 and T. congolense K45/1. Lane 1: T. simiae CP11. Lane 2: T. congolense K45/1. Molecular masses are shown in kiloDaltons.
Mass
Peptide sequence
453.2 544.7 668.3 751.8 450.2 666.3 707.8 426.3 614.3 617.82 549.8
1. AQTEGPFR 2. SNQQNFHSK 3. AQLAASQNDDFR 4. QWAGTPEAEWATR 5. EAPLGLTAK 6. TFWVVEQLPGR 7. SNLDSSAVATFFR 8. LASGGGHPR 9. LPWVEPSDER 10. DTDGPFSVQLR 11. HNAFVTLAAR
Sequences 1–4 were from protein bands at 64–68 kDa, sequences 5– 7 were from protein bands at 46–48 kDa and sequences 9–11 were from proteins at 35–38 kDa, all from positions approximately as shown in Fig. 5, panel A, lane 1.
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revealed the sequence GVDVATEAAAR in each band. This sequence was identical to amino acids 94–103 of the GARP sequence. Sequencing of tryptic peptides from a range of colloidal Coomassie blue-stained protein bands (approximately 64–68, 46–48 and 35–38 kDa) from T. simiae yielded several sequences (Table 1) none of which gave any matches after searching of the NCBI non-redundant or of Swiss-Prot databases. Mass spectrometry was thus successfully used to obtain peptide sequences from the parasite surface molecules, in contrast to gasphase microsequencing which failed to detect any N-terminal sequences.
4. Discussion Trypanosoma congolense and T. simiae are members of the same subgenus (Nannomonas) yet show different host ranges. T. congolense is infective for almost all domestic animals whereas T. simiae is almost exclusively a specific parasite of pigs. Both T. congolense and T. simiae can be transmitted by diverse species of Glossina and have similar developmental cycles in the fly. Migration of procyclic forms proceeds from the midgut to the proboscis where epimastigotes form and attach to the walls of the food canal. The parasites finally invade the hypopharynx where the animal-infective metacyclic forms differentiate. In contrast, members of the subgenus Trypanozoon (T. brucei spp.) develop in the salivary glands (Mulligan, 1970). A comparison of 2-D gel profiles of T. brucei spp. and T. congolense has previously shown that the proteomes of procyclic forms of these different species are remarkably different (Anderson et al., 1985). In the current study, the two-dimensional gel electrophoresis results with 35 S-methionine-labeled parasites showed that T. congolense Kilifi clone K45/1 and T. simiae CP11 procyclic proteomes were also drastically different. This is a bit of a surprise since these two species are members of the same subgenus and have the same developmental cycle. Nevertheless, these parasites may have evolved separately over many millions of years (Overath et al., 2001) and thus exhibit quite divergent protein compositions. It is of course possible that the proteins are still essentially the same although with different post-translational modifications or with slight differences in amino acid sequences which would manifest as size and charge shifts on the 2-D profiles. Nevertheless, despite their different proteomes, T. congolense and T. simiae procyclic forms share surface-exposed epitopes detected with mAbs in immunofluorescence reactions on living parasites (Beecroft et al., 1993; and in the work reported here). To further characterize these surface molecules of T. congolense and T. simiae procyclic forms, we used organic solvent extraction and reversed phase HPLC
with two different surface binding mAbs as probes to aid in their purification. Although immunoreactive molecules from both species of parasites eluted late in the HPLC gradient, a characteristic of high hydrophobicity, the profiles were reproducibly dissimilar indicating that the surface molecules of these two species were slightly different. The SDS–PAGE profiles and staining patterns of the HPLC-purified molecules were also unique to each species and indicated that although broadly similar, the molecules from the two species are biochemically different, with the T. simiae molecules being of higher apparent molecular masses. It was interesting that the staining profiles of the molecules showed characteristics of the profiles of the major surface glycoconjugates of T. brucei spp. and T. congolense, that is, they were broad, diffuse bands that did not stain well with Coomassie blue. Two major broad bands in each species were heavily labeled by treatment with biotin hydrazide, indicating that they contained large amounts of carbohydrate. Interestingly, in both species, both bands could be biosynthetically labeled with 3 H-ethanolamine, indicating that they contained terminal ethanolamines that are probably involved in attachment of polypeptides to lipid anchors. Though non-protein linked GPI anchors such as free GPI lipids and lipophosphoglycans have been described in protozoan parasites, these structures lack ethanolamine. The anchor structures may be different between T. simiae and T. congolense as only one of the bands (from T. congolense) bound to proaerolysin toxin. This toxin is known to bind specifically to GPI anchors. However, modifications of the core structure of GPIanchors by addition of sugars or phosphoethanolamine, or by acylation of inositol result in the loss of the binding of proaerolysin (Nelson et al., 1997), perhaps explaining the differential binding to anchors of the two trypanosome species. Amino acid analysis of pooled HPLC peaks revealed much higher levels of amino acids in the T. congolense fraction when compared to T. simiae. Amino acid analysis of the two bands from T. simiae after blotting of the SDS–PAGE separated HPLC fractions onto PVDF membrane, however, did show low levels of amino acids in each band. Several attempts at gas-phase N-terminal sequencing of each of these bands failed. The high abundance of carbohydrates may explain the low yields of amino acids and difficulty in sequencing although simple N-terminal blockage could explain the latter. Mass spectroscopic analysis of HPLC-separated proteins, detected by sensitive colloidal Coomassie brilliant blue staining, showed that the two major bands in T. congolense contained GARP, as expected, whereas no peptides matching GARP could be found in T. simiae. This result supports the observation that GARP genes were not detected by Southern blotting in the T. simiae CP 11 clone (this paper) and that the GARP protein could not be detected in immunoblotting experiments
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using an antiserum specific for GARP polypeptide (Mookherjee and Pearson, 2001). Sequencing of several peptides from T. simiae by Q-TOF mass spectrometry revealed several unique sequences, none of which matched any sequence in public databases. Taken together, the data suggest that the major HPLC-separated bands of T. simiae represent unique glycoproteins and that they share carbohydrate epitopes with each other and with GARP of T. congolense and that the T. simiae polypeptide sequences are different than the T. congolense-derived GARP sequence. Of relevance here is that novel non-GARP, carbohydrate-containing, GPI-anchored surface molecules have recently been detected in T. congolense (B€ utikofer et al., 2002). Interestingly, these authors also encountered great difficulty obtaining any protein sequences from the non-GARP glycoconjugates. This last observation is interesting and although a cause of much frustration in characterizing the surface procyclins of both T. congolense (and T. simiae; this paper), clearly indicates that an approach using carbohydrate microchemistry will be required for structure–function analysis of these molecules. To summarize, we have shown that several surfacedisposed, GPI-anchored molecules are present in both T. congolense and T. simiae and that these are glycoconjugates. Although these membrane molecules are immunologically similar, there are biochemical differences between them as reflected in their different HPLC elution and gel staining profiles, the absence of GARP in T. simiae and differential binding of proaerolysin toxin. It seems plausible that the carbohydrate epitopes shared between these two species of the subgenus Nannomonas may be involved in interactions with the tsetse vector since they both appear to have the same developmental cycle. If this is the case, then it appears that T. congolense and T. simiae, despite their widely different proteomes, have placed common carbohydrate structures on different carriers as a way to achieve the same function.
Acknowledgments We thank Dr. Isabel Roditi for the GARP probe, Dr. Peter B€ utik€ ofer Dr. Robert Burke, Dr. R.W. Olafson, and Jody Haddow and for technical advice, Dr. Tom Buckley for proaerolysin toxin and antiserum, Sandy Kielland for performing amino acid analyses, Lee Rafuse Haines for performing immunofluorescence microscopy and for reading the manuscript and helpful suggestions, Heather Down for help with preparation of the figures, and Morag Booy and Jennifer Chase for excellent technical help. N.M. was the recipient of a University of Victoria Student Fellowship. This work was supported by a Research Grant from the Natural Sciences and Engineering Council of Canada (NSERC).
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Rangarajan, D., Harvey, T.I., Barry, J.D., 2000. Characterisation of the loci encoding the glutamic acid and alanine rich protein of Trypanosoma congolense. Mol. Biochem. Parasitol. 105, 281–290. Richardson, J.P., Jenni, L., Beecroft, R.P., Pearson, T.W., 1986. Procyclic tsetse fly midgut forms and culture forms of African trypanosomes share stage- and species-specific surface antigens identified by monoclonal antibodies. J. Immunol. 136, 2259–2264. Richardson, J.P., Beecroft, R.P., Tolson, D.L., Liu, M.K., Pearson, T.W., 1988. Procyclin: an unusual immunodominant glycoprotein surface antigen from the procyclic stage of African trypanosomes. Mol. Biochem. Parasitol. 31, 203–216. Roditi, I., Carrington, M., Turner, M., 1987. Expression of a polypeptide containing a dipeptide repeat is confined to the insect stage of Trypanosoma brucei. Nature 325, 272–274. Roditi, I., Pearson, T.W., 1990. The procyclin coat of African trypanosomes (or the not-so-naked trypanosome). Parasitol. Today 6, 79–81. Roditi, I., Furger, A., Ruepp, S., Schurch, N., Butikofer, P., 1998. Unravelling the procyclin coat of Trypanosoma brucei. Mol. Biochem. Parasitol. 91, 117–130. Roditi, I., Clayton, C.E., 1999. An unambiguous nomenclature for the major surface glycoproteins of the procyclic form of Trypanosoma brucei. Mol. Biochem. Parasitol. 103, 99–100. Ruepp, S., Furger, A., Kurath, U., Renggli, C.K., Hemphill, A., Brun, R., Roditi, I., 1997. Survival of Trypanosoma brucei in the tsetse fly is enhanced by the expression of specific forms of procyclin. J. Cell. Biol. 137, 1369–1379. Sambrook, J., Fritsch, E.F., Maniatis, T., 1989. Molecular Cloning: A Lab Manual, second ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Stebeck, C.E., Baron, G.S., Beecroft, R.P., Pearson, T.W., 1996. Molecular characterization of the kinetoplastid membrane protein11 from African trypanosomes. Mol. Biochem. Parasitol. 81, 81–88. Tolson, D.L., Turco, S.J., Beecroft, R.P., Pearson, T.W., 1998. The immunochemical structure and surface arrangement of Leishmania donovani lipophosphoglycan determined using monoclonal antibodies. Mol. Biochem. Parasitol. 35, 109–118. Vassella, E., Den Abbeele, J.V., Butikofer, P., Renggli, C.K., Furger, A., Brun, R., Roditi, I., 2000. A major surface glycoprotein of Trypanosoma brucei is expressed transiently during development and can be regulated post-transcriptionally by glycerol or hypoxia. Genes Dev. 14, 615–626. Vickerman, K., Tetley, L., Hendry, K.A., Turner, C.M., 1988. Biology of African trypanosomes in the tsetse fly. Biol. Cell 64, 109–119. Zweygarth, E., R€ otcher, D., 1987. The occurence of Trypanosoma (Nannomonas) simiae in the cerebrospinal fluid of domestic pigs. Parasitol. Res. 73, 479–480.
Trypanosoma simiae and Trypanosoma congolense: surface glycoconjugates of procyclic forms—the same coats on different hangers? N. Mookherjee1 and T.W. Pearson* Department of Biochemistry and Microbiology, Petch Building, University of Victoria, P.O. Box 3055, Victoria, BC, Canada V8W 3P6 Received 20 November 2001; received in revised form 8 April 2002; accepted 15 April 2002
Abstract Organic solvent extraction, reverse-phase high performance liquid chromatography and enzyme-linked immunosorbent assay with surface binding monoclonal antibodies were used to isolate membrane molecules of procyclic culture forms of Trypanosoma simiae and Trypanosoma congolense. Gel electrophoresis of the purified molecules revealed two predominant molecular species from each parasite that were broadly similar yet showed different apparent molecular masses and staining characteristics. The molecules were shown to be glycosylphosphatidylinositol-lipid anchored glycoconjugates, rich in carbohydrates. Each moiety displayed surface-disposed carbohydrate epitopes that were recognized on the surface of both species of trypanosomes by monoclonal antibodies specific for procyclic parasites of the subgenus Nannomonas. The epitopes were previously shown to be displayed on the glutamic acid-alanine rich protein of T. congolense yet neither this protein, nor its encoding gene is present in T. simiae. The results indicate that although T. congolense and T. simiae share common carbohydrate surface epitopes, these are displayed on biochemically different molecules. We speculate that the surface disposed carbohydrate structures are involved in parasite–tsetse interactions since these species have the same developmental cycles in the insect vector. Index Descriptors and Abbreviations: African trypanosomes; procyclics, Trypanosoma simiae, Trypanosoma congolense, BSF, bloodstream forms; VSG, variant surface glycoprotein; GPI, glycosylphosphatidylinositol; GARP, glutamic acid alanine rich protein; mAb, monoclonal antibodies; PCF, procyclic culture forms; FBS, fetal bovine serum; KMP-11, kinetoplastid membrane protein-11; ELISA, enzyme-linked immunosorbent assay; SDS–PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; CBB, Coomassie brilliant blue; HPLC, high performance liquid chromatography; PVDF, polyvinylidene difluoride; MALDI-TOF MS, matrix-assisted laser desorption ionization time-of-flight mass spectrometry. ESI, electrospray ionization. Ó 2002 Elsevier Science (USA). All rights reserved.
1. Introduction Bloodstream forms (BSF) of African trypanosomes that are ingested by the tsetse fly differentiate into procyclic forms in the tsetse fly midgut (Vickerman et al., 1988). The transformation to procyclic forms results in the loss of BSF-specific variant surface glycoprotein *
Corresponding author. Fax: +250-721-8855. E-mail addresses: [email protected] (N. Mookherjee), [email protected] (T.W. Pearson). 1 Present address: Veterinary Infectious Disease Organisation (VIDO), 120 Veterinary Road, Saskatoon, Saskatchewan, Canada S7N 5E3; Fax: +306-966-7478.
(VSG) coat and expression of other stage-specific surface molecules, the procyclins (Mowatt et al., 1989; Richardson et al., 1988, 1987; Roditi et al., 1998). The procyclins have been extensively characterized in Trypanosoma brucei (Roditi et al., 1998) and have been shown to be essential for establishment of heavy infections in the tsetse fly (Nagamune et al., 2000; Ruepp et al., 1997). There are two major types of procyclins in T. brucei: the EP procyclins with extensive glutamic acid-proline repeats and the GPEET procyclins with glycine–proline–glutamic acid–glutamic acid–threonine repeats. Both types are glycosylphosphatidylinositol (GPI)-anchored proteins or glycoproteins and are encoded by multigene families. Both EP- and GPEET-
0014-4894/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII: S 0 0 1 4 - 4 8 9 4 ( 0 2 ) 0 0 0 2 3 - 1
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procyclins have several isoforms (Roditi and Clayton, 1999) that are expressed differentially depending on the trypanosome clone and on the culture conditions in vitro and in vivo at different times post infection (Vassella et al., 2000). It has been hypothesized that the procyclin coat plays a central role in the parasite life cycle, by its involvement in cell signaling which influences parasite differentiation and survival (Acosta-Serrano et al., 2001) or cell death (Pearson et al., 2000). Molecular interactions between procyclins and other tsetse molecules in the midgut have been hypothesized to aid in migration and homing of trypanosomes to different compartments in the fly vector (Roditi and Pearson, 1990). Another major function attributed to procyclins is protection of the parasite surface from tsetse proteases (Roditi and Pearson, 1990). In this regard, it has been recently shown that the N-terminal domains of procyclins are quantitatively cleaved in the tsetse midgut by tsetse proteases, whereas the C-terminal domains of procyclins are resistant (Acosta-Serrano et al., 2001). While procyclins have been characterized from T. brucei (subgenus Trypanozoon), the only procyclin analog characterized from the members of the subgenus Nannomonas is the glutamic acid-alanine rich protein (GARP) from T. congolense (Bayne et al., 1993; Beecroft et al., 1993). The genes encoding GARP are conserved among the T. congolense Savannah, Forest and Kilifi subgroups of the parasites (Asbeck et al., 2000) and are present in multiple copies (Rangarajan et al., 2000). Although the GARP gene was not detected in at least one clone of T. simiae (Garside and Gibson, 1995), surface directed monoclonal antibodies (mAbs) specific for T. congolense procyclic forms and which recognize an epitope on GARP and on other molecules (B€ utikofer et al., 2002) also bind to the surface of T. simiae (Beecroft et al., 1993). Since metacyclic trypanosomes of the subgenus Nannomonas (T. congolense, T. simiae, and T. godfreyi) develop in the proboscis of tsetse (Mulligan, 1970) it is tempting to suggest that this tissue tropism is influenced by the surface disposed, predominant carbohydrates on the parasites. Here we report a comparative analysis of the surface molecules of procyclic T. congolense and T. simiae, parasites that have a similar developmental cycle in the tsetse vector.
2. Materials and methods 2.1. Trypanosomes BSF of T. b. brucei 427.1 (Cross and Manning, 1973) and T. congolense K45/1 (derived from T. congolense STIB744) were obtained from Dr. R. Brun (Swiss Tropical Institute, Basel, Switzerland). BSF of T. simiae CP11 (Zweygarth and R€ otcher, 1987) were obtained from E. Zweygarth, Veterinary Laboratories, Kabete,
Kenya. Procyclic culture forms (PCF) of the above trypanosomes were derived from their corresponding BSF by transformation at 27 °C as previously described (Brun and Sch€ onberger, 1979). All PCF were maintained at 27 °C in a modified minimum essential medium containing 10–20% fetal bovine serum (FBS) (Fish et al., 1989). 2.2. Biosynthetic labeling of PCF with
35
[S]-methionine
Biosynthetic labeling of methionine-containing proteins in T. simiae and T. congolense PCF was performed as previously described (Pearson et al., 1987). 2.3. High resolution 2-D gel electrophoresis Multiple 2-D gel analysis using the ISO-DALT system (Anderson and Anderson, 1979) was performed as described by Pearson et al. (1987). For each parasite lysate 2 106 cpm were loaded onto the first dimension gel. The ampholines used were Pharmacia pH 3.0–10. Gradient gels of 5–15% acrylamide were used for the second dimension. The gels were dried and exposed to Kodak Biomax MR film (Eastman Kodak Company, Rochester, NY, USA) with intensifying screens for 6 days at )80 °C. The scanned autoradiographs were converted to JPEG files using Photoshop 5.5 graphics software (Adobe Systems, San Jose, CA). The autoradiograph profiles were analyzed using 2-D gel image analysis software, Melanie 3 (GeneBio, Geneva Bioinformatics SA, Geneva, Switzerland). 2.4. Southern blot analysis To determine whether the gene encoding GARP of T. congolense was present in T. simiae CP11, in-gel Southern blot analysis was performed with low-moderate stringency, using a PCR fragment corresponding to the T. congolense GARP coding region (Bayne et al., 1993) as the probe. The probe was obtained in the plasmid pGARP-PCR (Hehl et al., 1995) and was a gift from Dr. Isabel Roditi, Bern, Switzerland. The GARP probe was amplified from the plasmid by PCR using standard universal sequencing primers. Genomic DNA from PCF of T. simiae CP11, T. congolense K45/1 and T. b. brucei 427 was isolated from lysates of the parasites using standard methods (Sambrook et al., 1989). DNA (10 lg from each parasite) was digested in an Eppendorf microcentrifuge tube overnight at 37 °C with 100 U of the restriction enzyme Bam HI (New England Biolabs, Beverly, MA, USA) followed by a further 4 h digestion with another 100 U of enzyme at 37 °C. The digested DNA was ethanol precipitated, resuspended in 30 ll of distilled water and electrophoresed for 18 h at 10 V on a 0.6% agarose gel. The gel was denatured and neutralized using standard procedures (Sambrook et al., 1989) and
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259
dried using a BioRad model 483 slab dryer (BioRad Laboratories, Hercules, CA, USA). Prior to probing, the gel was rehydrated and treated with hybridization buffer (0.25 M Na2 HPO4 , pH 7.2/7% SDS) for 5 min at 55 °C (Bridge et al., 1998). The gel was probed overnight at 55 °C with 25 ng of the GARP PCR fragment endlabeled with 32 P dCTP (specific activity 3000 Ci/mM) and was washed with 0.25 M Na2 HPO4 , pH 7.2/7% SDS. Radioactivity in the gel was subsequently measured using a STORM 820 storage phosphor imaging system (Molecular Dynamics, Sunnyvale, CA, USA).
of procyclins from T. brucei spp. (Ferguson et al., 1993). The HPLC fractions were collected and screened by indirect ELISA (see below) with mAb TS 126, mAb TC 491, or mAb L98/L157 mixture as primary antibodies. Immunoreactive fractions were pooled, concentrated by vacuum evaporation (EYELA vapor mix S-10, Tokyo Rikakikai Company, Tokyo, Japan), and lyophilized. The lyophilized preparations were dissolved in 500 ll of 9% propan-1-ol in water (v/v) containing 0.1% Triton X100 and were stored frozen at )20 °C until further use.
2.5. Monoclonal antibodies and antisera
2.8. Enzyme-linked immunosorbent assay
Monoclonal antibody TC 491 (IgG3 ) is specific for procyclin-like molecules on the surface of T. congolense (Beecroft et al., 1993). The mAb binds most strongly to a T. congolense protease-resistant surface molecule (B€ utikofer et al., 2002) but also binds to epitopes of GARP (Beecroft et al., 1993). MAb TS 126 (IgM) was raised against living T. simiae PCF using immunization and screening procedures as previously described for T. congolense (Beecroft et al., 1993) except that the ClonaCell-HY system (StemCell Technologies, Vancouver, BC) was used for hybridoma growth. In this procedure the cell fusion mixture was diluted and plated in a semi-solid methylcellulose matrix containing HAT selective medium and B-cell growth factors, allowing single-step selection and cloning of hybridomas. MAbs L98 and L157 are both specific for kinetoplastid membrane protein-11 (KMP-11; Tolson et al., 1998) found in all kinetoplastid parasites (Stebeck et al., 1996). A rabbit antiserum specific for proaerolysin toxin of Aeromonas hydrophila was produced as previously described (Howard and Buckley, 1985).
Indirect enzyme-linked immunosorbent assay (ELISA) was performed essentially as previously described (Richardson et al., 1986). HPLC fractions (60 ll of each of the 2.9 ml fractions) from the octyl-Sepharose column (see above) were coated onto wells of ELISA plates by drying overnight at 37 °C. Ascites fluids containing mAb TS 126, mAb TC 491, or mAb L98/L157 mixture were used as primary antibodies at a dilution of 1:1000. A 1:2500 dilution of alkaline-phosphatase-conjugated goat anti-mouse IgG/IgM (Caltag, South San Francisco, CA, USA) was used as the secondary antibody. The substrate used was p-nitrophenyl phosphate (Sigma Chemical Company, Mississauga, ON, Canada). Absorbance at 405 nm was read after 20 minutes using an automated EIA plate reader (Model EL 310, Bio-Tek Instruments, Burlington, VT, USA).
2.6. Immunofluorescence Indirect immunofluorescence was performed on suspensions of live parasites (Pearson et al., 1981). Tissue culture supernatants containing mAb TS 126 or mAb TC 491 were used at a 1:4 dilution as the source of primary antibody. Alexa 488-labeled goat anti-mouse IgG (H + L) (Molecular probes, Eugene OR, USA) was used as secondary antibody. Immunofluorescence was observed using a Zeiss Standard microscope fitted with an epifluorescence attachment and an oil-immersion 63 X Neofluor objective. Photographs were taken using a digital camera. 2.7. Antigen purification Molecules were purified from T. simiae and T. congolense PCF by reverse-phase HPLC using a semi-preparative octyl-Sepharose column (1 cm 25 cm) and a procedure that was originally developed for purification
2.9. Polyacrylamide gel electrophoresis Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS–PAGE) was performed according to Laemmli (1970) using a Mini-Protean II minigel apparatus (BioRad Laboratories, Hercules, CA, USA). Unlabeled, biotin-labeled or radiolabeled HPLC-purified molecules from T. congolense and T. simiae PCF were separated using a 3% stacking gel and a 10% resolving gel at 100 V. The gels were subsequently stained with GelCode Blue (Pierce Chemical Company, Rockford, IL, USA), with silver stain (Merril et al., 1984) or StainsAll (Sigma Chemical Company, Mississauga, ON, Canada) (Green et al., 1973), or were used for immunoblotting, autoradiography, or fluorography. Rainbow high molecular weight colored markers (14.3–220 kDa, Amersham Pharmacia Biotech, Baie d’Urfe, Quebec, Canada) were run on each gel. For mass spectrometry analysis, gels were stained with colloidal Coomassie blue (see below). 2.10. In situ oxidation and labeling of glycoproteins SDS–PAGE-separated proteins were blotted onto PVDF membranes. Blots were washed extensively with
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PBS prior to oxidation of the oligosaccharide moieties of the glycoproteins with 10 mM sodium meta-periodate in 0.1 M sodium acetate buffer, pH 5.5, for 30 min at 0 °C in the dark. Blots were incubated with 15 mM glycerol for 5 min at 0 °C to quench the oxidation reaction and then were washed with PBS and incubated with 5 mM biotin hydrazide (EZ-Link, Pierce Chemical Company, Rockford, IL, USA) for 2 h at room temperature with agitation in the dark (O’Shannessy et al., 1987). Blots were washed extensively with PBS/ 0.1% Tween-20 (3 5 min, 1 15 min) and blocked with 5% skim milk powder (DIFCO, Becton Dickinson, Sparks, MD, USA) in PBS. Biotinylated molecules were detected using a 1/ 5000 dilution of streptavidin labeled with horseradish peroxidase (Cedarlane Laboratories Ltd., Hornby, ON, Canada). Blots were developed with SuperSignal Dura enhanced chemiluminescence substrate and exposed to Kodak Biomax MR film.
Signal Dura enhanced chemiluminescence substrate and exposed to Kodak Biomax MR film. 2.13. Amino acid microanalysis Amino acid microanalysis was performed on the pooled HPLC peaks from T. congolense and T. simiae PCF. Amino acid microanalysis was also performed on SDS–PAGE separated, HPLC-purified molecules from T. simiae PCF after blotting to PVDF membrane. In this case, bands were cut from unstained membrane after immunodetection of the bands in adjacent lanes. These PVDF bands were air dried and hydrolyzed in 6 N HCl at 165 °C for 45 min under argon gas. Amino acid microanalysis was performed using an Applied Biosystems model 420 derivatizeranalyzer by the University of Victoria Tripartite Microanalytical Center. 2.14. Protein microsequencing
2.11. Biosynthetic labeling of lipid anchors PCF (5 107 in 5 ml PCF medium) were added to 5 ml of fresh PCF medium, and radiolabeled with [13 H] ethan-1-ol-2-amine hydrochloride as described. Butanol extracts were prepared, lyophilized, and the dried material was dissolved in 50 ll of 9% (v/v) propan-1-ol (B€ utikofer et al., 1997). Radiolabeled proteins were separated by SDS–PAGE (10% resolving gels), the gels were soaked in Amplify (Amersham Pharmacia Biotech), dried, and exposed to Kodak Biomax MS film (Eastman Kodak Company, Rochester, NY, USA) with intensifying screens for 7 days at )70 °C. 2.12. Detection of proaerolysin-binding proteins Proaerolysin, the inactive precursor of the channelforming protein toxin aerolysin, binds specifically and with high affinity to several GPI anchors of cell surface proteins (Nelson et al., 1997). HPLC-purified molecules from T.simiae and T. congolense were separated by SDS–PAGE and electrophoretically transferred onto polyvinylidene difluoride membranes. The membranes were blocked using 5% skim milk powder in PBS/0.1% Tween 20 and were incubated with 50 lg of proaerolysin in 25 ml of blocking buffer for 1 h at RT. A 1/10,000 dilution of rabbit anti-aerolysin polyclonal antiserum was used as the primary antibody. Proaerolysin and antiserum specific for proaerolysin were obtained from Dr. J.T. Buckley, Dept. of Biochemistry, University of Victoria, BC, Canada. Anti-rabbit IgG/IgM labeled with horseradish peroxidase (Caltag, South San Francisco, CA, USA) was used as the secondary antibody at a 1/50,000 dilution. Blots were developed with Super-
PVDF membrane bands containing SDS–PAGE separated, HPLC-purified molecules from T. simiae PCF were placed directly in a gas-phase sequencer (model 470A, Applied Biosystems, Foster City, CA) and sequence analysis was performed by the University of Victoria Tripartite Microanalytical Center. 2.15. Peptide mass mapping by mass spectrometry For mass spectrometry, gels were stained with colloidal Coomassie brilliant blue G-250 by the method of Neuhoff et al. (1988) and stained gel bands were excised and the proteins reduced, alkylated, and trypsin-cleaved in-gel as previously described (Haddow et al., 2002). Peptide masses were determined using matrix-assisted laser desorption ionization time-of flight (MALDITOF) mass spectrometry as described previously (Haddow et al., 2002). The masses of the observed peptides were submitted to MS-Fit (Protein Prospector software package; http://prospector.ucsf.edu/) and Mascot (Matrix Science; http://www.matrixscience.com/) to perform the peptide mass fingerprint searches. 2.16. Peptide sequencing by mass spectrometry Nanospray ESI was used to introduce peptide ions into a PE-SCIEX Q-STRi quadrupole time-of-flight mass spectrometer (Applied Biosystems, Foster City, CA). as described (Haddow et al., 2002). Data were managed with Bioanalyst Software version 8.7 (PESCIEX, Boston, MA). Peptide fragmentation data searching was performed using the Mascot MS/MS Ions Search algorithm (Matrix Science; London, UK: http:// www.matrixscience.com/).
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3. Results 3.1. 2-D gel analysis of somes
35
S-methionine labeled trypano-
To assess the relatedness of the two members of the subgenus Nannomonas, methionine-containing proteins of T. simiae and T. congolense procyclic culture forms were compared by 2-D gel electrophoresis. The 2-D gel autoradiograph patterns of the 35 S-methionine labeled proteins of T.simiae and T. congolense PCF are shown in Fig. 1. The gel spots were identified by the Melanie 3 software and the profiles of T. simiae and T. congolense were superimposed. The T. simiae gel spots are indicated by ‘+’ and the T. congolense gel spots are outlined. Even though several major spot constellations were identical and were used as reference constellations by the Melanie 3 software, a gel automatch function was unable to superimpose the other protein spots. The composite image reveals that
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most of the labeled proteins migrated to different positions, indicating that the majority of the T. simiae and T. congolense labeled proteins are different from each other. 3.2. Southern blot analysis To determine whether the gene encoding GARP (originally identified in T. congolense) was also present in our T. simiae clone, hybridization of the T. congolense GARP probe to Bam HI-digested genomic DNA of PCF of T. simiae, T. congolense, and T. b.brucei, 427-01 was performed. The results are shown in Fig. 2. No hybridization was seen with DNA from T. simiae (lane 1) or from T. b.brucei (lane 3) even at lowmoderate stringency conditions, whereas a single band of approximately 700 bp was detected with genomic DNA of T.congolense (lane 2). The GARP gene was thus determined not to be present in T. simiae CP11 PCF.
Fig. 1. Superimposed, composite 2-D gel autoradiograms of 35 S-methionine-labeled T. simiae and T. congolense proteins. T. simiae CP11 gel spots are indicated by ‘+’. T. congolense K45/1 gel spots are represented by outlines. The overlapping spot constellations were recognized both visually, by overlapping the autoradiograms on a light box, and by Melanie 3 two-dimensional gel analysis software. The apparent molecular masses (in kiloDaltons) are indicated on the edge of the composite image.
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ELISA profiles of the fractions from a semi-preparative octyl-Sepharose reverse-phase HPLC column are shown in Fig. 4. This profile is representative of the results obtained in more than 10 experiments. Molecules detected with mAbs specific for the surface of the parasites eluted from both T. simiae (Fig. 4A) and T. congolense (Fig. 4B) between fractions #45 and #75 (range of 38% propanol to 56% propanol). It is clear that the HPLC-eluted molecules from both species cross-reacted with both of the mAbs. The kinetoplastid membrane protein-11 (KMP11) non-surface disposed membrane molecules eluted slightly before the major immunoreactive peak from T. simiae and at the beginning of the major antigen peak from T. congolense. It was thus apparent that surface reactive membrane molecules of both parasite species could be enriched by octyl-Sepharose reversed-phase chromatography. The surface mAb-immunoreactive HPLC fractions (minus those containing KMP-11) were pooled for further analysis. 3.5. SDS–PAGE analysis of HPLC-purified molecules
Fig. 2. Southern blot analysis of Bam-HI-digested genomic DNA of procyclic trypanosomes probed with GARP DNA from T. congolense K45/1. Lane 1: T. simiae CP11. Lane 2: T. congolense K45/1. Lane 3: T. brucei 427.01. The molecular size is shown in base pairs.
3.3. Immunofluorescence The anti-T. simiae mAb TS 126 and the anti-T. congolense mAb TC 491, both selected for reactivity with their respective PCF, were tested for cross-reactivity by surface immunofluorescence on living PCF. The results are shown in Fig. 3. Strong surface fluorescence on live PCF of both T. simiae and T. congolense was seen with mAb TS 126 (panels a and b) and with mAb TC 491 (panels c and d). No fluorescence was seen with a control mAb (anti-human transferrin) on either species of PCF (panels e and f). None of the three mAbs showed any fluorescence on T. brucei PCF (not shown). Both mAbs thus detected cross-reactive surface epitopes on trypanosomes of the subgenus Nannomonas. 3.4. HPLC separation of trypanosome molecules To prepare for biochemical characterization of the surface molecules detected by the cross-reactive, surface directed monoclonal antibodies, the relevant molecules of T. simiae and T. congolense PCF were isolated by organic solvent extraction and reversed-phase HPLC.
To analyze the molecules present in the HPLC-enriched immunoreactive material from T. simiae and T. congolense, SDS–PAGE gels were run and developed with three different stains. Representative results (selected from more than 10 different experiments) are shown in Fig. 5. Staining with GelCode Blue (Fig. 5A) showed a major broad band from 60 to 66 kDa for T. simiae (lane 1). This band was not present in the HPLCpurified material from T. congolense (lane 2). However, a major band at 50–52 kDa with a less prominent band centered at 44–46 kDa was seen with the molecules purified from T. congolense (lane 2). Stains-All (Fig. 5B) revealed a broad smear from 25 to 66 kDa with the highest intensity at 60–66 kDa for T. simiae (lane 1). With T. congolense (lane 2) the entire smear was shifted to a lower molecular mass with the peak intensity centered at 50 kDa. It was clear that the darkest intensities for both T. simiae and T. congolense corresponded to the major GelCode-Blue stained bands in Fig. 5A. For mass spectroscopic analysis, staining of SDS–PAGE gels was performed using highly sensitive colloidal Coomassie brilliant blue G-250. The staining pattern was similar to that obtained with Stains-All. Sections of these broad bands were cut and processed for mass spectroscopy (see below). Silver staining of the same material (Fig. 5C) revealed a complex pattern with both dark-staining bands and clear, unstained areas. The most prominent dark bands were a rough smear from 60 to 68 kDa in T. simiae (lane 1) and one centered at 46 kDa with T. congolense (lane 2). It was interesting that very preponderant clear (unstained) areas were revealed with silver staining, between 28 and 40 kDa with T. simiae (lane 1) and slightly lower with T. congolense (lane 2). Taken together, the staining results indicate that the
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Fig. 3. Indirect immunofluorescence analysis of anti-trypanosome monoclonal antibodies on living procyclic culture forms. Panels a, c, and e: mAbs TS 126, TC 491 and anti-HT respectively, on T. simiae CP 11 PCF. Panels b, d, and f: the same mAbs on T. congolense K45/1 PCF.
HPLC-enriched molecules from T. simiae are markedly distinct from those of T. congolense. 3.6. Biotin labeling of HPLC-purified PCF molecules To examine whether the HPLC-purified molecules from PCF of T. simiae and T. congolense were glycosylated, SDS–PAGE separated molecules on blots were oxidized with sodium meta-periodate and treated with biotin hydrazide. Biotin-labeled molecules were then detected with streptavidin-horseradish peroxidase. The results are shown in Fig. 6. Two major glycosylated bands were observed in the butanol extracts of the delipidated cell pellets from both parasites. With T. simiae, there was a broad, very dark smear at approximately 20–95 kDa (lane 1). With T. congolense, a less dark smear was seen from 20 to 95 kDa. This smear showed darker bands centered at approximately 30 kDa and 60 kDa (lane 2). The results clearly show that the HPLCenriched surface molecules from both T. simiae and T. congolense contain carbohydrates.
3.7. Biosynthetic labeling of lipid anchors To study whether the reverse-phase HPLC-purified molecules from T. simiae and T.congolense PCF were GPI-anchored, the parasites were biosynthetically labeled with ½1-3 H ethan-1-ol-2-amine hydrochloride. The cell counts after an overnight incubation were between 1:0–1:25 108 cells for both T. simiae and T. congolense. This was observed in several experiments and represents a doubling of cell numbers. Butanol extracts of the delipidated pellets contained 2:9 105 cpm (T. simiae) and 1:4 105 cpm (T. congolense). These labeled molecules were separated by SDS–PAGE and the gel was fluorographed. The results are shown in Fig. 7. A broad band centered at 66 kDa and another broad band between 25 and 40 kDa was seen with T. simiae (lane 1). A similar pattern was seen with T. congolense, although the bands were shifted slightly lower (lane 2). Both parasite species thus contained ethanolamine in their GPI anchors.
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3.8. Detection of GPI-anchored HPLC-purified molecules that bind to proaerolysin SDS–PAGE-separated HPLC-purified molecules from T. simiae and T. congolense PCF were analyzed for their ability to bind to proaerolysin, a molecule which has previously been demonstrated to bind to GPI anchor structures. The results are shown in Fig. 8. No proaerolysin-binding molecules were detected in the HPLC-purified fractions from T. simiae PCF (lane 1), whereas a molecule of approximately 60 kDa was detected in T. congolense PCF (lane 2), indicating that the anchor structures of the two trypanosome species are different. 3.9. Mass spectroscopic analysis of HPLC purifiedsurface molecules
Fig. 4. ELISA profile of T. simiae CP11 and T. congolense K45/1 PCF fractions eluted from a semi-preparative octyl-Sepharose HPLC column. A: T. simiae CP11. B: T. congolense K45/1. The primary mAbs were: TS 126, solid line. TC 491, dashed line. L98/L157 mixture gray line.
Although amino acid microanalysis confirmed that there was protein present in the HPLC-purified material from both T. simiae and T.congolense (data not shown), the samples could not be sequenced by gas-phase Nterminal sequencing, possibly due to interference by an abundance of carbohydrates. Therefore, in-gel tryptic digestion of the SDS–PAGE separated, HPLC-isolated protein bands was performed to obtain peptides for peptide mass mapping and for sequencing by MALDITOF and Q-TOF mass spectrometry, respectively. Although most of the protein bands gave good MALDITOF spectra, none yielded reliable protein identification after database searching with both MS Fit (Protein
Fig. 5. SDS–PAGE analysis of reverse-phase HPLC-purified molecules from PCF of T. simiae CP11 and T. congolense K45/1. (A) GelCode Blue stain. (B) Stains-All. (C) Silver stain. Lane 1: T. simiae CP11 PCF. Lane 2: T. congolense K45/1 PCF. The apparent molecular masses are shown in kiloDaltons.
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Fig. 6. Detection of biotin-labeled carbohydrates on blotted, SDS– PAGE separated reverse-phase HPLC-purified molecules from T. simiae CP11 and T. congolense K45/1. Lane 1: T. simiae CP11 PCF. Lane 2: T. congolense K45/1 PCF. Apparent molecular masses are shown in kiloDaltons.
Fig. 8. Detection of proaerolysin-binding molecules in SDS–PAGEseparated, HPLC-purified molecules from procyclic trypanosomes. Lane 1: T. simiae CP11 PCF. Lane 2: T. congolense K45/1 PCF. Apparent molecular masses are shown in kiloDaltons.
Prospector) and Mascot (Matrix Science) search algorithms. In contrast, peptide sequencing by Q-TOF mass spectrometry of the T. congolense major 50–52 kDa upper band and a fainter, lower 44–46 kDa band from this doublet (colloidal Coomassie blue-stained bands corresponding to those shown in Fig. 5, panel A, lane 2) Table 1 Peptide sequences obtained by Tandem nanospray MS/MS analysis of tryptic peptides from HPLC-purified molecules of T. simiae
Fig. 7. Fluorograph of SDS–PAGE separated, [1-3 H] ethan-1-ol-2amine hydrochloride labeled, reverse-phase purified molecules from T. simiae CP11 and T. congolense K45/1. Lane 1: T. simiae CP11. Lane 2: T. congolense K45/1. Molecular masses are shown in kiloDaltons.
Mass
Peptide sequence
453.2 544.7 668.3 751.8 450.2 666.3 707.8 426.3 614.3 617.82 549.8
1. AQTEGPFR 2. SNQQNFHSK 3. AQLAASQNDDFR 4. QWAGTPEAEWATR 5. EAPLGLTAK 6. TFWVVEQLPGR 7. SNLDSSAVATFFR 8. LASGGGHPR 9. LPWVEPSDER 10. DTDGPFSVQLR 11. HNAFVTLAAR
Sequences 1–4 were from protein bands at 64–68 kDa, sequences 5– 7 were from protein bands at 46–48 kDa and sequences 9–11 were from proteins at 35–38 kDa, all from positions approximately as shown in Fig. 5, panel A, lane 1.
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revealed the sequence GVDVATEAAAR in each band. This sequence was identical to amino acids 94–103 of the GARP sequence. Sequencing of tryptic peptides from a range of colloidal Coomassie blue-stained protein bands (approximately 64–68, 46–48 and 35–38 kDa) from T. simiae yielded several sequences (Table 1) none of which gave any matches after searching of the NCBI non-redundant or of Swiss-Prot databases. Mass spectrometry was thus successfully used to obtain peptide sequences from the parasite surface molecules, in contrast to gasphase microsequencing which failed to detect any N-terminal sequences.
4. Discussion Trypanosoma congolense and T. simiae are members of the same subgenus (Nannomonas) yet show different host ranges. T. congolense is infective for almost all domestic animals whereas T. simiae is almost exclusively a specific parasite of pigs. Both T. congolense and T. simiae can be transmitted by diverse species of Glossina and have similar developmental cycles in the fly. Migration of procyclic forms proceeds from the midgut to the proboscis where epimastigotes form and attach to the walls of the food canal. The parasites finally invade the hypopharynx where the animal-infective metacyclic forms differentiate. In contrast, members of the subgenus Trypanozoon (T. brucei spp.) develop in the salivary glands (Mulligan, 1970). A comparison of 2-D gel profiles of T. brucei spp. and T. congolense has previously shown that the proteomes of procyclic forms of these different species are remarkably different (Anderson et al., 1985). In the current study, the two-dimensional gel electrophoresis results with 35 S-methionine-labeled parasites showed that T. congolense Kilifi clone K45/1 and T. simiae CP11 procyclic proteomes were also drastically different. This is a bit of a surprise since these two species are members of the same subgenus and have the same developmental cycle. Nevertheless, these parasites may have evolved separately over many millions of years (Overath et al., 2001) and thus exhibit quite divergent protein compositions. It is of course possible that the proteins are still essentially the same although with different post-translational modifications or with slight differences in amino acid sequences which would manifest as size and charge shifts on the 2-D profiles. Nevertheless, despite their different proteomes, T. congolense and T. simiae procyclic forms share surface-exposed epitopes detected with mAbs in immunofluorescence reactions on living parasites (Beecroft et al., 1993; and in the work reported here). To further characterize these surface molecules of T. congolense and T. simiae procyclic forms, we used organic solvent extraction and reversed phase HPLC
with two different surface binding mAbs as probes to aid in their purification. Although immunoreactive molecules from both species of parasites eluted late in the HPLC gradient, a characteristic of high hydrophobicity, the profiles were reproducibly dissimilar indicating that the surface molecules of these two species were slightly different. The SDS–PAGE profiles and staining patterns of the HPLC-purified molecules were also unique to each species and indicated that although broadly similar, the molecules from the two species are biochemically different, with the T. simiae molecules being of higher apparent molecular masses. It was interesting that the staining profiles of the molecules showed characteristics of the profiles of the major surface glycoconjugates of T. brucei spp. and T. congolense, that is, they were broad, diffuse bands that did not stain well with Coomassie blue. Two major broad bands in each species were heavily labeled by treatment with biotin hydrazide, indicating that they contained large amounts of carbohydrate. Interestingly, in both species, both bands could be biosynthetically labeled with 3 H-ethanolamine, indicating that they contained terminal ethanolamines that are probably involved in attachment of polypeptides to lipid anchors. Though non-protein linked GPI anchors such as free GPI lipids and lipophosphoglycans have been described in protozoan parasites, these structures lack ethanolamine. The anchor structures may be different between T. simiae and T. congolense as only one of the bands (from T. congolense) bound to proaerolysin toxin. This toxin is known to bind specifically to GPI anchors. However, modifications of the core structure of GPIanchors by addition of sugars or phosphoethanolamine, or by acylation of inositol result in the loss of the binding of proaerolysin (Nelson et al., 1997), perhaps explaining the differential binding to anchors of the two trypanosome species. Amino acid analysis of pooled HPLC peaks revealed much higher levels of amino acids in the T. congolense fraction when compared to T. simiae. Amino acid analysis of the two bands from T. simiae after blotting of the SDS–PAGE separated HPLC fractions onto PVDF membrane, however, did show low levels of amino acids in each band. Several attempts at gas-phase N-terminal sequencing of each of these bands failed. The high abundance of carbohydrates may explain the low yields of amino acids and difficulty in sequencing although simple N-terminal blockage could explain the latter. Mass spectroscopic analysis of HPLC-separated proteins, detected by sensitive colloidal Coomassie brilliant blue staining, showed that the two major bands in T. congolense contained GARP, as expected, whereas no peptides matching GARP could be found in T. simiae. This result supports the observation that GARP genes were not detected by Southern blotting in the T. simiae CP 11 clone (this paper) and that the GARP protein could not be detected in immunoblotting experiments
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using an antiserum specific for GARP polypeptide (Mookherjee and Pearson, 2001). Sequencing of several peptides from T. simiae by Q-TOF mass spectrometry revealed several unique sequences, none of which matched any sequence in public databases. Taken together, the data suggest that the major HPLC-separated bands of T. simiae represent unique glycoproteins and that they share carbohydrate epitopes with each other and with GARP of T. congolense and that the T. simiae polypeptide sequences are different than the T. congolense-derived GARP sequence. Of relevance here is that novel non-GARP, carbohydrate-containing, GPI-anchored surface molecules have recently been detected in T. congolense (B€ utikofer et al., 2002). Interestingly, these authors also encountered great difficulty obtaining any protein sequences from the non-GARP glycoconjugates. This last observation is interesting and although a cause of much frustration in characterizing the surface procyclins of both T. congolense (and T. simiae; this paper), clearly indicates that an approach using carbohydrate microchemistry will be required for structure–function analysis of these molecules. To summarize, we have shown that several surfacedisposed, GPI-anchored molecules are present in both T. congolense and T. simiae and that these are glycoconjugates. Although these membrane molecules are immunologically similar, there are biochemical differences between them as reflected in their different HPLC elution and gel staining profiles, the absence of GARP in T. simiae and differential binding of proaerolysin toxin. It seems plausible that the carbohydrate epitopes shared between these two species of the subgenus Nannomonas may be involved in interactions with the tsetse vector since they both appear to have the same developmental cycle. If this is the case, then it appears that T. congolense and T. simiae, despite their widely different proteomes, have placed common carbohydrate structures on different carriers as a way to achieve the same function.
Acknowledgments We thank Dr. Isabel Roditi for the GARP probe, Dr. Peter B€ utik€ ofer Dr. Robert Burke, Dr. R.W. Olafson, and Jody Haddow and for technical advice, Dr. Tom Buckley for proaerolysin toxin and antiserum, Sandy Kielland for performing amino acid analyses, Lee Rafuse Haines for performing immunofluorescence microscopy and for reading the manuscript and helpful suggestions, Heather Down for help with preparation of the figures, and Morag Booy and Jennifer Chase for excellent technical help. N.M. was the recipient of a University of Victoria Student Fellowship. This work was supported by a Research Grant from the Natural Sciences and Engineering Council of Canada (NSERC).
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