From the Department of Pathology and the Sandler Center, University of California, San Francisco, California 94143, the
Department of Pharmaceutical Chemistry, Mass Spectrometry Facility and the ¶ Liver Center, University of California, San Francisco, California 94143-0446, and the || San Francisco Veterans Affairs Medical Center, San Francisco, California 94121
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ABSTRACT |
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The life cycle of S. mansoni is complex (3) (Fig. 1). The initiation of infection of the human host by schistosome parasites involves penetration of skin by a multicellular larva (0.1 mm) called a cercaria(ae). Cercariae have forked tails that propel them through fresh water. Depending upon their specific vertebrate host, cercariae can respond to a variety of stimuli, including motion, light, and shadow, chemical gradients, and heat. Upon contact with human skin, cercariae are stimulated by the lipid on the surface of skin to begin penetration. Initial penetration involves mechanical entry into the superficial, cornified layer of skin, which presents little barrier in the aquatic environment. However, further entry requires degradation of intercellular bridges between epidermal cells, the dermal/epidermal basement membrane, and the extracellular matrix of the dermis. Ultimately the larvae, which have now shed their tails and are called schistosomula, enter small vessels in the superficial dermis where they complete their life cycle as described above.
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The only consistently effective experimental vaccine against schistosome infection is that produced by irradiation of cercariae. Identifying the protein repertoire released during the initial stage of the skin invasion is key to both understanding the pathogenesis of infection and ultimately preventing it.
We carried out proteomic analysis of the secretions of cercariae stimulated to invade and transform into schistosomula by two independent techniques (4, 7, 8). In addition to identifying the major protein components released during this initial stage of infection, our analysis provides insights into how "background" protein species can be identified with mass spectrometry technology in a complex "ecosystem" of target organism, snail host, investigator, and laboratory environment.
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EXPERIMENTAL PROCEDURES |
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Biomphalaria glabrata snails are used as intermediate hosts and are maintained in a BSL2 laboratory in accordance with all approved biosafety protocols. Cercariae are obtained using a light induction method described previously (9). Cercarial secretions were prepared according to two methods reported previously (10) in response to human skin lipid and from cultures of mechanically transformed larvae. Proteins released without stimulation were also collected. Specifically these samples were prepared as follows.
Cercariae were "shed" from their host snails in glass culture dishes overnight (9). In the first method, which recapitulates the biological stimulus, cercariae were placed in a Fisher Petri dish coated with human skin lipid. The cercariae were monitored microscopically and allowed to secrete for 1.52 h into water warmed to 37 °C by immersion of the Petri dish in a water bath. Cercarial bodies and tails were then removed from the secretions by centrifugation at 3000 rpm, and the secreted protein mixture was lyophilized and stored at 20 °C. This sample is referred to as skin lipid-induced "cercarial secretion."
An alternative method for inducing secretion was through mechanical tail shearing to transform the cercariae into schistosomula (11, 12). This provides a cleaner protein sample known to contain acetabular gland secretions (11). Cercariae that were shed in aquarium water were chilled and allowed to settle in a 50-ml centrifuge tube, and the aquarium water was exchanged twice with cold distilled water by decantation/resuspension followed by gentle centrifugation at 3000 rpm to collect the cercariae. The cercariae were then sheared through a small bore syringe until >90% of the larval tails were released and transferred to a 10-cm Petri dish containing 50% serum-free schistosome culture medium 169 (13) at room temperature for 1.52 h. Finally the plate was swirled to remove the schistosomula and tail debris by pipette. The conditioned medium was collected and pooled, while the acetabular secretions, visible in a dissecting microscope as vesicles, were retained on the plate surface. These vesicles could then be released from the plate surface by scraping using a cell scraper (Corning Costar, Acton, MA) and rinsing with a few milliliters of 0.1% SDS solution.
First Stage Preparation by SDS-PAGE
One-dimensional SDS-PAGE was performed on the cercarial secretion samples using NOVEX Tris-glycine 420% acrylamide gels from Invitrogen and SeeBlue Plus 2 standards from Invitrogen to calibrate the molecular weight range. The gels were stained by silver stain according to the method by Shevchenko (14) with modifications that reduce background staining (15).
In-gel Digestion and LC-MS/MS Analysis
The SDS-PAGE gel protein bands were excised, reduced with dithiothreitol, alkylated with iodoacetamide, and then subjected to in-gel digestion1 with side chain-protected porcine trypsin (Promega, Madison, WI) (16, 17). The resulting peptides were extracted and then analyzed by on-line liquid chromatography/mass spectrometry using an HPLC system consisting of a Famos autoinjector and an Eksigent nanoflow pump coupled to a quadrupole-orthogonal acceleration-time-of-flight hybrid tandem mass spectrometer, a QSTAR XL (Applied Biosystems, Foster City, CA). The reversed-phase chromatography was controlled with Eksigent software to develop a 550% acetonitrile gradient in 30 min using 0.1% formic acid as the ion pairing agent at a 350 nl/min flow rate. Data were acquired in information-dependent acquisition mode: 1-s mass measurements were followed by 3-s CID experiments for which the multiply charged precursor ions were computer-selected and the collision conditions were adjusted to the charge state and the m/z values of the precursor ions. CID data were analyzed using Analyst QS service pack 6 software (Applied Biosystems) with the Mascot script 1.6b4 (Matrix Science, London, UK). Parameters used in the Mascot script were as follows: AutoCentroid for the TOF: 20-ppm merge distance, 10-ppm minimum width, 50% percentage height, and 100-ppm maximum width; PeakFinding for Spectrum: 0.5% default threshold, 400-gauss filter, and a gaussian filter limit of 10. Information-dependent acquisition survey scan centroid parameters were as follows: automatic charge state determination from survey scan, 50% percentage height, and 0.02-amu merge distance. MS/MS averaging of information-dependent acquisition dependents was as follows: reject spectra if less than eight peaks, 0.5-Da precursor mass tolerance for grouping, and 10 maximum and 1 minimum cycles between groups. MS/MS data centroid and threshold parameters were as follows: remove peaks with <0% of highest intensity, centroid all MS/MS data, no smoothing, 50% height percentage, and 0.05-amu merge distance.
Database searches were performed using Mascot Server version 2.0.01 (18, 19), and the MS-Tag and MS-Pattern modules of the internal Protein Prospector server version 4.11 (20) were applied to individual peptide sequences and CID data. Searches were performed first on the National Center for Biotechnology Information non-redundant (NCBInr) data bank (September 8, 2004), and the results were parsed into a working database. The Mascot Server search parameters were as follows. Only tryptic peptides were considered with one missed cleavage allowed. Variable modifications included carbamidomethylation or propionamidylation (i.e. acrylamide addition) of Cys residues, protein N-acetylation, Met oxidation, and pyroglutamate formation from N-terminal Gln residues. Mass accuracy was within 100 ppm in MS and 0.2 Da for CID data. Peptide sequences matched to species other than schistosomes by Mascot were further BLAST searched against the expressed sequence tags available from The Institute for Genomic Research (TIGR) S. mansoni genome project in version 5.0 (www.tigr.org/tdb/tgi/smgi/). If the peptides were identically matched to schistosome sequences, the Mascot scores were transferred. Gene Ontology (GO)2 annotations were assigned based on sequence similarity searches against the GO annotated proteins in the Swiss-Prot and TrEMBL databases at European Bioinformatics Institute calculated using the GOblet server (21).
Serine Protease Activity of the Cercarial Secretions
Activity was measured using a fluorescence end point assay detecting proteolytic cleavage of specific substrates for cercarial elastase (also known as acetabular protease) versus background snail serine proteases: succinyl-AAPF-aminomethylcoumarin and carbobenzoxy-(Z)-FPR-aminomethylcoumarin, respectively, as described previously (22, 23). Total fluorescence was monitored for 6 h (excitation, 355 nm; emission, 460 nm) on a Molecular Devices Flex Station. Protein concentration was determined using the Bradford assay, and the activity values are reported in relative fluorescence units per second per microgram of protein.
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RESULTS |
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At the detection level of the QSTAR XL system used, it was possible to identify proteins from the laboratory environment in the lipid-induced sample. These included the snail digestive "tryptases" called serine proteases and ß, snail hemoglobins, and a developmentally regulated albumin gland gene characterized as part of the snail immune response to schistosome infection (30). Human (investigator) proteins included lysozyme C, a prolactin-induced protein, tear lipocalin 1, skin-related keratins, and psoriasin. Photosynthetic proteins such as ribulose-1,5-bisphosphate carboxylase/oxygenase, and chlorophyll aligned with sequences from Lactuca sativa, the lettuce used as a food source for the B. glabrata snails. These and other photosynthetic plant proteins as well as serine protease ß, but not the developmentally regulated albumin gland gene protein, were found in a sample of aquarium water conditioned by uninfected snails (data not shown).
The lipid-induced cercarial secretions contained the three S. mansoni cercarial elastase (CE) gene isoforms that are known to be expressed (1a, 1b, and 2a) (22). The peptide sequences found are shown in an alignment of the CE isoforms in Fig. 4, showing 54, 42, and 57% sequence coverage, respectively. N-terminal prodomains for these enzymes are absent; the first peptides detected in the sequence are immediately adjacent to a known prodomain processing site at Leu27 (CE1a numbering). As confirmation for these three isoforms, CID mass spectra for example isoform-unique peptides are shown in Figs. 57. CE peptides were found in bands 19 (Fig. 2, left panel). The lowest molecular weight bands contain peptides from the C terminus of the protein, whereas band 9 contained the full-length catalytic domain of the protease. The presence of CE2a in band 12, i.e. at molecular mass 40 kDa, was also unambiguously established from a tryptic peptide, 119QTLSGFDITVMLAQMVNLQSGIR142 (data not shown).
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To identify background proteins released without any stimulation to invade, cercariae incubated in water alone were also analyzed (Table II). As expected, there were reduced amounts of protein relative to lipid-stimulated cercariae (0.47 µg/10 µl by Bradford assay compared with 1.7 µg/10 µl). The schistosome proteins that yielded the most abundant peptides in this sample were abundant cytosolic proteins, actin, enolase, Sm20.8, thioredoxin, and triose-phosphate isomerase (31). Background laboratory environment proteins were present here as well: chlorophyll a/b-binding protein, ribulose-1,5-bisphosphate carboxylase/oxygenase, snail serine proteases and ß, the developmentally regulated snail albumin gland gene, and bovine serum albumin.
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GO annotations for molecular function, biological process, and cellular component could be assigned for many of the schistosome-derived proteins using the program GOblet, which transfers GO annotations based on sequence similarity searches against the TrEMBL and Swiss-Prot databases (Table IV). The percentages of cercarial secretome proteins found in each GO category are compared with the percentages found for all the full-length S. mansoni proteins listed in the NCBInr data bank. GO categories where there is an interesting enrichment of proteins in the cercarial secretions sample over all schistosome proteins are "binding function" (including the calcium-binding proteins) and "enzymatic activity" (including the hydrolytic enzymes). The percentage of chaperone activity was enriched more than twice over that generally observed in the NCBInr data bank schistosome proteins. Among the biological processes, "physiological processes" and "metabolism" seemed greatly enhanced in this sample. Finally the apparent cellular component of 60% of these proteins is intracellular as would be expected for the numerous glycolytic proteins identified.
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DISCUSSION |
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We carried out a proteomic analysis of the secretory products of cercariae induced to invade by two well characterized experimental protocols. In the first, human skin lipid, the natural stimulant for cercarial invasion, induced invasive behavior in vitro and led to identification of a spectrum of proteins released from three potential cercarial sources: the large acetabular glands known to secrete proteases during invasion, the smaller head glands thought to play a role in late stages of vessel entry, and proteins released from turnover of the surface tegumental membrane complex.
Table I shows 72 proteins identified through LC-MS/MS analysis of lipid-induced secretions. A number of calcium-binding proteins and the cercarial elastase (also known as cercarial protease and acetabular gland protease), identified previously as acetabular gland secretions, were present. Absent from these samples were many of the tegument-bound antigens previously reported: Sm25, Sm23, Sm22, Sm15, Sm13, and Sm8 (29, 32). The only example of this type of protein found in lipid-induced secretions was the fatty acid-binding protein Sm14. Many of these proteins have transmembrane motifs, and therefore it seems likely that the contribution of the shed membrane-associated tegumental proteins to lipid-induced cercarial secretions is minimal in comparison with acetabular gland derivatives (33).
Other proteins that were abundant in this sample were soluble glycolytic proteins such as triose-phosphate isomerase, GAPDH, and phosphoglycerate kinase. There are three possibilities why these "cytosolic" proteins appear in secretions. First, they may merely represent proteins that have "leaked" from cercariae because of "artificial" damage during lipid stimulation. However, careful microscopic analysis of larvae during this collection procedure showed no significant morphologic damage, and the small group of proteins does not match the proteome of an extract of whole cercariae reported by Curwen et al. (31). The second possible source is the syncytium, or cytosolic component of the tegument, also reported to be shed as "vesicles" with tegumental membrane by developing larvae (5, 10). GST, phosphofructokinase (34), fructose-bisphosphate aldolase (35), and phosphoenolpyruvate carboxykinase (36) have all been localized to the Sm tegument. Very recently an analysis of the tegumental subproteome of schistosomes was reported (37). Of the proteins identified in this study, 95 were found in the tegument preparations as indicated in Tables I III.
A third possible source of cytosolic proteins is the secretory acetabular glands. The acetabular glands are in fact a set of cells in the posterior portion of the cercaria head. Cell processes extend to the anterior of the larva to serve as "ducts" for passage of secretory material. Because these glands are in fact cells that release their cytoplasmic contents during secretion, at least some of the glycolytic enzymes identified may be from this cellular source. The acetabular cells are indeed a major volume of the cercaria (6).
The exact source of these cytosolic components including glycolytic and metabolic enzymes is a key issue for laboratories working on development of the subunit schistosome vaccine. Curwen et al. (31) recently reported a proteomic analysis of soluble sonicates from several stages of S. mansoni. The authors identified the 40 most abundant soluble proteins across the schistosome life cycle and reached the important conclusion that these primarily represent cytosolic enzymes, which appear to vary little with transition from stage to stage. They make the compelling argument that such proteins are less likely to be suitable vaccine components as it is unlikely for them to be "seen" by the host immune system in intact larvae or adult. Although our analysis of cercarial secretions raises the possibility that some of these glycolytic enzymes may be released from sources like acetabular cells, it is nevertheless imperative for groups working on specific vaccine candidates to confirm location within developing larvae and whether or not such proteins are accessible to an induced immune response. Taking into consideration the conclusions of Curwen et al. (31), it is instructive to note the striking differences between the GO category shown in Table IV for presumed secretion-related proteins versus all the non-fragmentary S. mansoni proteins listed in the NCBI data bank. There is an enrichment of binding function in the cercarial secretions, specifically metal ion nucleotide and protein binding categories. There are also enriched enzymatic activities including hydrolases (like the acetabular gland protease, isomerase, kinase, and lyase activities). The percentage of chaperone activity is enriched more than twice over that generally observed in the NCBI data bank of schistosome proteins. This may indicate that chaperone proteins are concentrated within secretion sources like the acetabular glands to facilitate or maintain folding of secreted proteins that are densely "packed" in acetabular gland vesicles (4).
In the absence of any stimulation, 15 proteins were identified. Two of these were snail digestive enzymes (see discussion below). The schistosome proteins identified represent a subset of those released by "shedding" of the tegument (e.g. GST), as discussed above, or merely of high abundance (enolase). Some of these proteins match those in the "soluble" sonicate analysis of Curwen et al. (31) and therefore have likely leaked from damaged organisms.
In the lipid-induced secretion analysis, an important observation was the identification of contaminating proteins from the laboratory environment due to the high sensitivity of the QSTAR XL system. This serves as a caveat for investigators to carefully characterize proteins to separate those that represent elements of the biological phenomenon being studied versus those that may invariably come from environmental contamination. The latter are more easily identified as the sensitivity of LC-MS/MS increases despite careful attention to sample preparation. In our analysis, one such group represented proteins from the snail host in which the cercariae develop. Although not schistosome proteins, these provide important biological information. First it is clear that the snail digestive enzymes (snail tryptases) are released as cercariae emerge from the hepatopancreas adjacent to the snail digestive tract. In addition, a protein that is a component of the snails own defense response to developing parasites was identified (developmentally regulated albumin gland protein). The methods of analysis were so sensitive we could even detect proteins from the lettuce on which the snails feed.
The sensitivity of QSTAR XL also meant that trace amounts of protein from the investigator preparing the samples could be detected. This included keratin from sloughed epidermal cells during collection of skin lipid. Proteins were also identified from human tears, presumably from microscopic lachrymal gland droplets that exited the technicians eye as samples were prepared and observed.
Finally proteins were identified that represent trace contamination from the laboratory environment. Principal among these was bovine serum albumin. We were able to identify this as a laboratory contaminant because, although our laboratory primarily uses BSA in preparation of tissue culture media, a second laboratory from which we obtained snails uses human serum albumin, which was the trace contaminant when those snails were used. This indicates how readily serum albumin can contaminate a laboratory environment and, with the increased sensitivity of LC-MS/MS, can contaminate protein samples as well.
To minimize environmental contamination and directly identify the protein contents of the acetabular gland secretions, a second secretion collection method was used. This involves mechanically shearing the tails off the cercariae, which has been shown to stimulate and induce invasion behavior, specifically release of acetabular gland contents (11, 12). By this method, the number of schistosome-related proteins relative to snail proteins increased substantially. Using an internal standard of protease activity known to be released by cercariae (cercarial elastase) versus snails (snail tryptases), the change in ratio of these enzyme activities showed that the mechanical shearing method indeed gave a more direct analysis of the acetabular gland proteins. Acetabular secretions appeared on the plate as small vesicles, the form in which they are released from the acetabular cells before they rupture in the host (4). An interesting observation was that the acetabular gland protease isolated from the intact vesicles showed no autoproteolysis products but was present as an active, mature catalytic domain or proform prior to its release. N-terminal signal peptide sequences were found in all predicted sequences of the cercarial elastase species, calreticulin, SPO-1, endoplasmin, protein-disulfide isomerase, GAPDH, and prohibitin using two different motif search methods, InterPro (38) and SignalIP (39).
Proteomic analysis of isolated acetabular gland vesicles also validated previous reports of specific proteins residing in these organelles by immunolocalization. Aside from the cercarial elastase, paramyosin and SPO-1 (Sm16) were also identified. Paramyosin is a very immunogenic protein originally studied as a potential vaccine component. It is hypothesized to be involved in immune evasion by "trapping" host immunoglobulins or complement components (40). We had previously identified paramyosin as a major protein component of acetabular gland secretions in a limited proteomic analysis involving lipid-induced secretion followed by ion exchange chromatography.3 For the current analysis, paramyosin was cleaved or processed into smaller fragments, resulting in its appearance across multiple bands in SDS-PAGE separation. This may be due to its co-localization or co-secretion with the acetabular gland protease (cercarial elastase). SPO-1 (also known as Sm16) is a cercarial protein reported as abundantly present in acetabular secretions (41, 42). It is thought to also play a role in anti-inflammatory mechanisms of invading schistosome larvae (42).
Proteomic analysis of cercarial secretions induced by two independent methods identifies a spectrum of proteins that can be mined for potential anti-schistosome vaccine components. Furthermore, identification of functional proteins such as cercarial elastase, calcium-binding proteins, and paramyosin provides clues or validation of proposed mechanisms of host skin invasion and immune evasion. Finally sorting of environmental contaminants from schistosome proteins serves to alert investigators that the increasing sensitivity of LC-MS/MS may invariably result in identification of "environmental" contaminants. In our case, these came from the "biological ecosystem" of schistosome and snail, the investigator preparing the sample, and the laboratory environment.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Published, August 10, 2005
Published, MCP Papers in Press, August 18, 2005, DOI 10.1074/mcp.M500097-MCP200
1 The University of California San Francisco Mass Spectrometry in- gel digestion procedure is on line at ms-facility.ucsf.edu/ingel.html.
2 The abbreviations used are: GO, Gene Ontology; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; CE, cercarial elastase.
3 E. Hansell and J. H. McKerrow, unpublished data.
* This work was supported by the Sandler Family Supporting Foundation, by National Center for Research Resources, National Institutes of Health Grants RR001614, RR015804, and RR012961 to the University of California San Francisco (UCSF) Mass Spectrometry Facility (Director A. L. Burlingame), and by a Veterans Affairs merit award (to J. H. M.). Core support was from the UCSF Liver Center.
S The on-line version of this article (available at http://www.mcponline.org) contains supplemental material.
** To whom correspondence should be addressed: Dept. of Pathology, University of California, QB3, Box 2550, McKerrow 508B, San Francisco, CA 94143. Tel.: 415-476-2940; Fax: 415-514-3165; E-mail: jmck{at}cgl.ucsf.edu
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REFERENCES |
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