Mar 11, 1992 - from the Burroughs Wellcome Fund (to L. H. T. V. D. P.), and by. National Institutes of Health Grant R01-AI-10627-21 (to D. D.). The costs of ...
Vol , 267. No. 26. Issue of September 15,, pp. 18459-18465,1992 Printed in U.S.A.
OF BIOLOGICAL CHEM!STRY THEJOURNAL
0 1992 by The American Society for Blochemistry and Molecular Biology, Inc.
Analysis of a 43-kDa Glycoprotein from the Intracellular Parasitic Nematode Trichinella spiralis” (Received for publication, March 11, 1992)
Demetris K.VassilatisSQ, Dickson Despommierll, David E. MisekSII , Ramona I. Polverell, Allen M. Gold**, and Lex H. T. Vander PloegStSS From the $Departmentof Genetics and Development, the 7Division of Tropical Medicine, School of Public Health, and the **Department of Biochemistry and Molecular Biophysics, Columbia University, New York, New York10032
The L1 larvaeof the parasitic nematode Trichinella meats containing infective L1 (pre-adult) larvae. After passpiralis invade skeletal muscle and initiate a process sage through the stomach, the L1larvae migrate to thesmall that has been interpreted to represent skeletal muscle intestine, where they invade the columnar epithelium and dedifferentiation. In this process, the infected region undergo four molts over a 30-hour period to reach sexual of the muscle cellis converted into a unique structure, maturity (1-4). The adultsmate within the intestinalepithecalled the Nurse cell. The nematode T. spiralis can lium (5), and larvae are born 5-10 days later. The newborn survive for tens of years within the cytoplasm of the larvae exit the epithelium from the basolateral surface and Nurse cell and secretes proteins into cytoplasm the that migrate through the bloodstream and lymphatics (6,7). Tranare believed to play a role in mediating the Nurse cell sient infections of various organs including the heart, brain, formation or maintenance. We have cloned a cDNA retina choroid in the eye, and liver have been reported, but encoding the T.spiralis-derived, 43-kDasecreted pro- their primary destination is skeletal muscle. When anewborn tein. Structural analysis of the predicted 344-amino larva enters a skeletalmuscle syncitium it induces permanent acid sequence revealed an N terminally located signal peptide and a potential helix-loop-helix motif in the alterations, leading to the formation of the Nurse cell, in main body of the protein. Antibodies raised against the which it can survive for up to 30 years (8-11). T. spiralis lives 43-kDa recombinant protein were used in immunocy- in thecytoplasm of the Nurse cell. Infection results in loss of tolocalizations of T. spiralis-infected skeletal muscle myofiber proteins (day 4-8 postintramuscular invasion (11, sections. These antibodies strongly stained the Nurse 12)),muscle-specific gene inactivation (13), enlargement and cell nuclei and the nematode itself. Specific, though proliferation of muscle cell nuclei (day 6 postintramuscular slightly weaker staining also occurred in theNurse cell invasion (14)), and alterations in the levels of certain enzycytoplasm. In Western blots, the antibodies react with maticactivities (such as phosphofructokinase), as well as the 43-kDa protein but also detected at least two other secretion of collagen (day 10 postintramuscular invasion (11, T.spiralis-secreted proteins. DNA hybridizations re- 15)) and thepossible secretion of angiogenic factors (day 12 veal at least one additional 43-kDa-related sequence postintramuscular invasion (11)).Growth and differentiation encoded in the T.spiralis genome. We conclude that of T. spiralis and theformation of the Nurse cell are virtually either the 43-kDa protein and/or a closely related 43- complete within 20 days postintramuscular invasion (16). kDa family member is secreted into the muscle and Induction of Nurse cell formation depends upon the T. spiralis translocates to the muscle-derived nuclei. This model larvae, and hence it is reasonable to hypothesize that the may provide insights intothe mechanisms involved in worm directs the process via secretion of proteins and metabNurse cell formation.
The parasitic nematode Trichinella spiralis is infective to a wide range of hosts including mice, rats, swine, and humans. Host infection begins upon ingestion of raw or undercooked *This workwas supported by agrant from the John D. and Catherine T. MacArthur Foundation (to L. H. T. V. D. P.), by a grant from the Burroughs Wellcome Fund (to L. H. T. V. D. P.), and by National Institutes of Health Grant R01-AI-10627-21 (to D. D.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. The nucleotide sequence(s) reported in this paper has been submitted to theGenBankTM/EMBL Data Bankwith accession number($ M95499. 8 Current address: Dept. of Genetics and Molecular Biology, RY8OY-255, Merck Sharp & Dohme Research Laboratories, Rahway, NJ 07065. 1) Current address: Dept. of Signal Transduction,Parke-Davis Pharmaceutical Research Division, 2800 Plymouth Rd., Ann Arbor, MI 48105. $3 To whom correspondence should be addressed. A Burroughs Wellcome Scholar in Molecular Parasitology.
olites. T. spiralis contains specialized secretory cells called stichiocytes, in which at least 20-30 proteins are synthesized and stored in vesicles (17-19). These proteins are enriched in the S3 (large particle) fraction from L1, muscle-derived larvae. Pooled fractions of S3 proteins further purified by preparative isoelectric focussing have been shown to induce protection against subsequent infection (18). Although these proteins have been characterized in terms of their apparentmolecular weights, glycosylation patterns, and isoelectric points, their structures and potential functions in the larvae or in Nurse cell formation remain unknown. An abundant 43-kDa glycoprotein is presentin the S3 fraction and secretion of theL1 larva. Antibodies raised against the purified 43-kDa protein recognized numerous protein bands and stained proteins that localized to thenuclei and to the cytoplasm of infected muscle cells (20). These antibodies reacted almost exclusively with the carbohydrate moieties of the protein (21) and hence the identity of the T. spiralis-secreted protein(s) that translocate to the nuclei of the infected muscle cell remained undetermined. We have cloned the cDNA encoding the 43-kDa glycoprotein. Structural analysis of the deduced 344-amino acid se-
18459
18460
Trichinella spiralis43-kDa Glycoprotein
quence revealed a possible helix-loop-helix domain that shares 46% amino acid identity with the Drosophila protein emc and 38% with the myogenic program negative regulator protein Id in thefirst 13-aminoacid helix (22,23). Immunolocalizations using antibodies raised against a fusion protein containing the 43-kDa amino acid sequence gave strongstainingin muscle cell-derived nuclei in infected cells. Our data indicate that the 43-kDa protein or a related family member(s) is secreted by T. spiralis while living in thecytoplasm of skeletal muscle after which the protein(s)translocate(s) to the nuclei of the infected muscle cell. MATERIALS ANDMETHODS
T. spiralis Infections-L1 muscle larvae were isolated from stock infected mice and rats by peptic digestion according to Despommier et al. (24). Oral infections were administered to anesthetized mice or rats with the aid of a 1-ml tuberculin syringe fitted with an 18-gauge blunted needle. Mice routinely received 500 viable larvae, whereas rats received 5000 viable larvae. Infections were allowed to proceed for a minimum of 35 days before animals were sacrificed. Synthesis of Degenerate Anti-sense Oligonucleotides-Anti-sense oligonucleotides were synthesized on an Applied Biosystems model 380A DNA Synthesizer based upon sequence data obtained from Nterminal and internal peptides of the 43-kDa protein (21). First, degenerate oligonucleotides were synthesized based on the partial protein sequence from the Nterminus of the purified, reduced, pyridylethylated 43-kDa protein (21). The following N-terminal amino acid sequence was chosen: Asp-Asp-Thr-Glu-Trp-Phe. The anti-sense sequence chosen was 5' A/GAACCAC/TTCA/G/TGTG/ ATCG/ATC 3' (oligonucleotide pool 43k-2). The 43k-3 oligonucleotide pool, derived from the amino acid sequence Met-Phe-Gly-AsnGlu-Thr of a CNBr fragment of 43 kDa had the nucleotide sequence 5' GTC/TTCA/GTTG/T/CGCCG/AAACAT 3'. Preparation of RNA and Poly(A+) RNA from T. spiralis L l Larvae and Adult Worms-L1 muscle larvae and adult worms wereharvested as previously described (24), flash-frozen in liquid nitrogen, and then stored at -80 "C until used. Approximately 2 g of frozen worms were suspended in 4 M guanidinium isothiocyanate, 14% P-mercaptoethanol, 100 mM Tris-HC1, pH 7.5, dissociated by mixing with a Polytron (on average, 3 X 30 s), and then centrifuged for 5 min at 12,000 X g in order to pellet insoluble debris. The solution was overlaid on a 3ml cesium chloride cushion ( r = 1.67 g/ml) in SW41 tubes and centrifuged at 36,000 rpm for 18 h. The RNA pellet was resuspended in 10 mM Tris-HC1, pH 7.5, containing 100 pg/ml proteinase K, extracted with phenol, chloroform, isoamyl alcohol, precipitated with ethanol, and stored in ethanol at -20 "C (25, 26). Poly(A+)RNA was purified from total RNA by oligo(dT) fractionation. Preparation of Genomic DNA from T. spiralis L l Larvae-Approximately 2 g of frozen T. spiralis L1 larvae were placed in a mortar(on dry ice) and ground until all larvae were broken (as determined by light microscopy). Broken larvae were placed in a small Erlenmeyer flask and lysis buffer (100 mM Tris-HC1, pH 7.5; 200 mM NaCl, 100 mM EDTA, 1% 0-mercaptoethanol, 1.5%SDS')was added. The solution was incubated at 37 "C until larvae were lysed (normally about 2 h) and then extracted with phenol twice, carefully to avoid shearing the genomic DNA. The DNA was ethanol-precipitated and lightly spooled onto a Pasteur pipette, after which it was washed in 70% ethanol and resuspended in 10 mM Tris-HC1, pH 7.5, 1 mM EDTA (TE) containing 50 pglml RNase A followed by incubation at 37 "C for30 min. Proteinase K was added to 100 pglml, and the solution was incubated for an additional 15 min a t 37 "C. The DNA was extracted with phenol twice, precipitated with ethanol, and resuspended in 4 ml of TE. In order to separate DNA from glycogen, ethidium bromide was added to theDNA to a final concentration of 40 pg/ml. A solution of cesium chloride was prepared so that 1part of DNA added to 4 parts of cesium chloride solution would give a final density of 1.67 g/ml. This solution was centrifuged in a SW50.1 rotor at 30,000 rpm for 40 h. The banded DNAwas visualized using a hand-held UV light, carefully removed from the tube using a sterile Pasteur pipette, extracted twice with isoamyl alcohol, ethanol-precipitated, resusThe abbreviations used are: SDS, sodium dodekyl sulfate; kb, kilobase(s); nt, nucleotide(s).
pended in TE, and stored at -20 "C until used. Preparation of cDNA Libraries-T. spiralis cDNA libraries were prepared according to themethod of Gubler and Hoffman (27) using the cDNA Synthesis System Plus kit (Amersham Corp.), following the protocol included in the kit. The newly synthesized cDNA was methylated using EcoRI methylase (New England Biolabs, Beverly, MA). Dephosphorylated EcoRI linkers (New England Biolabs) were phosphorylated with [32P/ATP, and then ligated onto newly synthesized cDNA.Linkers were cut back with the restriction enzyme EcoRI, and cDNA was separated from free linkers by running the mixture over a Sepharose CL-4B column equilibrated in 10 mM Tris-HCl, pH 7.5,O.l mM EDTA, 300 mM NaC1. The cDNA was ligated onto X g t l l (Stratagene, La Jolla, CA), packaged in Gigapack Gold packaging extract (Stratagene), and amplified in Y1090 Escherichia coli (Promega Biotec) grown in LB medium containing 10 mM MgSO, and 100 pg/ml ampicillin. Screening ofcDNA Libraries-T. spiralis cDNA libraries were titered, plated, and screened with modifications of published protocols. Briefly, a T. spiralis X gtll cDNA library was plated on Y1090 E. coli, and approximately 120,000plaques were screened in duplicate using degenerate 32P-labeledoligonucleotide probes described above. Hybridizations were done a t 25 "C in 6 X NET (1 X N E T 150 mM NaCl, 1mM EDTA, 15 mM Tris-HC1,pH 7.5,lX Denhardt's solution, 2% dextran sulfate, 0.02% sodium pyrophosphate, 0.1% SDS) with 40 pg of tRNA/ml (Sigma) added as carrier. The filters were washed at 44 "C in 6 X SSC, 0.5% Triton X-100 with several buffer changes and exposed at -80 "C on Kodak XR-5 x-ray film. Several washes at higher stringencies (up to 55 "C) were performed to compare and improve signal-to-noise ratios. All positive clones that could be obtained were rescreened until single plaques could be isolated. Positive clones were picked and resuspended in 1 ml of sterile SM buffer (50 mM Tris-HC1, pH 7.5, 100 mM NaC1, 10 mM MgS04,2% gelatin) and stored with 50 pl of chloroform at 4 "C. Preparation of DNA from Positive X Clones-X phage from positive clones was amplified overnight in Y1090 bacteria grown in LB medium (containing 10 mM MgSO, and 100 pg ampicillin/ml). Bacterial debris was removed by centrifugation, and fresh Y1090 bacteria were added. The phage was again allowed to amplify overnight. Bacterial debris was removed by centrifugation, and then DNase I (25 pg/ml) and RNase A (75 pg/ml) were added, and the solution was incubated at 37 "C for 1 h. Phage was precipitated with polyethylene glycol, resuspended in SM buffer, and extracted twice with chloroform after which EDTA was added to a final concentration of10mM. The sample was extracted with phenol three times, precipitated with ethanol, and resuspended in 10 mM Tris-HC1, pH 7.5. Subcloning of Insert DNA from X Clones-Purified X DNA was digested with a 4-fold excess of the restriction enzyme EcoRI and insert DNA band isolated from 1.0% low melting point agarose gels (GIBCO-BRL). The insert was subcloned into pucl8 and used to transform E. coli DH5a bacteria. Northern Blots, Southern Blots, and DNA Sequencing-Restriction enzyme fragments, purified from the digestedplasmid, were subcloned into M13 mp19 and used for sequencing according to the dideoxy chain termination method (28). In order to generate overlapping clones, DNA was cleavedwith specific restriction endonucleases and ligated into the appropriate M13 mp19 vector. T. spiralis genomicDNA and plasmid DNAwere digested to completion by restriction endonucleases, and the digestion products were fractionated on agarose gels. After transfer to nitrocellulose filters (29), hybridizations with 32P-labeledprobes were performed as described (30). After hybridization, filters were washed to 0.1 X SSC, 0.1% SDS at65 "C prior to exposure using Kodak XAR-5 x-ray film. Low stringency Southern hybridizations were done at 50 "C in 6 X NET overnight, and washed at 55 "C in 6 X SSC, 0.1% SDS. The filters were exposed on Kodak XAR-5 x-ray film. Northern analysis of T. spiralis total and poly(A+) RNA was performed according to the procedure of Boedtker (31). Briefly, 1-2 pg of poly(A+)RNA or 5-10 pg of total RNA was run in each lane of a 1.0% agarose gel containing 2.2 M formaldehyde. After transfer to nitrocellulose, the filters were prehybridized and hybridized with 32Plabeled probes (30). Posthybridization washes were performed at 0.1 X SSC, 0.1% SDS at 65 "C prior to exposure of Kodak XAR-5 x-ray film. Expression and Isolation of Recombinant Proteins 313 and C-95A 0.9-kb Fnu4HI fragment of the 43-kDa cDNA, encoding the secreted form of the protein minus its 9 C-terminal amino acids, was subcloned into the blunt-ended Sal1 restriction site of the bacterial expression vector pGEMEX-1 (Promega Biotec). In this vector, the
Trichinella spiralis 43-kDa Glycoprotein
18461
cDNAisexpressedas a fusionproteinwithgene10 of the T7 400c 300c 350c bacteriophage.The plasmid was used to transform E. coli strain JM109 (DE) (Promega Biotec). Transformed cells were grown in 100 ml of LB medium with ampicillin at 37 "C until an Amnm of 0.2-0.5, was added to a after which isopropyl-1-thio-8-D;-galactopyranoside final concentration of0.5mM. The cells were further incubated at 37 "C for 3 h. The fusion protein was separated by preparative SDSpolyacrylamide gelelectrophoresis and visualized by negatively staining the gel with Progreen Staining System (Integrated Separation Systems).The band containing the fusion protein was cut from the gel, andthe protein was electroeluted in Tris-glycine-SDS buffer (190 1.3 kb mM glycine, 25 mM Tris, 0.1.% SDS). For expression of the 95 C-terminal amino acids, a FspZ/Sau3A 400-base pair fragmentof the 43-kDa cDNA was subclonedinto the blunt-ended EcoRI/BamHI restriction enzyme sites of the expression vector Path-111. The 95 C-terminal amino acids of 43-kDa protein would be expressed under the control of the Trp promoter and fused to the C terminus of the TrpE protein. The plasmid was used to transform E. coli strain HB101. The cells were grown at 37 "C in M9 minimal media and were induced by the addition of 0-indoleacrylic FIG. 1. RNA analysis of T. spiralis L1 larvae poly(A+)RNA acid (20 pg/ml). The TrpE-C-95 fusion protein was purified by the (2 pgllane) hybridized at 26 O C in 6 X NET with the '*P-endmethod described forthe 313 fusion protein. labeled 43k-2 oligonucleotide. The differentpanelsrepresent Preparation of Antisera-Two adult New Zealand White rabbits posthybridizationalwashes in 6 X SSC at varying temperatures. The were injected intradermally with approximately 100pg of the fusion washing temperatures are indicatedabove each Northern blot. protein mixedwithcompleteFreund's adjuvant.Fouradditional boostinginjections of 100 pg of protein inincompleteFreund's adjuvant weregiven to the animals at 2-week intervals. The first positive upon rescreening. One clone hybridizing to both43kboosting injection was given 6 weeks after the first immunization. 2 and 43k-3oligonucleotide pools was chosen,and itsnucleoPreimmune serum was collected prior to the immunizations. Sera tide (nt) sequence was determined (Fig. 2). The cDNA measwere kept at 4 'C after the addition of 0.1% sodium azide. IgG/IgA ured 1250 nt and encoded a 344-aminoacid open reading fractions were isolated using ammonium sulfate precipitation (32). frame anda 70-nucleotide poly(A) tail.The size of this cDNA Immunocytolocalizatwns-Slides with paraffin-embedded sections of 15-day-, 35-day-, and 6-month-old infected mouse tongue, thigh, therefore closely matches that of the 1.3-kb mRNA identified and diaphragm muscle were used in immunocytolocalizations. The in the Northern blot in Fig. 1, and the nucleotide sequence paraffin was removed from the sections by three 3-min emersions in obtained must represent almost thecomplete mRNA. The predicted 344-amino acid open reading frame starts xylenes, followed by serial hydration in ethanol/water baths (loo%, 95%, 90%, 80%, 7075, 50%, 30%, and sterile water). Sections were with a translation initiationcodon (35; Fig. 2). The nucleotide blocked with 10% horse serum. Antisera or purified IgG/IgA were sequence of the cDNA extended another 6 n t 5', and addiincubated at 1:lOO to 1:lOOO dilutions in 0.1 M PBS, pH 7.2, at 4 "C tional ATG translation initiationcodons could not be identiovernight. A horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (Sigma) was reacted at a 1:2000 dilution in 0.1 M fied in this region (nucleotidesequence 5' TTCGTT 3'). acid sequence,obtained from the PBS, pH7.2, at room temperature for 1 h. The reactions were Since the N-terminal amino developed with 0.5 mg/ml DAB (Sigma)and 0.003% H202(Sigma)in purified 43-kDa peptide, started with an alanine (codon start0.01 M PBS, pH7.6. Sections were examined bylight microscopy and ing at nucleotide position 1 in Fig. 2), it is reasonable to photographed on Kodak Kodacolor slide film or Kodak Tri-X black- assume that we have indeed identified the ATG translation and-white film. Western Blots-Samples were subjectedto SDS-polyacrylamide gel initiation codon and an immediately adjacent signal peptide, electrophoresis fractionation and electrotransferred to nitrocellulose which had beencleaved from thepurified protein. The predicted amino acid sequence of the 43-kDa cDNA sheets as described by Towbin et al. (33). A horseradish peroxidaseconjugated goat anti-rabbit secondary antibody was used for detecwas compared with the peptide sequence obtained from the tion. The remainderof the procedure was carried outas described by purified 43-kDa protein. All of the six peptides that had been Tsang et al. (34). sequenced (21), representing a total of 112 amino acids, were present in the predicted amino acid sequence of the 43-kDa RESULTS cDNA (amino acids according to Gold et al. (21) are depicted Isolation and Characterization of T.spiralis 43-kDa cDNAin boldface in Fig. 2). Only a single discrepancy existed a t I n order toisolate cDNA clones encoding the 43-kDa protein, amino acid position 81 (amino acid 1 is the translation inititwo sets of degenerate antisense oligonucleotide pools (43k-2 atorMet), where thepeptide sequence gave a threonine, and 43k-3; see "Materials and Methods" for details) were whereas thecDNA sequence showeda methionine codon. We of the peptide synthesized based on the partial peptidesequence of the 43- assume that this results from misinterpretation kDa protein (21). Since data indicating preferential codon sequencing due to low yield, since theproposed threonine was usage of T.spiralis are unavailable, oligonucleotides with all the last aminoacid of the sequenced peptide (Fig. 2). In vitro possible codons for the above amino acids were incorporated expression of the 43-kDa cDNA in rabbitreticulocyte lysates into each pool. The only consideration that was applied to showed a single polypeptideband of the expected sizein SDSdecrease thedegeneracy of the pools was the predicted infre- polyacrylamide gel electrophoresis (data notshown). We conquent occurrence of -CG- dinucleotides in many eukaryotic clude that the cDNA clone indeed encoded the 43-kDa progenomes. Hybridization of thesesynthetic oligonucleotide tein. probes to RNA in Northern blots revealed that both synthetic The predicted amino acid sequence of the 43-kDa protein oligonucleotide pools (43k-2 and 43k-3) detected a similarly revealedseveral interesting features. First, the translation sized 1.3-kb mRNA in T.spiralis L1 larvae poly(A+) RNA initiation methioninecodon is followed by a basic amino acid, (Fig. 1,hybridizations for 43k-2 pool only shown). Arg, and a stretch of hydrophobic amino acids indicating the A T.spiralis cDNA library consistingof 120,000 individual presence of a signal peptide. This finding is consistentwith recombinant clones was screened, using 32P-labeled 43k-2 as the fact that the 43-kDa protein is secretedby the nematode. Second, there are two potential N-linked glycosylation sites a probe. We selected five putative positivelyhybridizing clones for further screening. Of these, three proved to be at amino acids24 and 117 (Fig. 2). These two N-glycosylation
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FIG.2. Nucleotide sequence and deduced amino acid sequence of the 43-kDa cDNA. The one-letter amino acid code is used. Numbering of nucleotides starts with the first nucleotide of the first amino acid (alanine) found a t the N terminus of the secreted form of the 43-kDa protein. The amino acids corresponding to previously sequenced peptides are indicated in boldface. Two potential N-linked glycosylation sites are boxed. The putative helix-loop-helix motif is boxed, and thetwo potential amphipathic helices are shaded. The potential poly(A) addition signal AATAAA is underlined. sites areconsistentwith the two-step deglycosylation observed following treatment of the peptide with endoglycosidase F (21). In the sequenced peptides, the putatively glycosylated asparagines could not be identified, indicating that most likely theyare both glycosylated. Third,structural analysis of the peptide backbone revealed the presence of two strong amphipathic helices, which conform to potentialhelixloop-helix motifs (Fig. 3A). Interestingly, these motifs have been found in several proteins, among which are several that are involved in control of muscle gene expression (36). Helix 1shares 46% amino acid identity with the negative regulator of Drosophila neuronal development emc (22). In addition, helix 1 shares 38% amino acid identity with the muscle
.-
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FIG.4. Southern blot of T. spiralis genomic DNA (5 pgl lane) digested with restriction enzymes. Lanes 1-4, EcoRI, PuuII, ScaI, and BstEII, respectively. Size standards are shown on the left. The Southern blot was hybridized with the 43-kDa cDNA and washed at 65 "C and 0.1 X SSC (panel A ) or at 55 "C and 6 X SSC (panel B ) . The low intensity hybridizing bands are indicated with arrowheads in the ScaI restriction enzyme digestion only in panel A, whereas additional bands in panel B are indicated with asterisks. A t this exposure, the additional bands identified in panel A are not yet visible in panel B .
differentiation negative regulator protein Id (23;Fig. 3B). Helix 2 has no obvious homologies with known helix-loophelix proteins. Another featureof the 43-kDa predicted amino acid sequence is that thefour C-terminal amino acids (CPYS) are consistent with the CXXX isoprenylation motif (37). After we had analyzed the 43-kDa cDNA clone, a similar T. spiralis-derived cDNA sequence was reported by Su et al. (38). The predicted amino acid sequence of this cDNA, which did not include the predicted signal peptide sequence, revealed two amino acid substitutions when compared with the predicted amino acid sequence described here. Other sequences with significant homology could not be detected in searches through the database. Genomic Organization of the 43-kDa Gene-Hybridization of the 43-kDa cDNA with T. spiralis genomic DNA at high stringency revealed a single restriction enzyme fragment only, indicative of a single gene encoding the 43-kDa protein (Fig. 4A).Additional weakly hybridizing bands can also be seen in some digests (bands labeled with arrowheads, Fig. 4 A , lane 3).
Trichinella spiralis 43-kDa Glycoprotein l
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FIG.5. A, Northern blots of size-separated t o t a l T. spiralis L1 larvae RNA ( l a n e I ) or adult ( l a n e a) RNA (10 pgllane) hybridized with the 43-kDa cDNA. Expression of the 43-kDa gene is observed only in the L1 larvae. The approximate size of the hybridizing band is indicated on the left of the panel. B, the panel inA was rehybridized with 7 '. spiralis-derived cDNA clone 17.2, which detects a 700-base pair RNA, to confirm that approximately equal amounts of RNA had been loaded in each lane.
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The intensity of the additional bands does not increase when hybridizations are performed at lower stringencies, indicating that these fragments encode a small region of absolute identity that could bea small exon. Alternatively, since our laboratory strain of T. spiralis is not cloned, the possibility also exists that a different strain, with polymorphisms at the 43-kDa locus, contaminates the population. In hybridizations at reduced stringencies (6 x NET, 50 "C), an additional band of almost equal intensity also hybridized with the 32P-labeled43-kDa cDNA (Fig. 4B; bands are indicated with asterisks). The additional hybridizing band indicates the possible presence of another gene in the genome of T. spiralis, with homology to the43-kDa cDNA. Developmental Expressionof the 43-kDa Gene-The expression of the 43-kDa gene was restricted to the pre-adult L1 larvae of T. spiralis. Northern blots of total RNA from adult worms (Fig. 5A, lane a) and L1larvae (6 monthsold; Fig. 5A, lane 1) were hybridized with 32P-labeled43-kDa cDNA, and the expected 1.3-kb mRNA wasdetected in the L1larvae but not in adult worms. The presence of RNA in the lane with RNA from adult worms was verified by rehybridization of the blot with another T. spiralis cDNA (Fig. 5B)? The precise time of onset of the 43-kDa gene expression is not known. Immunocytolocalization studies on synchronously infected muscle cellsusing antiglycosylated 43-kDa sera show the first appearance of staining in thestichiocyte cells of larvae at day 7 postintramuscular infection (20). However, these antisera exhibit extensive cross-reactivity with several other stichiocyte-derived proteins, and the exact onset of 43-kDa gene expression is therefore still obscure. Several Proteins Are Detected in Western Blots, withAntiserum Raised against Recombinant 43-kDa Proteins-Previous studies to determine the localization of the 43-kDa protein in infected skeletal muscle suffered from a lack of specificity of the antibodies, which detected numerous bands in Western blots (Fig. 6A, lane 3 ) . Most of these bands had been attributed to cross-reacting carbohydrate epitopes (21). We therefore raised antibodies against recombinant 43-kDa proteins purified from E. coli. These antibodies should have a markedly improved specificity when compared with antibodies raised against the purified glycosylated 43-kDa protein and should be diagnostic for the cellular location of the 43kDa protein. We expressed two fusion proteins inE. coli. One
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FIG.6. A , Western blot treated with a 1to 1000 dilution of purified IgG/IgA of antiserum raised against the purified, glycosylated form of the 43-kDa protein. The lanes contain 0.5 pg each oE endoglycosidase F-treated, deglycosylated43-kDa protein (lane I ) , glycosylated 3 ) . The 43-kDa protein (lane 2 ) , and S3 fraction (1.0pg;lune reactivity of these antibodies with the deglycosylated formof the 43kDa protein (band at 32 kDa) is greatly reduced, indicating that the major epitopes recognized by these antibodies are on N-linked carbohydrate chains. The estimated sizes of the glycosylated and deglycosylated forms of the 43-kDa are indicated on the left. B, Western blot with identical lanes as in panelA reacted with a 1to 100 dilution of C95-recombinant protein antiserum. This antiserum reacts with equal efficiency with both the deglycosylated (lane 1) and the glycosylated (lane 2) forms of the 43-kDa protein and specifically and exclusively with the 43-kDa protein in the S3 fraction (lane 3). C, determination of specificity of the anti-313 fusion protein serum. Western blots with equal amounts (0.2 pg) of purified glycosylated 43-kDa protein (lanes I ) , 0.2 pg of deglycosylated 43-kDa protein (lunes 2 ) , 1.25 pgof S3 fraction of T.spiralis-secretedproteins(lanes 3 ) ,and 1.25 pgof endoglycosidaseF-treated S3 (lanes 4 ) . The apparent sizes of the glycosylated 43-kDa (43k) and thedeglycosylated 43kDa (32k) are indicated on the left. Three identical Western blots were reacted with the anti-313 fusion protein serum at l : l O , 1:100, and 1:lOOO dilutions (as indicated below each panel). The anti-313 fusion serum reacts primarily with the glycosylated and deglycosylated forms of the 43-kDa protein but cross-reacts (even at high dilutions of the antiserum) with at least two other T.spiralis-secreted proteins (marked with arrows).
with 95 C terminally located amino acids of the 43-kDa protein fused to the C terminus of the TrpE gene of E. coli, anda second313-aminoacid fusion protein representing almost the entire 43-kDa protein, except for the signal peptide and the last nine C terminally located amino acids,fused downstream of gene 10of bacteriophage T7. Antiserum was raised against the 95-amino acid C-terminal domain. This serum detected only the 43-kDa protein in S3 lysates (Fig. 6B,lane 3 ) . Preimmune serum did not show any staining (data not shown). These data suggest that the 43kDa protein is a single protein that does not exist as a large family of heterogeneously modified forms in the S3 protein fraction. The multitude of bands that can be detected with the antiserum raised against the purified, glycosylated 43kDa protein (Fig. 6A, lane 3 ) therefore probably either results from cross-reactive epitopes in regions of the 43-kDa protein
18464
43-kDaspiralis Trichinella
other than the 95 C-terminal amino acids or from crossreactive carbohydrate epitopes shared with other proteins. Antibodies raised against the 313-amino acid fusion protein (anti-313 fusion) also detected the purified 43-kDa glycoprotein (Fig. 6C, lanes 1 ) and the deglycosylated form of the purified 43-kDa protein (Fig. 6C, lanes 2). Immunoadsorbed anti-313 fusion antiserum (adsorbed on E. coli-bacteriophage T7-gene 10 lysates immobilized on nitrocellulose filters to removegene10-specific antibodies) gave the same results, whereas preimmune serum did not show reactivity (data not shown). The anti-313 fusion sera primarily reacted with the 43-kDa protein in the S3 fraction of L1 larvae (Fig. 6, lanes 3), as shown by the effect of increased dilution of the antiserum (Fig. 6C). However, this serum cross-reacts with at least two other T. spiralis proteins contained in the S3fraction (Fig. 6C, lanes 3, bands identified with arrowheads). Deglycosylation of the S3 proteins reduced their size, but did not result in theproduction of a single reactive protein band (Fig. 6C, lanes 4 ) . We, therefore, assume that theantiserum raised against the recombinant 313 fusion protein detects either several forms of the 43-kDa protein that differ in the processing of their C-terminal end or additional proteins bearing immunological similarities in their peptide backbone. Cytolocalizatwn of the 43-kDa Protein in Infected Skeletal Muscle-Immunocytolocalizations using the anti-313 fusion serum, or purified IgG/IgA fractions, on sections obtained 15 days after synchronous infection or 35 days and 6 months after oral infections, showed staining of the cytoplasm and the muscle-derived nuclei of the Nurse cells (Fig. 7, B and C, data for the 35-day infections shown only;several nuclei have been highlighted with arrows; please note that significant staining of the nematode is only observed in sections that include the stichiocytes; compare with Fig. 8A for preimmune serum; due to the fixation, the worm is separated from the cytoplasm). Immunoadsorbed anti-313 fusion protein serum (adsorbed on E. coli bacteriophage T7 gene 10 lysates) gave identical results (data notshown). We have been unsuccessful, so far, in obtaining well defined staining of the Nurse cell nuclei and cytoplasm with the antiserum raised against the fusion protein encoding the 95 C-terminal amino acids. This could be due to the lower titer of this antiserum or masking of the epitope(s) present in the 95 C-terminal amino acids duringthe fixation techniques. Alternatively, a processed form of the 43-kDa protein or the product of another gene (see Fig. 4B) may locate to the cytoplasm and/or nuclei of the Nurse cell. We tentatively conclude that antibodies raised against the 43-kDa recombinant protein identify a T. spiralis-derived protein(s) that is/are secreted into the cytoplasm of the infected muscle cell, at least one of which translocates to the muscle-derived nuclei of the Nurse cell.
Glycoprotein b
FIG. 7. Immunocytolocalizations using rabbit anti-313 recombinant IgG/IgA. Mouse muscle sections obtained 35 days postinfection with 7’. spiralis were used. A horseradish peroxidase-conjugated goat anti-rabbit antiserum was used for detection of antibody. Staining was performed with diaminobenzidine and HZ02.A, preimmune IgG/IgA at 1:500 dilution. The uninfected muscle, the Nurse cell, and the worm do not stain. Staining of a region on the right is due to clustered red blood cells that haveendogenousperoxidase activity. B, anti-BlB-IgG/IgA at a 1:500 dilution. The Nursecell cytoplasm and the muscle cell-derived Nurse cell nuclei (indicated by arrows) stain strongly. In this particular section, the worm does not stain since there are no stichiocytes in this region of the nematode. C, region of another Nurse cell from the same experiment as shown in panel €3, but at a higher magnification. The stained nuclei are indicated by arrows. In this section, T.spiralis stichiocytes also stain, whereas cytoplasmic staining in this region of the Nurse cell is almost absent.
sequence revealed a possible helix-loop-helix motif with homology to the negative regulator of myogenic differentiation Id and theDrosophila protein e m . Antibodies raised against the recombinant 43-kDa protein detect protein(s) in the nuclei and cytoplasm of infected skeletal muscle. However,these antibodies detected up to three bands, including the 43-kDa protein, any one of which may be localized to the skeletal DISCUSSION muscle nuclei; the identity of the nuclearly located protein We have cloneda cDNA that encodes the 43-kDa glycopro- therefore remains uncertain. We also cannot exclude the less tein of the nematode T. spiralis. The gene for the 43-kDa likely possibility that theT. spiralis infection induced expresprotein is expressed in the L1 larvae of infected skeletal sion of a nuclear protein encoded by skeletal muscle, which muscle (this paper), and the 43-kDa protein can be purified shares epitopes with the T. spiralis-derived 43-kDa protein. from in vitro secretions and the S3fraction of L1 larvae that Because of these uncertainties, we tentatively conclude that have been isolated from skeletal muscle (21). We, therefore, either the 43-kDa protein itself, or the product of a related consider it reasonable to assume that the 43-kDa protein is gene, is localized to thenuclei of infected muscle cells. This research was initiated because of our interest in the indeed synthesized by the L1larvae in the Nurse cell and that mechanisms by which the intracellular nematode T. spiralis it is secreted into the cytoplasm of infected skeletal muscle. The predicted 344-amino acid sequence of the 43-kDa gene induces formation of the Nurse cell. The possibility that the indicates that thiscDNA encodes a secreted glycoprotein with 43-kDa protein, or a closely related family member of this a typical signal peptide and two potential N-linked carbohy- protein, locates to the nucleus of the affected myocyte and drate moieties. Structural analysis of the predicted amino acid the homology of the 43-kDa protein with helix-loop-helix
Trichinella spiralis 43-kDa Glycoprotein
18465
16. Des ommier, D. D., Aron, L., and Turgeon, L. (1975) Exp. Parasitol. 37, 198-116 17. Despommler, D. D., and Muller, M. (1976) J. Parasitol. 62,775-785 18. Despommier, D. D., and Laccetti, A. (1981) Ex . Parasitol. 61,279-295 19. Despommier, D. D., and Laccetti, A. (1981) J. fkasitol. 67,332-339 20. Despommier, D. D., Gold, A. M., Buck, S. W., Capo, V., and Silberstein,D. (1990) Exp. Parasztol. 71,27-38 Acknowledgments-We thank Dr. S. Le Blancq for critical reading 21. Gold, A.M., Despommier, D. D., and Buck, S. W. (1990) Mol. Biochem. Parasitol. 41, i87-196 of the manuscript and S. Buck for helpful discussion. 22. Garrell, J. and Modollel, J. (1990) Cell 61, 39-48 23. Benezra, k., Davis, R. L., Lockshon, D., Turner, D. L., and Weintraub, H. REFERENCES (1990) Cell 61,49-59 24. Despommier, D. D., Campbell, W. E., and Blair, L. S. (1977) Parasitology 1. Villella, J. B. (1958) J. Parasitol. 44% 41 74,109-119 2. Ali Khan, 2. (1966) J. Purasitol. 62, 248-259 25. Auffray C , and Rougeon F. (1980) Eur. J. Biochem, 107,,303-314 3. Kozek. W. J. (1971) J . Purasitol. 67. 1015-1028 26. Gl@hL+.,'Crkvenjakov, k., and Byus, C. (1974) Bzochemlstry 13, 26334. Kozek; W. J. (1971j J. Purasitol. 67;1029-1038 2637 27. Gubler, U., and Hoffman B. J. (1983) G e m Amst ) 26 263269 5. Gardiner, C. H. (1976) J. Purasitol. 62,865-870 28. Sanger F., Coulsen, A. k., Barrell, B. G., Qmith, A. j. H.: and Roe, B. 6. Basten, A., and Beeson, P. B. (1970) J. Exp. Med. 131, 1288-1304 (1986) J. Mol. Biol. 1 4 3 , 161-178 7. Harley, J. P., and Gallicchico, V. (1971) Exp. Purasitol. 30, 11-12 Southern E. (1975) J. Mol. Biol. 98,503-517 8. Despommier, D. D. (1976) Musculature: Ecological Aspects of Parasitology 29. 30. Jeffre s k.J. and Flavell, R. A. (1977) Cell 12, 429-439 (Kennedy, C. R., ed) pp. 270-285, Elsevier-North Holland, Amsterdam 31. Boedtkr, H. (l971),Bioehim.Biophys. Acta 240, 448-453 9. Ribas-Mujal, D., and Rivera-Pomar, J. M. (1968) Virchows Arch. A 346, 32. Harboe H. and In Ild A (1973) In A Manual o Quantztatiue Immunoelec154-168 and A p lications (Axefsen, N. H. Kroll, J., and trop&re&: Met%& 10. Stewart, G. L. (1973) Thesis, Rice University, Houston Weeke B., eds) p 161-164, eniversitetforla et, Oslo, dorway 33. Towbin, k., Staeheyin, T., and Gordon, J. (1959) Proc. Natl. Acad.Sci. 11. Despommier, D. D. (1975) Am. J. Pathol. 78,477-496 U. S. A. 76,4350-4354 12. Jasmer. D. P., Bohnet, S.. and Prieur, D. J. (1991) Exp. Parasitol. 72,32134. Tsang, V. C. W., Peralta, J.M., and Simons,A. R. (1983) Methods Enzymol. 331 13. Jasmer, D. P. (1990) Exp. Parasitol. 70,452-465 14. Despommier, D., Symmans, W. F., and Dell, R. (1991) J. Parasitol. 77, 290-295 15. Teppema, J. S., Robinson, J. E., and Ruitenberg, E. J. (1973) Parasitology 66, 291-296
proteins provide us with the intriguingoption that these secreted proteins might function in modulating gene expression of the affected muscle cell.
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