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Proteins Exposed at the Adult Schistosome Surface Revealed by Biotinylation

2005; Elsevier BV; Volume: 5; Issue: 2 Linguagem: Inglês

10.1074/mcp.m500287-mcp200

1535-9484

Bryony Braschi, R. Alan Wilson,

Malaria Research and Control

The human blood-dwelling parasite Schistosoma mansoni can survive in the hostile host environment for decades and must therefore display effective strategies to evade the host immune responses. The surface of the adult worm is covered by a living syncytial layer, the tegument, bounded by a complex multilaminate surface. This comprises a normal plasma membrane overlain by a secreted bilayer, the membranocalyx. Recent proteomic studies have identified constituents of the tegument, but their relative locations remain to be established. We labeled the most exposed surface proteins using two impermeant biotinylation reagents that differed only in length. We anticipated that the two reagents would display distinct powers of penetration, thereby producing a differential labeling pattern. The labeled proteins were recovered by streptavidin affinity and identified by tandem mass spectrometry. A total of 28 proteins was identified, 13 labeled by a long form reagent and the same 13 plus a further 15 labeled by a short form reagent. The parasite proteins included membrane enzymes, transporters, and structural proteins. The short form reagent additionally labeled some cytosolic and cytoskeletal proteins, the latter being constituents of the intracellular spines. Only a single secreted protein was labeled, implying a location between the plasma membrane and the membranocalyx or as part of the latter. Four host proteins, three immunoglobulin heavy chains and C3c/C3dg, a fragment of complement C3, were labeled by both reagents indicating their exposed situation. The presence of the degraded complement C3 implicates inhibition of the classical pathway as a major element of the immune evasion strategy, whereas the recovery of only one truly secreted protein points to the membranocalyx acting primarily as an inert protective barrier between the immune system and the tegument plasma membrane. Collectively the labeled parasite proteins merit investigation as potential vaccine candidates. The human blood-dwelling parasite Schistosoma mansoni can survive in the hostile host environment for decades and must therefore display effective strategies to evade the host immune responses. The surface of the adult worm is covered by a living syncytial layer, the tegument, bounded by a complex multilaminate surface. This comprises a normal plasma membrane overlain by a secreted bilayer, the membranocalyx. Recent proteomic studies have identified constituents of the tegument, but their relative locations remain to be established. We labeled the most exposed surface proteins using two impermeant biotinylation reagents that differed only in length. We anticipated that the two reagents would display distinct powers of penetration, thereby producing a differential labeling pattern. The labeled proteins were recovered by streptavidin affinity and identified by tandem mass spectrometry. A total of 28 proteins was identified, 13 labeled by a long form reagent and the same 13 plus a further 15 labeled by a short form reagent. The parasite proteins included membrane enzymes, transporters, and structural proteins. The short form reagent additionally labeled some cytosolic and cytoskeletal proteins, the latter being constituents of the intracellular spines. Only a single secreted protein was labeled, implying a location between the plasma membrane and the membranocalyx or as part of the latter. Four host proteins, three immunoglobulin heavy chains and C3c/C3dg, a fragment of complement C3, were labeled by both reagents indicating their exposed situation. The presence of the degraded complement C3 implicates inhibition of the classical pathway as a major element of the immune evasion strategy, whereas the recovery of only one truly secreted protein points to the membranocalyx acting primarily as an inert protective barrier between the immune system and the tegument plasma membrane. Collectively the labeled parasite proteins merit investigation as potential vaccine candidates. The trematode Schistosoma mansoni is a long lived parasite of the human hepatic portal system, infecting millions of people in Africa and South and Central America (1World Health OrganizationTDR Strategic Direction for Research: Schistosomiasis. World Health Organization, Geneva2002Google Scholar). Infection of the mammalian host occurs by direct cercarial penetration through the skin followed by transformation into schistosomula. These then undertake a protracted intravascular migration to the hepatic portal vein where they begin to feed on blood, mature, and pair. The male carries the female up the mesenteric blood vessels to the gut wall where she begins to deposit eggs. A proportion of these pass through the tissue and reach the gut lumen to continue the life cycle, whereas others are washed downstream to the liver where they initiate the granulomatous inflammation that characterizes the disease. That schistosomes can survive for 30–40 years in humans (2Harris A.R. Russell R.J. Charters A.D. A review of schistosomiasis in immigrants in Western Australia, demonstrating the unusual longevity of Schistosoma mansoni.Trans. R. Soc. Trop. Med. Hyg. 1984; 78: 385-388Google Scholar) attests to their possession of an effective immune evasion strategy. The mature worm is covered by a syncytial cytoplasmic layer, the tegument, attached to underlying cell bodies by narrow cytoplasmic connections. The nuclei, ribosomes, endoplasmic reticulum, and Golgi apparatus are located in these cell bodies, and their vesicular products, the discoid bodies and multilaminate vesicles, traffic to the tegument syncytium via the connections. Early ultrastructural studies showed that the apical surface of the tegument has a complex multilaminate appearance (3Hockley D.J. McLaren D.J. Schistosoma mansoni: changes in the outer membrane of the tegument during development from cercaria to adult worm.Int. J. Parasitol. 1973; 3: 13-25Google Scholar) that has been interpreted as a plasma membrane overlain by a trilaminate secretion, which was termed a membranocalyx by analogy with the glycocalyx of eukaryotic cells (4Wilson R.A. Barnes P.E. The formation and turnover of the membranocalyx on the tegument of Schistosoma mansoni.Parasitology. 1977; 74: 61-71Google Scholar). The membranocalyx is formed when the bounding membrane of the multilaminate vesicle fuses with the apical plasma membrane of the tegument to release its sheetlike contents. These unfold and flow laterally across the plasma membrane. The membranocalyx has some unusual properties including the ability to sequester glycolipids from host erythrocytes (5Goldring O.L. Clegg J.A. Smithers S.R. Terry R.J. Acquisition of human blood group antigens by Schistosoma mansoni.Clin. Exp. Immunol. 1976; 26: 181-187Google Scholar). Although initial in vitro observations suggested a rapid turnover, in vivo studies using erythrocyte antigens as a marker indicated a half-life of approximately 5 days (6Saunders N. Wilson R.A. Coulson P.S. The outer bilayer of the adult schistosome tegument surface has a low turnover rate in vitro and in vivo.Mol. Biochem. Parasitol. 1987; 25: 123-131Google Scholar). Given a slow turnover, it is plausible that the membranocalyx provides a physical barrier protecting the underlying plasma membrane, which possesses normal cellular functions, from immune attack. Methods were devised more than 30 years ago to detach the tegument surface complex for compositional analysis (7Roberts S.M. MacGregor A.N. Vojvodic M. Wells E. Crabtree J.E. Wilson R.A. Tegument surface membranes of adult Schistosoma mansoni: development of a method for their isolation.Mol. Biochem. Parasitol. 1983; 9: 105-127Google Scholar, 8Oaks J.A. Cain G.D. Mower D.A. Raj R.K. Disruption and removal of the tegument from Schistosoma mansoni with triton X-100.J. Parasitol. 1981; 67: 761-775Google Scholar, 9Bennett J.L. Seed J.L. Characterization and isolation of concanavalin A binding sites from the epidermis of S. mansoni.J. Parasitol. 1977; 63: 250-258Google Scholar). However, despite separation of the protein constituents by electrophoresis (10Roberts S.M. Boot C. Wilson R.A. Antibody responses of rodents to a tegument membrane preparation from adult Schistosoma mansoni.Parasitology. 1988; 97: 425-435Google Scholar, 11Dalton J.P. Strand M. Mangold B.L. Dean D.A. Identification of Schistosoma mansoni glycoproteins recognized by protective antibodies from mice immunized with irradiated cercariae.J. Immunol. 1986; 136: 4689-4694Google Scholar), little progress was made in obtaining their identities. The advent of proteomics has provided the tools to link proteins with their encoding cDNA. Simultaneously the generation of a large S. mansoni expressed sequence tag database (12Verjovski-Almeida S. DeMarco R. Martins E.A. Guimaraes P.E. Ojopi E.P. Paquola A.C. Piazza J.P. Nishiyama Jr., M.Y. Kitajima J.P. Adamson R.E. Ashton P.D. Bonaldo M.F. Coulson P.S. Dillon G.P. Farias L.P. Gregorio S.P. Ho P.L. Leite R.A. Malaquias L.C. Marques R.C. Miyasato P.A. Nascimento A.L. Ohlweiler F.P. Reis E.M. Ribeiro M.A. Sa R.G. Stukart G.C. Soares M.B. Gargioni C. Kawano T. Rodrigues V. Madeira A.M. Wilson R.A. Menck C.F. Setubal J.C. Leite L.C. Dias-Neto E. Transcriptome analysis of the acoelomate human parasite Schistosoma mansoni.Nat. Genet. 2003; 35: 148-157Google Scholar) and release of the draft genome sequence (www.SchistoDB.org) allow us to assign putative functions to many of the transcripts/genes. Two recent studies have reported on tegument protein composition. In the first, an inventory was compiled of proteins present in the whole tegument syncytium relative to the worm body (13van Balkom B.W. van Gestel R.A. Brouwers J.F. Krijgsveld J. Tielens A.G. Heck A.J. van Hellemond J.J. Mass spectrometric analysis of the Schistosoma mansoni tegumental sub-proteome.J. Proteome Res. 2005; 4: 958-966Google Scholar), but the approach provided no information about the position of the constituents within the complex structure. In the second, we reported on the composition of a surface membrane preparation highly enriched by density gradient centrifugation and then subjected to a differential extraction procedure. We were able to identify many components and classify them into cytosolic, cytoskeletal, membrane, and secreted categories (14Braschi S. Curwen R. Ashton P. Verjovski-Almeida S. Wilson RA. The tegument surface membranes of the human blood parasite Schistosoma mansoni: a proteomic analysis after differential extraction.Proteomics. 2006; (in press)Google Scholar). However, it was not possible to determine the location of individual constituents within the surface complex and hence to say which are potentially accessible to the external environment of the parasite (including the host immune system). We report here the use of impermeant biotinylation reagents to label live adult worms in vitro. We then recovered the labeled proteins by streptavidin affinity for identification by tandem MS from which we inferred their relative accessibility. The results were interpreted in the context of immune evasion. Details of schistosome isolate, life cycle maintenance, and recovery of 7-week-old adult worms were as described previously (14Braschi S. Curwen R. Ashton P. Verjovski-Almeida S. Wilson RA. The tegument surface membranes of the human blood parasite Schistosoma mansoni: a proteomic analysis after differential extraction.Proteomics. 2006; (in press)Google Scholar). The scheme from parasite labeling through recovery of biotinylated proteins for MS is set out as a flow chart (Fig. 1). We first established the reaction conditions using BSA as a model protein before applying them to the parasite. Worms were washed five times in Hanks' balanced salt solution (Invitrogen), and any that showed damage were removed under 10× magnification. Two sulfo-NHS 1The abbreviations used are: NHS, N-hydroxysuccinimide; 1-D, one-dimensional; CRP, complement-regulatory protein; SB 3-10, N-decyl-N,N-dimethyl-3-ammonio-1-propane sulfate; strep-HRP, streptavidin conjugated to horseradish peroxidase; bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol. -biotin reagents, differing in the length of their spacer arms (Table I), were used to label worms, namely sulfo-N-succinimidyl-6-(biotinamido) hexanoate (long form reagent; EZ-Link™ sulfo-NHS-LC-biotin) and sulfo-N-succinimidobiotin (short form reagent; EZ-Link sulfo-NHS-biotin; Pierce). For each experiment the parasite load from 40 mice, ∼2000 worm pairs (no attempt was made to separate male and female worms), was incubated in 15 ml of Hanks' balanced salt solution containing 890 μm labeling reagent for 30 min at 4 °C with gentle agitation (15Davies S.J. Pearce E.J. Surface-associated serine-threonine kinase in Schistosoma mansoni.Mol. Biochem. Parasitol. 1995; 70: 33-44Google Scholar). The labeling solution was removed, and any unbound reagent was quenched using RPMI 1640 medium (Invitrogen) containing free amino acids. Worms were then washed three times in RPMI 1640 medium, plunged into liquid nitrogen, and stored at −80 °C with protease inhibitors (protease inhibitor mixture, Sigma). Uniformity of labeling and integrity of the surface membrane was examined by exposing a sample of live, labeled worms in RPMI 1640 medium to streptavidin-conjugated FITC (Sigma) for 30 min at room temperature. After incubation the worms were washed three times in RPMI 1640 medium and optically sectioned using a Zeiss LSM 510 meta confocal microscope (Zeiss UK Ltd., Welwyn Garden City, Herts, UK).Table IProperties of the two biotinylation reagents used to label the S. mansoni surface Open table in a new tab The tegument was removed from labeled worms by the freeze/thaw/vortex method (7Roberts S.M. MacGregor A.N. Vojvodic M. Wells E. Crabtree J.E. Wilson R.A. Tegument surface membranes of adult Schistosoma mansoni: development of a method for their isolation.Mol. Biochem. Parasitol. 1983; 9: 105-127Google Scholar), and the released membrane material was pelleted at 1000 × g for 30 min at 4 °C to yield the starting material. The pellet was subjected to five sequential cycles of extraction with 200 μl of different solvents of increasing strength followed by centrifugation at 100,000 × g for 1 h. Each cycle of the extraction procedure was performed three times, and the resulting supernatants were pooled to give a sample volume of 600 μl. The solubilizing regimes were: Extract 1, 40 mm Tris, pH 7.4, vortexed for 1 min and allowed to stand for 20 min on ice; Extract 2, 5 m urea (BDH, VWR International, Dorset, UK), 2 m thiourea (BDH, VWR International) in 40 mm Tris, pH 7.4, 25 °C; Extract 3, 5 m urea, 2 m thiourea, 4% CHAPS (Sigma), 2% N-decyl-N,N-dimethyl-3-ammonio-1-propane sulfate (SB 3-10; Sigma) in 40 mm Tris, pH 7.4, 25 °C; Extract 4, 0.1% SDS, 1% Triton X-100 in 40 mm Tris, pH 7.4, 25 °C; Extract 5, 0.2% SDS, 1% Triton X-100 in 40 mm Tris, pH 7.4, 25 °C. Protease inhibitor mixture (Sigma) was added to each pooled supernatant prior to isolation of the extracted labeled molecules. The biotinylated material in each of the five extracts was isolated by incubation with 60 μl of a prewashed slurry of streptavidin immobilized onto agarose beads (ImmunoPure® immobilized streptavidin, Pierce) for 2 h at room temperature with head-over-head mixing. The streptavidin-bound complex was pelleted by centrifugation for 1 min at 3000 × g, and the supernatant (unbound material) was recovered. The beads were then washed for 5 min in 500 μl of 0.1% SDS, 1% Triton X-100 in PBS by head-over-head mixing at room temperature to remove any further unbound material and pelleted as before. This wash procedure was repeated four times. The bound material was recovered from the washed streptavidin beads by addition of 100 μl of 2% SDS, heating to 90 °C for 10 min, vortexing at maximum speed for 2 min, and pelleting of denuded beads by centrifugation as above. The supernatant containing the biotinylated macromolecules was recovered, the procedure was repeated, and supernatants were combined to yield 200 μl for each of the five extracts. Proteins from each of the five extracts were precipitated with 10% TCA, 80% acetone and solubilized in SDS sample buffer plus reducing agent (Invitrogen). They were separated by 1-D SDS-PAGE using 7-cm NuPAGE 4–12% bis-Tris gels (Invitrogen) followed by SYPRO Ruby (Bio-Rad) staining and image capture using Molecular Imager FX (Bio-Rad). The biotin content of the bound and unbound material after the incubation with streptavidin-agarose beads was examined by Western blotting. An aliquot of the samples was separated by 1-D SDS-PAGE and transferred onto a PVDF membrane using an XCell II blot module (Invitrogen). The membrane was blocked in 4% casein, probed with streptavidin conjugated to horseradish peroxidase (strep-HRP; Amersham Biosciences), and TMB peroxidase substrate (Kirkegaard & Perry Laboratories) was added to visualize labeled bands. The SDS-PAGE gels of the bound material were restained with Coomassie BioSafe (Bio-Rad), and each protein band was excised, digested with trypsin, and subjected to LC-MS/MS as described previously (14Braschi S. Curwen R. Ashton P. Verjovski-Almeida S. Wilson RA. The tegument surface membranes of the human blood parasite Schistosoma mansoni: a proteomic analysis after differential extraction.Proteomics. 2006; (in press)Google Scholar). Sections of gel lanes where no protein staining was visualized were also analyzed for potential protein content. Briefly peptides were separated on a reversed-phase Monolith column (LC Packings, Dionex, Sunnyvale, CA) using an Ultimate nanoflow HPLC system (LC Packings) and spotted directly onto a MALDI target plate using a Probot (LC Packings). A 4700 Proteomics Analyzer with TOF-TOF optics (Applied Biosystems, Framingham, MA) was used to obtain fragmentation spectra, which were processed by GPS Explorer software version 2.0 (Applied Biosystems) to provide peak lists. These were submitted to MASCOT version 1.9 (16Perkins D.N. Pappin D.J. Creasy D.M. Cottrell J.S. Probability-based protein identification by searching sequence databases using mass spectrometry data.Electrophoresis. 1999; 20: 3551-3567Google Scholar) (Matrix Science, London, UK) and searched against the National Center for Biotechnology Information non-redundant (NCBInr) database as well as the S. mansoni genome and transcriptome databases (www.SchistoDB.org/and cancer.lbi.ic.unicamp.br/schisto6/, respectively). Mass tolerance was set to ±0.3 Da for both the precursor and product ion spectra, and search parameters allowed for a maximum of three missed tryptic cleavage sites, the carbamidomethylation of cysteine, the possible oxidation of methionine, and the possible modification of lysine and N-terminal residues by the biotinylation reagents. A protein was considered to be identified when two or more peptides, each with a GPS-generated confidence interval of above 99%, were positively matched in the database. Putative functions were assigned using BLAST (17Altschul S.F. Madden T.L. Schaffer A.A. Zhang J. Zhang Z. Miller W. Lipman D.J. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs.Nucleic Acids Res. 1997; 25: 3389-3402Google Scholar) against annotated proteins in the GenBank™ database; scores with an expect value <1e−16 were regarded as a significant match. Potential signal sequences and glycosylation sites were predicted using SignalP version 3.0 and NetNGlyc/NetOGlyc version 1.0, respectively (www.cbs.dtu.dk/services/), and transmembrane domains were predicted using HMMTOP (www.enzim.hu/hmmtop/). When the BSA was subjected to the complete procedure from labeling to tandem MS and compared with unmodified BSA, no dramatic alterations in peptide mass fingerprints were observed. From the labeled BSA, we identified 14 peptides, six of which (43%) contained a biotinylated lysine residue (peptide details can be found in the supplemental table). The extent of labeling of live adult worms was assessed using FITC-conjugated streptavidin. A sharply demarcated line of surface staining was observed on whole mounts (Fig. 2). Optical sectioning through the worm body confirmed that the pattern of staining was confined to the surface with no penetration into internal tissues. No biotin reagent or streptavidin had entered the gut during incubation. The labeled tegument surface, detached from worm bodies, was extracted to recover proteins according to their differential solubility. Labeled proteins were then retrieved from each of the five extracts using streptavidin-agarose beads, and the bound material was separated by SDS-PAGE. The SYPRO Ruby-stained images revealed a sharp pattern of bands with strong similarities between the patterns labeled by the two biotin reagents (Fig. 3a). Overall more proteins were retrieved after labeling with the short form rather than the long form reagent. In both instances, the Tris and 0.2% SDS, 1% Triton X-100 extracts (lanes i and v, respectively) appeared devoid of protein with the exception of a single band of ∼15 kDa, which was also present in other extracts. Conversely the other three extractions (lanes ii–iv) recovered more complex mixtures of labeled material. Thus, after urea/thiourea treatment (lane ii), nine bands were visible in the long form-labeled extract, and 15 were visible in the short form-labeled extract. After urea/thiourea/CHAPS/SB 3-10 treatment (lane iii) only three bands were present in both long and short form extracts; the ∼70-kDa species were absent from lane ii. After 0.1% SDS, 1% Triton X-100 treatment (lane iv), five bands were present in the long form and nine were present in the short form extracts; apart from the ∼15- and ∼70-kDa species, the remainder were unique. To verify that the proteins recovered from the beads were biotinylated, an aliquot was separated by SDS-PAGE, blotted onto a PVDF membrane, and then probed with strep-HRP. Each protein band visualized by SYPRO Ruby could be detected as a matching band on the Western blot (Fig. 3b) with the exception of the aforementioned ∼15-kDa protein, which was absent from several lanes. Conversely additional bands of labeled material were detected only on the Western blots. In particular, after short form labeling, a whole array of new bands appeared, especially noticeable in the high molecular weight region of the Tris extract (lane i), whereas the blot of long form-labeled material more closely resembled its corresponding SYPRO Ruby-stained gel (Fig 3b). These data indicate that the sensitivity of biotinylated protein detection by blotting and enzymatic amplification of the signal is much greater than staining with SYPRO Ruby. The supernatant recovered after each of the five extracts that had been incubated with streptavidin beads (i.e. the unbound material) was profiled for both protein and biotin content by SDS-PAGE and Western blotting, respectively. The SYPRO Ruby-stained 1-D gel revealed large numbers of protein bands, reducing in complexity from extract 1 to extract 5 (Fig. 4a, lanes i–v). The blotted material showed traces of biotinylation in experiments with both reagents (Fig. 4b) that was more evident when the short form was used. In both cases the labeled material was almost exclusively confined to the urea/thiourea extract (lane ii) with only the faintest traces visible in 0.1% SDS, 1% Triton X-100 extract (lane iv). Notably the few biotinylated protein bands in the unbound material were also detected in the gels and blots of the bound material (Fig. 3). Bands detected on 1-D separations of fractions recovered from the agarose beads were excised and trypsinized, and the resulting peptide mixture was fractionated by reversed-phase chromatography before identification by tandem MS. In total, 117 peptide fragmentation spectra were matched to genome sequences/expressed sequence tags, but only one revealed the presence of a modified lysine residue (peptide details can be found in the supplemental table). A total of 28 identities was obtained plus streptavidin in all five extracts (Table II). These comprised 13 proteins in samples recovered after use of the long form reagent and the same 13 plus a further 15 after use of the short form reagent. It should be noted that almost invariably a single gel band contained two or more proteins, making it impossible to draw inferences about the relative amounts of individual constituents in the preparation. Furthermore some proteins were identified in several gel bands of different molecular weights.Table IIProteins identifiedProtein nameaThe putative identity gained by BLAST analysis against the GenBank™ database.Accession no.bAccession numbers beginning with Sm correspond to the S. mansoni genome database (www.SchistoDB.org), and those beginning with gi correspond to the NCBInr database.Band number(s)cBand number equating with protein bands in Fig. 3a. Where no number is given (—), the identity was not found in that sample.Long formShort formHost proteins IgM heavy chaingi‖7004847 IgG1 heavy chaingi‖440121710 IgG3 heavy chaingi‖1304160710 Complement C3 (C3c/C3dg fragment)gi‖2817578669Secreted protein Sm29Sm09193812Membrane structural proteins Tetraspanin B (TE736)Sm04463—20, 22, 25 Tetraspanin D (CD63-like tetraspanin)Sm1236616, 1714, 20, 24, 27, 29 AnnexinSm039874, 84, 7, 12 DysferlinSm104332, 3, 53, 5, 6, 8Membrane enzymes CalpainSm08542—7, 11, 22, 23, 26 Alkaline phosphataseSm0096211, 149, 17, 21, 23 ATP-diphosphohydrolaseSm127451520, 22 PhosphodiesteraseSm0345812, 1518, 20, 21, 22Membrane (other) 200-kDa Surface proteinSm03865—2Transporters Voltage-dependent anion channelSm00707—13 Sodium/potassium transporter (SNaK1)Sm08331—21Cytoskeletal proteins FimbrinSm13240—8 ActinSm01276—11 SeverinSm04123—11Cytosolic proteins Glycerol-3-phosphate dehydrogenase 2Sm08702—7 Heat shock protein 70Sm09042—8 Carbonic anhydrase 4Sm04975—23No homology UnknownSm11517—6 UnknownSm01030—8 UnknownSm13096—10 UnknownSm00749—17 UnknownSm11921914, 15 UnknownSm073921513, 20, 21, 22Protein from agarose beads Streptavidingi‖13641561, 10, 13, 18, 191, 16, 19, 28, 30a The putative identity gained by BLAST analysis against the GenBank™ database.b Accession numbers beginning with Sm correspond to the S. mansoni genome database (www.SchistoDB.org), and those beginning with gi correspond to the NCBInr database.c Band number equating with protein bands in Fig. 3a. Where no number is given (—), the identity was not found in that sample. Open table in a new tab Four host proteins, three distinct immunoglobulin heavy chains and complement component C3, were consistently recovered from SYPRO Ruby-stained gels after surface biotinylation. The heavy chains for IgG1 and IgG3 were present in a single band of an apparent molecular mass of 55 kDa. The four peptide hits for IgG3 originated in constant domains CH1, CH2, and CH3 (two peptides), whereas six peptides for IgG1 originated in the variable domain framework regions VH1 and VH3 and the constant domains CH2 and CH3 (two in each). The heavy chain for IgM was present in a gel band of apparent molecular mass 75 kDa with peptide hits originating in the constant domains CH2 and CH4. Complement component C3 was identified in a gel band of apparent molecular mass 69 kDa, and the two peptide hits both originated in the α chain C3dg region; the apparent molecular mass is consistent with the polypeptide recovered being the partial degradation product of the C3 molecule comprising C3c/C3dg. The 24 schistosome proteins could be grouped according to their putative functions (Table II). The long form reagent labeled one protein, Sm29, apparently secreted on the basis of SignalP analysis of the amino acid sequence. Three membrane enzymes, alkaline phosphatase, phosphodiesterase, and diphosphohydrolase, capable of hydrolyzing organic phosphates were recovered as well as three structural membrane proteins, annexin, dysferlin, and the tetraspanin D (CD63-like tetraspanin). The short form reagent retrieved a 200-kDa membrane-bound protein, termed "Surface protein" in the original description, plus one further tetraspanin (B; tetraspanin TE736) involved in membrane structure. A membrane protease, calpain, was also labeled, as were two transporters, the sodium/potassium transporter (SNaK1) and a voltage-dependent anion channel. The short form reagent also appeared to have greater penetrating power, labeling three cytoskeletal proteins, actin, fimbrin, and severin, and three cytosolic proteins, carbonic anhydrase 4, heat shock protein 70, and glycerol-3-phosphate dehydrogenase 2. A further four schistosome proteins of unknown function were also labeled by the short form reagent (Table II). To determine the external accessibility of tegument surface proteins, we incubated live adult schistosomes with impermeant biotinylation reagents. The two reagents used label proteins with exposed amine groups either at lysine residues or at the N terminus. Because of their sulfo group, they are water-soluble and therefore excluded by the lipid bilayer, preferentially labeling secreted proteins and membrane proteins with extracellular loops or domains. It was necessary to keep the duration of the reaction short (30 min) because labeling had to take place in an amino acid-free balanced salt solution rather than culture medium, increasing the possibility of worm degradation. We first optimized the methods for labeling, recovery of proteins, and their identification by tandem MS using purified BSA. The retrieval of biotinylated molecules from agarose-immobilized streptavidin is challenging because of the exceptionally high binding constant of the complex (Kd ∼ 10−15 m (18Green N.M. Avidin.Adv. Protein Chem. 1975; 29: 85-133Google Scholar)). We found that boiling in 2% SD