- Email: [email protected]
Mimicry of elastin repetitive motifs by Theileria annulata sporozoite surface antigen
Molecular and Biochemical Parasitology, 53 (1992) 105-112 © 1992 Elsevier Science Publishers B.V. All rights reserved. / 0166-6851/92/$05.00
105
MOLBIO 01748
Mimicry of elastin repetitive motifs by Theileria annulata sporozoite surface antigen R o g e r Hall a, Philip D. H u n t a, M a r k C a r r i n g t o n b, David S i m m o n s c, Susanna Williamson d, R o b e r t P. M e c h a m e and A n d r e w Tait f "Department of Biology, University of York, York, UK; bDepartment of Biochemistry, University of Cambridge, Cambridge, UK; ClCRF Laboratories, Institute of Molecular Medicine, John Radcliffe Hospital, Oxford. UK; dCentrefor Tropical Veterinary Medicine, University of Edinburgh, Roslin, UK; ~Jewish Hospital of St. Louis, Washington University Medical Center, St. Louis, MO, USA; and rWellcome Unit for Molecular Parasitology, University of Glasgow, Glasgow, UK (Received 15 November 1991; accepted 7 February 1992)
Theileria annulata is an important pathogen of cattle in the tropics. The gene sequence of a sporozoite surface antigen (SPAG-I) is reported. Data is also presented demonstrating that SPAG-I is synthesised as a large precursor. This antigen, which is a candidate for inclusion in a subunit vaccine, shows a remarkable degree of molecular mimicry to the extracellular matrix protein elastin. It contains both repetitive motifs PGVGV and VGVAPG. lmmunofluorescence using a monoclonal antibody against VGVAPG confirmed that this peptide is expressed on sporozoites as predicted. The presence of VGVAPG is particularly interesting since this is the ligand for elastin receptors on a range of cell types, including macrophages/monocytes which are a major class of host target cells. It is proposed that this antigen represents the ligand whereby T. annulata recognises its host cells. Key words: Theileria annulata; Molecular mimicry; Subunit vaccine; Receptor-ligand; Elastin; Sporozoite antigen
Introduction
In order to penetrate target cells intracellular parasites must undergo a recognition event, presumably via an interaction between ligands on the parasite surface and receptors on the exterior of the target cell (for reviews see refs. 1,2). The infective stage (sporozoite) of the apicomplexan protozoan parasite, Theileria annulata, a major pathogen of cattle in Asia, southern Europe and north Africa, preferenCorrespondence address: R. Hall, Department of Biology, University of York, Heslington, York, YOI 5DD, UK
Note: Nucleotide sequence data reported in this paper have been submitted to the GenBankT M data base with the accession number M63017 Abbreviations: PBS, phosphate-buffered saline; BSA, bovine serum albumin; mAb, monoclonal antibody
tially invades BoLA class II bearing cells, particularly monocytes/macrophages and B cells [3,4]. The molecular details of the iigand-receptor interactions involved in T. annulata invasion are unknown, but some aspects of the process have been elucidated [5,6]. Similar results are available for the closely related species T. parva [7]. Recently we have identified an antigen (SPAG-1) on the surface of T. annulata sporozoites using a monoclonal antibody which blocks sporozoite penetration [8]. This antigen is not only a candidate for the development of a vaccine but also a putative ligand for recognition of the target leucocytes. Our results based on the complete cDNA sequence encoding this molecule, and immunofluorescence analysis demonstrate the presence of 2 regions showing remarkable homology to the repetitive domain in bovine elastin, an extracellular matrix
106
protein [9]. We also present data demonstrating that the protein is produced as a precursor which is subsequently processed to produce the mature forms of the molecule.
Materials and Methods
Parasite material, cDNA synthesis, cloning and sequencing. Total RNA was extracted exactly as described [8] from the salivary glands of 2day fed ticks (Hyalomma anatolicum anatolicum) infected with T. annulata (Hissar) [10]. cDNA was synthesised [11] and ligated to Bst XI adaptors and inserted into the vector pCDM8 [12]. The resulting library was screened with the Eco RI insert derived from 2gtll-SRl [8] and of several positive clones, one (pCSPAG7) with a 2.8-kb insert was selected for sequence analysis using the chain termination method [13]. A combination of random 'shotgun' cloning [14] into M13mpl8 and an ordered approach using specially constructed oligonucleotide primers was used to generate the full sequence. Sequence analysis was performed using the University of Wisconsin Genetics Computer Group software [15] run on a VAX computer. Indirect immunofluorescence. This was performed on T. annulata (Hissar) sporozoites exactly as previously described [16]. Briefly, Percoll-purified sporozoites [17] were fixed with 3.7% formaldehyde in PBS and spotted onto multiwell slides (Hendley, Essex) and left to dry. Individual wells were incubated with 10 #1 of monoclonal antibody diluted 1/100 in PBS followed by 10 /~1 of 1/40 diluted fluorescein labelled anti-mouse IgG (Sigma). They were examined on a fluorescence microscope and photographed. The monoclonal antibodies used were 1A7 anti-SPAG [8], BA4 anti-elastin peptide VGVAPG [18], and 5El which is directed against an antigen found in the merozoite and piroplasm (intra-erthrocytic) stages [19]. Cell-free protein synthesis, immunoprecipitation
and gel electrophoresis. Cell-free translation was performed using the nuclease treated reticulocyte lysate system [20]. Briefly a 10 pg aliquot of RNA, extracted from T. annulata (Hissar) sporozoite infected tick salivary glands [8], was translated in 100 pl of translation cocktail in the presence of 50 pCi [35S]methionine at a specific activity of 1000 Ci mmol --~ (DuPont-NEN). Immunoprecipitation of the translation products was performed with the rabbit anti-2gtil SRI fusion protein [8] as described [21]. The immune complexes were separated by SDS-PAGE [22] and the bound antigen was visualised by fluorography after treating the gel with En3Hance (Amersham). Results
Sequence analysis. The complete coding nucleotide sequence, derived from a cDNA clone, of a previously characterised sporozoite surface antigen, SPAG-1, from T. annulata [8], is shown in Fig.1. There is a single open reading frame extending for 2721 nucleotides encoding a predicted 91.9-kDa polypeptide of 907 amino acids. The region underlined near the Cterminus represents the sequence defined in the original 2gtll clone (2gtil-SR1), and contains the epitope for monoclonal antibody IA7 [8]. There are several remarkable features about the predicted amino acid sequence (summarised in Fig 2(i)). The most notable is the presence of 2 regions containing the pentapeptide PGVGV (module 2, Fig. 2(i)) repeated several times (shown boxed in Fig.l). A repetitive region containing the identical pentapeptide unit occurs in elastin from several diverse sources including cow, chicken, and man [9,23,24]. The alignments [15] with bovine elastin shown in Fig.2(ii) show this feature very clearly. The first block of repeats (residues 179-236) is the most similar to the elastin structure, having exactly the same number of units (11), and differing in only 3 positions (Fig. 2(ii)a), due to the presence of an extra glycine in unit 7 and extra terminal alanines in
107 1
GTTTTAAAAAGGAGTAACATTGGAATTTAAATTTCATTTTCCAACACTCAACGATGAATATTATACACTTTCTGTTGACCATTCCGGCTA
90
M N I I H F L L T I P A I T T T T T G T A T C T G G A G C G G A C A A G A T G C C T G C C ~ A G A A A G TTC T A G A A C C T C T A A A C C C A G T C C C C T A G T A A C C C T A G A A T C G G C G G T A A F V S G A D K M P A G E S S R T S K P S P L V T L E S A V T CAC AACC TTCAAAGGAC C C ATTCAAGAC AATTAGTGCC TTGTCAAAAGCAACAAAAGTATGGAAGTCAGCGGTA TCAG TATCAGGTGAC T Q P S K D P F K T I S A L S K A T K V W K S A V S V S G D S CTAAGAC TGTACCTACTCCAGTTTCGGAACCAATGATCACTCGATCTTTTCAAGAAC CAGTATCTCAAGAACTTGAATTCCAATCAGATA K T V P T P V S E P M I T R S F Q E P V S Q E L E F Q S D T
270
361
CTGAAATTAATGAGTCAGGATCCGGTTCAGATGAGGATGAGGATGACGATGACGATGAGGAGGAAGAAGAAGACGATAAATCTACCTCAT
450
451
E I NLE S G S G CTAA~CGGAAAAGGCAGCCCAAAAGC K N G K G S P K
540
541
TATCACAAACTGGATTC-C~ACCAAGTGGTTCCCACGCTCAACAAGATCCCGGTGTAGGTGTTCCAGGAGT~TGTTCCAGGAGTAGGTG
91 181 271
631 721 811 901 991 1081 1171 1261 1351 1441 1531 1621 1711 1801 1891 1981 2071 2161 2251 2341 2431 2521 2611 2701 2791
D E D E D D D D D E E E E E D D K S T S S TCAGCCTC-GAGTATCTTCAAGCAGTACATCCTCAGCAAGTC CAACATCTCCAAC TACAACAT A Q P G V S S S S T S S A S P T S P T T T L
180
360
S
630
S Q T G L G P S G S H A Q Q DIP G V G V P G V G V P G V G V l TTC CAGGAGTAGGTGTTCC AGGAG TAGGTGTTCCAGGTGTAGGTGTGC CAGGTGTAGGAGGTGTTCC CGGAGTTGGCGTTGCACCAGGGG 720 IP G V G V P G V GV P G V G V P G V G G V PG V G V A P G V J TAGGTGTTCCAGGAGTTC-G TGTTGCAC C AGGTGTAGGTGTTGGAGC TGATAGTAGTGGATTGCCTGGAAGTC~TGGTC TTGGAGCAGGAG 810 IG V P G V G V A P G V G V GIA D S S G L P G S G G L G A G A C A A A G G C T G G G A A A G G T C A A G G A T C T G G T C T A C A G G G A C C A G G A G G T G T T G G A G T A G TAC C T G G T G T A G G T G T A G C A G C T T C TTC T T C T T 900 K A G K G Q G S G L Q G P G G V G V V IP G V G V IA A S S S S CAC CAGGAAAACCTC CAGGAGTAGGAGCAGGAGTTATGC C TGGAGTTGGTGTA CGAGCACAAGGAGGAGTAATAATTGGTGCGCCAGGAG 990 P G K P P G V G A G V M . IP G V G V IRA Q G G V I I G A P G V T A G C A G G T G T G C C A G G A G G A A A G C C A G G A C A A C C A G TATC T C A A G A A C T T G A A C T G A A A T C A G A C A C T G A A A T T A A T G A G T C A G G T TC C A 1080 A G V P G G K P G Q P V S Q E L E L K S D T E I NLE S G S S G T T C A G A A G G G G A A G A C G A T G A C G A T G A A G A A G A G G A A G A A G A A A A T A A A T C T A C C T CATC T A A A G G A G C A G G A G G A A A G G C T G G A A A A G 1170 S E G E D D D D E E E E E E N K S T S S K G A G G K A G K G GTCAAGGATCTGTATCACCAGGAGGAGGATCC TCAGCAAGTCAAACATCTCCAACTACAACACCACAATCTGGC TTGGCATCAAGTGGTT 1260 Q G S V S P G G G S S A S Q T S P T T T P Q S G L A S S G S CTCATGCTCAACAAAGTCC TCAACAAGATCCAGCGC CTAGTAAACCTAGTGGAGGAGGTGTGCCAGGAGT'I~AGTTCCTC, GTGTTGGCG 1350 H A Q Q S P Q Q D P A P S K P S G G G VIP G V G V P G V G V I TTC C CGGTGTTGGAG TAC CAGGAG TAGGAGTTGCGC CGGGAGTTGGTGTTGTAC CTGGAGTAC~TG C A A C A A C T T C TTC A T C A T C A A 1440 IP G V G V P G V G V A P G V G V V P G V G G IA T T S S S S T CAACTTC AACTTCAACTTCAACTACTACTACTACTACAAC TTCATCAGGAAAA CCTTCAGACCAAGGAAGCCATGGTACTTCTCCAAGAA 1530 T S T S T S T T T T T T T S S G K P S D Q G S H G T S P R N ATGCAGTAACCAGACAAACTGACTCAATATCAGGAC C C A T A C C A T C A C C A G G A G A T C C A A G A G C A A T T A C TC-GA C A A A T G G G T G A A G G A G 1620 A V T R Q T D S I S G P I P S P G D P R A I T G Q M G E G E AAAC,G T T T G C T G T A C A G T T C C T G G G A G A T T T T A A A C C A A A A C C A A G G A G A T A T G A A G G A C A A G G A A C A G A T G C A G T A A A A C TAAAA CAAT 1710 R F A V Q F L G D F K P K P R R Y E G Q G T D A V K L K Q F T C A T T T T CGAAGAC, G T C A A A T C G C T G G T G C A A A C C T T A A T A A A C C T T A A A T T A G C A A T T G C A A A C G A C T T T G T T G A A A T C A G T G A A A A G T 1800 I F E E V K S L V Q T L I N L K L A I A N D F V E I S E K L T G A A A A A G A A A A A T C A A A A T T A C G TA C C G A A A T T A A A G T T G T T A A A A G G A G A A C A A T T T G A CAC C A A A C A G A A G G T A G C C A A C G T A C T A A 1890 K K K N Q N Y V P K L K L L K G E Q F D T K Q K V A N V L K AAGC43TT C A A T T C T C T G T A C T T C G T A T T T T T T A T G A A C C T T A A C CTAGC G A A A G A A G T T A A C A A A C C G G A A G A A T T G G C A G A A T T T C T T T 1980 G F N S L Y F V F F M N L N L A K E V N K P E E L A E F L W GGAAACTAAA TACAA TCCC AGATAAAGTAGGAAGAGAATTTGAGTTAGCAATAGAAAAAAC TAAAGGTTCAGAGAAAAAGAAGGAA TTAG 2070 K L N T I P D K V G R E F E L A I E K T K G S E K K K E L E
AAGAAGCATTTAATTCAATAC~TTAGGTTTCAAAATA@CACAGTACGCAACAAATGACATCCTCTCAAGTATAACAAATTCAGTCTACT E A F N S I G L G F K I A Q Y A T N D I L S S I T N S V Y S CCC TGATAAAACTAAAGAATTTTGGAGATGATTTTGTTACCGAAGTAAGAAAGTCAC TGCAAATGGTTC CACAC CAAAAGAACCTAAACG L I K L K N F G D D F V T E V R K S L Q M V P H Q K N L NL G GAT CAGC ATTTATAGTCAAAATCT CAGAAATAATCAACAAAAAAGGAACAGAAGATC AGGATCAAACAT CAGGAAGTGGGTCAAAAGGAA S A F I V K I S E I I N K K G T E D Q D Q T S G S G S K G T CAGAAGGAGGA TCAC TAAGGG~ AAGATTTGACAGAAGAAGAAGTTTTGAAAGTTC TGGATGAA C TAG TGAAGGATGTAAG CGAAGAAC E G G S L R G Q D L T E E E V L K V L D E L V K D V S E E H ATGTTGGAATAGGAGATTTAAGTGACCCAAGTAGCAGAACACCAAATGCAAAACCAGCCGAACTTGGAC CTTCACTAGTGATACAAAATG V G I G D L S D P S S R T P N A K P A E L G P S L V I Q N V TACCGTCAGACCCCTCAAAAGTGACACCAACACAGCCTTCAAATTTGC~ACAAGTACCAACAACAGGGCCGGGGAALCGGGACGGATGGAA P S D P S K V T P T Q P s N L P Q V P T T G P G N G T D G T CAA CAAC AGGA C CAGGTGGAAACGGGGAAGGAC.GCAAAGA TTTGAAGGAAGGAGAAAAGAAAGAAGGATTATTT CAAAAGAT CAAAAACA T T G ~ ~ G N G ~ q G K D U ~< ~ G E K K E $ ~ ~ Q K ~ K ;~ A A C T C T T G G G C T C A G G A T T C G A A G T C G C A A G T A T T A T T A T A C C A A T G A C A A C A A T C A T A T T CAGC A T A G TC C A C T A A A A C T A A A A A C A C A L L G S G F E V A S I I I P M T T I I F S I V H * ACTAACCACAC TAAT TTATAATATACAAAAAAAAA 2825
2160 2250 2340 2430 2520 2610 2700 2790
Fig. 1 c D N A sequence of 7". annulata sporozoite surface antigen (SPAG-I). The predicted amino acid sequence is shown below. The PGVGV repeat regions showing homology to elastin are boxed. The C-terminal region encoded by the insert of 2gtl I-SRI, used to isolate this clone, is underlined. Putative N-linked glycosylation sites are marked with arrowheads.
108 {])
-~
2
I
2
3
mmmmm
i
"
I'
1: D/E regions: amino acids 92-144 and 323-364 2: [PGVGV]n : amino acids 179-236 and 424-456 2: T/S region : amino acids 458-478 4:1A7 epitope : between amino acids 778-891
(ii) a s
179
PGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGGVPGVGVAPGVGVPGVGVAPGVGV
,,rlllllllJllllllllillil E
335
S
588
E
i010 648 1070 708 1130
S
424 335
I I illllll
236
lllltll,;l
IIIII
PGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGV.GVPGVGV.PGVGVPGVGV.PGVGV
389
CCCGGTGTAGGTGTTCCAGGAGTTGGTGTTCCAGGAGTAGGTGTTCCAGGAGTAGGTGT'? II II II il II II II II II II I. I, II II I I ] I I I I I :I CCAGGAGTTGGAGTCCCTGGTGTCGGGGTCCCTGGTGTCGGGG'F'FCCAGGTGTCGGGGTC
647 II
II 1069
C CAGGAGTAGGTGTTCCAGGTGTAGGTGTGCCAGGTGTAGGAGGTGTTCCCGGA,<;TTGGC I II II If I I I I I I I I I I I I! II I, IIIII II II II 'I IIII: CCT(;GTGTCC~;GGTTCCAGGTGTCGGAGTCCCAGGTGTT. . .G G A G T C C C A G G T G T T G G A GTTGCACCAGGGGTAGGTGTTCCAGGAGTTGGTGTTGCACCAGGTGTAGGq~q"I' II 'I II II rl II ~I li I!II II II !IIII GTC...CCTGGTGTTGGGGTCCCTGGTGLFI'G'GCGTC...CCTGGTGTTGGAGTT
PGVGVPGVGVPGVGVPGVGVA
IIIIIIIIIIIIIIIIII E
I II
.... P G V G V .... V P G V G G A
!:
P G V G V P G k iG V P G V G V P G V G V P G k
IIIII
707 ] 129
761 II
:II 1183
456
lilll.:
IGV.m G V G V P G V G V P G V G V P
375
{iii) 92
PVSQELEFQSDTEINESGSGSDEDEDDDDDEEEEEDDKSTSSK
IIIIII1:.111111
324
:::111111111::11111} (c PVSQELELKSDTEINESGSSS--EGEDDDDEEEEEENKSTS~K
]~4
I,Izl
264
Fig. 2. Structural features of SPAG-I. (i) Cartoon of the polypeptide SPAG-I. The modules marked with a I represent a directly repeated peptide sequence whose precise composition is shown in (iii) below and which contain the D/E regions. Module 2 represents the regions containing the PGVGV and VGVAPG repeats. Box 3 represents the T/S region. Box 4 represents the region containing the IA7 epitope. (ii) Alignment of the PGVGV repeat regions encoded by SPAG-1 with the homologous region in bovine elastin. In all cases the top line (S) represents the Theileria sequence and the lower line (E) the bovine sequence. (a) Comparison of the amino-terminal PGVGV repeat. (b) Comparison of the DNA sequence encoding the N-terminal PGVGV repeat. (c) Comparison of the more C-terminal PGVGV repeat. The VGVAPG motif is underlined. (iii) Comparison of the internal repeat sequence designated as module 1 in (i) above. The D/E regions are underlined. For more details see text. The alignments were made using the BESTFIT programme on the U W G C G package [15].
units 8 and 10. It is interesting that this remarkable degree of homology at the protein level is not reflected to the same extent at the nucleotide level, since over this region there is a high degree of third base wobble (Fig. 2(ii)b). Over the region compared in Fig. 2(ii)b there are 43 silent third base differences of which 18
show A/T for G/C substitutions whilst only 3 show the reverse. This is consistent with the known A/T richness of the Theileria genome [25]. The second region of PGVGV repeats (residues 424--456, Fig. 2(ii)c) consists of only 6 units, the first 3 being perfect and the last 3 being slightly degenerate. The fourth unit,
109
of threonine (13 residues) and serine (9 residues) and thus called the T/S region (designated as module 3 in Fig. 2(i)). At the amino terminus there is a stretch of 18 mainly hydrophobic amino acids (residues 1-18), which is predicted by the S I G N A L P E P programme [15] to be a signal peptide. At the C-terminus there is a 24-residue hydrophobic domain (residues 883-907) which could be a membrane anchor. There are also 5 potential N-linked glycosylation sites marked with an arrowhead (Fig. 1). Overall the protein is relatively rich in glycine (15.1%), serine (11.8%), proline (8.3%) and valine (8.9%). There are no cysteine residues and hence no disulphide bonds. Codon usage also shows marked bias in favour of codons with an A in the third position (analysis not shown).
containing a terminal alanine residue, is identical to units 8 and 10 in the first repeat region. These units, with an additional alanine residue, may be highly significant since in all 3 cases a hexapeptide of the form V G V A P G can be generated (underlined in Fig.2 (ii) a). This hexapeptide also occurs in elastin, outside the repeat region, and has been demonstrated to be chemotactic and a ligand for the elastin receptor identified on a range of cells including fibroblasts and monocytes [26-28], (see Discussion). A number of other features may be noted from this sequence (Fig. 2). There appears to have been an internal duplication event such that the sequences designated as modules 1 and 2 occur as tandem repeats towards the amino terminal half of the molecule. Module 2 contains the PGVGV repeats just described. The 2 versions of module 1 are almost perfect matches over 41-43 amino acids and the alignments are shown in Fig. 2(iii). Within each of the versions of module 1 is a region of extreme negative charge consisting entirely of glutamic and aspartic acid residues (shown underlined in Fig. 2(iii)). The more amino terminal of these contains 9 aspartates and 7 glutamates; the other consists of 4 aspartates and 7 glutamates. These modules are thus designated as D/E regions. Immediately downstream of the second block of PGVGV repeats is a region of 22 polar amino acids consisting
A
Monoclonal antibody to the VGVAPG elastin motif recognises sporozoites. To extend the findings from our sequencing data, and confirm that the hexapeptide V G V A P G was present, as predicted, on sporozoites, an immunofluorescence assay was undertaken using a monoclonal antibody, BA4, which specifically recognises this peptide [18]. The results are shown in Fig 3A. As can be seen, the sporozoites are clearly labelled and thus express this determinant. Positive (anti-sporozoite monoclonal I A7) and negative (antimerozoite monoclonal 5El) controls are
B
C
Fig. 3. Indirect immunofluorescence assay on sporozoites. (A) BA-4 anti VGVAPG monoclonal [18]; (B) IA7 anti-sporozoite monoclonal (positive control); Panel C. 5E 1 anti-merozoite and piroplasm monoclonal (negative control). The sporozoites in the positive panels (A and B) are identified as fluorescent specks. The photographs were taken at x 400 magnification.
I10
1
2
-
115
Cell-free protein synthesis demonstrates that S P A G - I is synthesisised as a precursor. In our previous characterisation of this antigen we demonstrated that it was complex and that at least 4 peptides (85, 70, 63 and 54 kDa) carried the epitope defined by mAb I A7, in extracts of purified sporozoites [8]. We were unclear whether these represented independent gene products or were the result of processing from a common precursor. To resolve this issue we translated sporozoite m R N A in a reticulocytelysate cell-free system and immunoprecipitated the product with a rabbit antiserum against thc original 2gtll-SRl fusion protein. These results are illustrated in Fig. 4 and clearly show that the primary translation product is a single major polypeptide with an apparent Mr of 115 000. This coincides with the size of the largest band revealed by Western blotting using material containing immature sporozoites [8]. We interpret this to mean that the products observed on maturc sporozoites are derived by proteolytic processing from this common precursor (see discussion). We note that there is a size discrepancy between the primary translation product (115 kDa) and the predicted size, 91.9 kDa. A similar anomaly was present in the 2gtll-SR1 fusion protein and we assume that these variations are due to the unusual structural features and amino acid composition of the molecule [8].
Discussion
Fig. 4. Cell-free translation and immunoprecipitation of SPAGI. Lane I, immunoprecipitation with pre-immune rabbit serum; lane 2, immunoprecipitation with rabbit antiserum to the ).gtl I-SRI fusion protein [8]. Arrow, size in kDa.
shown in Figs 3B and 3C respectively and react as expected. Specificity controls performed on slides of merozoites (an extra-cellular stage in the blood) using the same dilutions as in the figure demonstrated that BA4 did not react whilst 5El did stain as expected (data not shown).
In this paper we describe the gene for a surface antigen expressed on the infective (sporozoite) stage of T. annulata. The most remarkable feature of this sequence is the presence of 2 regions mimicking the elastin repetitive structure consisting of tandem arrays of the pentapeptide PGVGV [9,23,24]. In addition the hexapeptide VGVAPG, which occurs twice in bovine elastin, occurs 3 times in the repetitive domains in SPAG-I. Molecular mimicry by parasites is a fascinating phenomenon for which there are essentially 2 explanations which are not mutually exclusive (extensively reviewed in
I11
ref. 29). In the first place one can imagine that the parasite mimics the host in order to escape the immune response. On the other hand mimicry of functional molecules which the parasite can exploit to its own advantage is a particularly compelling idea. Both these explanations can be advanced for the mimicry we observe in the SPAG-1 antigen of T. annulata. In particular, we can speculate that SPAG-1 functions as a ligand for host cell recognition. The VGVAPG peptide has been shown to be chemotactic for monocytes and most significantly binds to the elastin receptor identified in these cells [26-28], which are a major class of target cells for T. annulata. It is interesting to note that in culture the sporozoite can infect fibroblasts [30] which also carry high affinity elastin receptors [27]. Use of the elastin receptor by T. annulata sporozoites could explain to some extent the host cell specificity of this parasite. In this regard it is noteworthy that Theileria parva does not significantly invade monocytes, but rather prefers T-cells [3,4] and we would predict that this parasite will not carry ligands for the elastin receptor on its surface. If SPAG-1 is a ligand for host target cells, then it becomes tempting to suggest that functional ligand domains are revealed during processing of this antigen from a l l5-kDa precursor into the 4 polypeptides detected by Western blotting with mAb 1A7 on the mature sporozoite [8]. Obvious candidates as functional sequences are the 3 VGVAPG elastin ligand peptides. It is clear that the processing must involve cleavages towards the aminoterminal end of the molecule, since the observed mature products all retain the 1A7 epitope known to reside near the C-terminus [8]. We have not resolved the fate of the Nterminal fragments, but since many, if not all of them would be likely to carry at least one of the elastin repeats we predict that they will be retained on the surface exposing the proposed ligand sequences. The extensive mimicry we describe here is on a previously unobserved scale, although there are many interesting examples of mimicry of host components by viruses and parasites
[29,31]. For example, in the malaria system there is similarity between the extracellular matrix glycoprotein thrombospondin and 2 Plasmodium proteins, the circumsporozoite antigen [32] and TRAP, a blood stage protein [33]. The exact significance of this similarity is not clear but it has been noted that the liver cells possess a thrombospondin receptor and it is tempting to speculate that this is a receptorligand system for the entry of malaria parasites into the liver parenchyma. The degree of similarity is not so extensive as for the elastin mimicry we have observed. However it may be that mimicry of host matrix proteins is a more common feature in host-parasite interactions than has previously been recognised. Finally, in the design of sub-unit vaccines it is important to bear these considerations in mind since vaccination with determinants that mimic the host could have unfortunate autoimmune consequences.
Acknowledgements This work was supported by the Agricultural and Food Research Council, the Wellcome Trust and the Overseas Development Administration. We thank Duncan Brown, Pamela Knight, Howard Baylis and Brian Shiels for helpful discussion. For provision of parasite material we are indebted to Duncan Brown, Alan Walker, June Fletcher and all the staff of the Protozoology Division, Centre For Tropical Veterinary Medicine, University Of Edinburgh. For excellent technical assistance we are grateful to Anne Megson and Susan McKellar.
References 1 Tait, A. and Sacks, D.L. (1988) The cell biology of parasite invasion and survival. Parasitol. Today 4, 228234. 2 Perkins, M.E. (1989) Erythrocyte invasion by the malarial merozoite: recent advances. Exp. Parasitol. 69, 94-99. 3 Spooner, R.L., Innes, E.A., Glass E.J. and Brown, C.G.D. (1989) Theileria annulata and T. parva infect and transform different bovine mononuclear cells. Immunology 66, 284-288.
112 4 Glass, E.J., Innes. I-.A.. Spooner, R.I,. and Brown. C G . I ) . (1989) Infection of bovine monocyte.:macrophage populations with Theileria annulata and Theih,ria parva. Vet. lmmunol. Immunopathol. 22, 355 368 5 Jura. W.G.Z.O.. Brown, ( ' . G D . , and Kelly. B. (1983) Fine structure and invasivc behaviour of the early developmental stages of Theileria annuhtta in vitro. Vet. Parasitol. 12, 31 -44. 6 Jura, W.G.Z.O. (1984) Factors affecting the capacity of lTwih,ria atmulala sporozoites to invade bovine peripheral blood lymphocytes. Vet. Parasitol. 16. 215 223. 7 Shaw, M.K., Tilney, I,.G. and Mousoke. A.J. (1991) The entry of Theileria parva sporozoites into bovine lymphocytes: evidence for MH(" class 1 involvement. J. Cell Biol. 113, 87 101. 8 Williamson, S., Tait. A., Brown, I).. Walker, A., Beck, P., Shiels, B., Fletcher. J. and Hall, R. (1989) Theih,ria annulata sporozoite surface antigen expressed in Fscherichia coli elicits neutralizing antibody. Proc. Natl. Acad. Sci. USA 86, 4639 4643. 9 Raju, K. and Anwar, R.A. Primary structures of bovine elastin a.b. and c deduced from the sequences o f c l ) N A clones. (1987) J.Biol. Chem. 262, 5755 5762. 10 (Jill, BS.. Bhattacharyula. Y. and Kaur, D. (1976) I m m u n i z a t i o n against bovine tropical thcilcriosis (7heileria annulata infections). Res. Vet. Sci. 21. 146 149. II Gubler. U. and Hoffman, B.J. (1983) A simple and very efficient method for generating eDNA libraries. C~cne 25, 263 269. 12 Seed, B. (1987) An LFA-3 eDNA encodes a phospholipid-linked m e m b r a n e protein homologous to its receptor CD2. Nature 329. 840 842. 13 Sangcr, F.. Nicklcn. S. and Coulson, A.R. (1977) DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74, 5463 5467. 14 Bankier, A.T.. Weston. K.M and Barrell. B.G. (1987) Random cloning and sequencing by the MI3/dideoxynucleotide chain termination method. Methods Enzymol. 155. 51 93. 15 Dcvereux, J. (1989) Genetics computer group sequence analysis software package. Nucleic Acids Res. 12. 387 395. 16 Williamson. S.M. (1988) PhD Thesis. University of F,dinburgh. 17 Walker, A.R. and McKcllar. S.B. (1983) Observations on the separation of Theileria sporozoites from ticks. Int. J. Parasitol. 13, 313-318. 18 Wrenn. D.S, Griffin, G.L., Senior, R.M and Mecham, R.P. (1986) Characterization of biologically active domains on elastin: identification of a monoclonal antibody to a cell recognition site. Biochemistry. 25. 5172 5176. 19 Glascodine. J.. Tetley, I,., Tait, A., Brown. C.G.D. and Shiels. B. (1990) Developmental expression of a
7"heileria annulata mcrozoite antigen. Mol. Biochen~. Parasitol. 40. 105 112. 20 Pelham, It. and Jackson. R. (1976) An efficient mRNA dependent translation system from reticulocyte lysates. Fur. J. Biochem. 67, 247 256. 21 HarIow. E. and Lane, I). (1988) Antibodies. A l,aboratory Manual, Chapter I I. Cold Spring Harbor Laboratory, ('old Spring Harbor, NY. 22 l,aemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680 682. 23 Bressan. G.M., Argos, P. and Stanley, K.K. (1987) Repeating structure of chick tropoelastin revealed by complementary cl)NA cloning. Biochemistry 26, 1497 15O3. 24 Fazio, M.J., Olsen, D.R., Kauh. E.A., Baldwin. C.T.. Indik. Z.. Ornstein-Goldstein, N., Yeh, H.. Rosenbloom. J. and Uitto, J. (1988) Cloning of full-length elastin cl)NAs from a human skin fibroblast recombimint eDNA library: further elucidation of ahernative splicing utilizing exon-specific oligonucleotides. J. Invest. Dermatol. 91, 458464. 25 AIIsop, B. and Allsop, M.T.E.P. (1988) Theileria parva genomic I)NA studies reveal intra-spccific sequence diversity. Mol. Biochem. Parasitol. 28. 77 84. 26 Mecham, R.P., Hinek, A., Entwistle, R., Wrenn, I)S., Griffin, G. and Senior, R.M. (1989) Biochemistry 28, 3716 3722. 27 Blood, C.tI., Sasse, J.. Brodt, P.A. and Zetter, B.R. (1988) Identification of a tumour cell receptor for VGVAPG, an elastin derived chemotactic peptide. J. ('ell. Biol. 107, 1987 1993. 28 Robert. 1,.. Jacob, M.P., Fulop. T.. Timar, J and Ilornebeck, W. (1989) Elastonectin and the elastin receptor. Pathol. Biol. 37, 736 741. 29 Damian, R.T. (1989) Molecular mimicry: parasite evasion and host defense. Curr. Top. Microbiol. lmmunol. 145. 101 115. 30 Brown, C.G.D. and Gray. M.A. (1981) Infection of bovine fibroblastic cell lines with 7heileria parva and 7annulata. Trans. R. Soc. Trop. Med. Hyg. 75, 323 324. 31 Oldstone. M.B.A. (1989) Molecular mimicry as a mechanism and as a probe uncovering etiologic agent(s) of autoimmune disease. Curr. Top. Microbiol. lmmunol. 145, 127 135. 32 Goundis. D. and Reid, K.B.M. (1988) Properdin, the terminal complement components, thrombospondin and the circumsporozoite protein of malaria parasites contain similar sequence motifs. Nature 335, 82 85. 33 Robson. K.J.H., Hall, J.R.S., Jennings, M.W.. Harris, T.JR.. Marsh. K.. Newbold, C.I., Tate, V.E. and Weatherall, D.J. (1988) A highly conserved amino-acid sequence in thrombospondin, properdin and in proteins from sporozoites and blood stages of a human malaria parasite. Nature 335. 79 82.
105
MOLBIO 01748
Mimicry of elastin repetitive motifs by Theileria annulata sporozoite surface antigen R o g e r Hall a, Philip D. H u n t a, M a r k C a r r i n g t o n b, David S i m m o n s c, Susanna Williamson d, R o b e r t P. M e c h a m e and A n d r e w Tait f "Department of Biology, University of York, York, UK; bDepartment of Biochemistry, University of Cambridge, Cambridge, UK; ClCRF Laboratories, Institute of Molecular Medicine, John Radcliffe Hospital, Oxford. UK; dCentrefor Tropical Veterinary Medicine, University of Edinburgh, Roslin, UK; ~Jewish Hospital of St. Louis, Washington University Medical Center, St. Louis, MO, USA; and rWellcome Unit for Molecular Parasitology, University of Glasgow, Glasgow, UK (Received 15 November 1991; accepted 7 February 1992)
Theileria annulata is an important pathogen of cattle in the tropics. The gene sequence of a sporozoite surface antigen (SPAG-I) is reported. Data is also presented demonstrating that SPAG-I is synthesised as a large precursor. This antigen, which is a candidate for inclusion in a subunit vaccine, shows a remarkable degree of molecular mimicry to the extracellular matrix protein elastin. It contains both repetitive motifs PGVGV and VGVAPG. lmmunofluorescence using a monoclonal antibody against VGVAPG confirmed that this peptide is expressed on sporozoites as predicted. The presence of VGVAPG is particularly interesting since this is the ligand for elastin receptors on a range of cell types, including macrophages/monocytes which are a major class of host target cells. It is proposed that this antigen represents the ligand whereby T. annulata recognises its host cells. Key words: Theileria annulata; Molecular mimicry; Subunit vaccine; Receptor-ligand; Elastin; Sporozoite antigen
Introduction
In order to penetrate target cells intracellular parasites must undergo a recognition event, presumably via an interaction between ligands on the parasite surface and receptors on the exterior of the target cell (for reviews see refs. 1,2). The infective stage (sporozoite) of the apicomplexan protozoan parasite, Theileria annulata, a major pathogen of cattle in Asia, southern Europe and north Africa, preferenCorrespondence address: R. Hall, Department of Biology, University of York, Heslington, York, YOI 5DD, UK
Note: Nucleotide sequence data reported in this paper have been submitted to the GenBankT M data base with the accession number M63017 Abbreviations: PBS, phosphate-buffered saline; BSA, bovine serum albumin; mAb, monoclonal antibody
tially invades BoLA class II bearing cells, particularly monocytes/macrophages and B cells [3,4]. The molecular details of the iigand-receptor interactions involved in T. annulata invasion are unknown, but some aspects of the process have been elucidated [5,6]. Similar results are available for the closely related species T. parva [7]. Recently we have identified an antigen (SPAG-1) on the surface of T. annulata sporozoites using a monoclonal antibody which blocks sporozoite penetration [8]. This antigen is not only a candidate for the development of a vaccine but also a putative ligand for recognition of the target leucocytes. Our results based on the complete cDNA sequence encoding this molecule, and immunofluorescence analysis demonstrate the presence of 2 regions showing remarkable homology to the repetitive domain in bovine elastin, an extracellular matrix
106
protein [9]. We also present data demonstrating that the protein is produced as a precursor which is subsequently processed to produce the mature forms of the molecule.
Materials and Methods
Parasite material, cDNA synthesis, cloning and sequencing. Total RNA was extracted exactly as described [8] from the salivary glands of 2day fed ticks (Hyalomma anatolicum anatolicum) infected with T. annulata (Hissar) [10]. cDNA was synthesised [11] and ligated to Bst XI adaptors and inserted into the vector pCDM8 [12]. The resulting library was screened with the Eco RI insert derived from 2gtll-SRl [8] and of several positive clones, one (pCSPAG7) with a 2.8-kb insert was selected for sequence analysis using the chain termination method [13]. A combination of random 'shotgun' cloning [14] into M13mpl8 and an ordered approach using specially constructed oligonucleotide primers was used to generate the full sequence. Sequence analysis was performed using the University of Wisconsin Genetics Computer Group software [15] run on a VAX computer. Indirect immunofluorescence. This was performed on T. annulata (Hissar) sporozoites exactly as previously described [16]. Briefly, Percoll-purified sporozoites [17] were fixed with 3.7% formaldehyde in PBS and spotted onto multiwell slides (Hendley, Essex) and left to dry. Individual wells were incubated with 10 #1 of monoclonal antibody diluted 1/100 in PBS followed by 10 /~1 of 1/40 diluted fluorescein labelled anti-mouse IgG (Sigma). They were examined on a fluorescence microscope and photographed. The monoclonal antibodies used were 1A7 anti-SPAG [8], BA4 anti-elastin peptide VGVAPG [18], and 5El which is directed against an antigen found in the merozoite and piroplasm (intra-erthrocytic) stages [19]. Cell-free protein synthesis, immunoprecipitation
and gel electrophoresis. Cell-free translation was performed using the nuclease treated reticulocyte lysate system [20]. Briefly a 10 pg aliquot of RNA, extracted from T. annulata (Hissar) sporozoite infected tick salivary glands [8], was translated in 100 pl of translation cocktail in the presence of 50 pCi [35S]methionine at a specific activity of 1000 Ci mmol --~ (DuPont-NEN). Immunoprecipitation of the translation products was performed with the rabbit anti-2gtil SRI fusion protein [8] as described [21]. The immune complexes were separated by SDS-PAGE [22] and the bound antigen was visualised by fluorography after treating the gel with En3Hance (Amersham). Results
Sequence analysis. The complete coding nucleotide sequence, derived from a cDNA clone, of a previously characterised sporozoite surface antigen, SPAG-1, from T. annulata [8], is shown in Fig.1. There is a single open reading frame extending for 2721 nucleotides encoding a predicted 91.9-kDa polypeptide of 907 amino acids. The region underlined near the Cterminus represents the sequence defined in the original 2gtll clone (2gtil-SR1), and contains the epitope for monoclonal antibody IA7 [8]. There are several remarkable features about the predicted amino acid sequence (summarised in Fig 2(i)). The most notable is the presence of 2 regions containing the pentapeptide PGVGV (module 2, Fig. 2(i)) repeated several times (shown boxed in Fig.l). A repetitive region containing the identical pentapeptide unit occurs in elastin from several diverse sources including cow, chicken, and man [9,23,24]. The alignments [15] with bovine elastin shown in Fig.2(ii) show this feature very clearly. The first block of repeats (residues 179-236) is the most similar to the elastin structure, having exactly the same number of units (11), and differing in only 3 positions (Fig. 2(ii)a), due to the presence of an extra glycine in unit 7 and extra terminal alanines in
107 1
GTTTTAAAAAGGAGTAACATTGGAATTTAAATTTCATTTTCCAACACTCAACGATGAATATTATACACTTTCTGTTGACCATTCCGGCTA
90
M N I I H F L L T I P A I T T T T T G T A T C T G G A G C G G A C A A G A T G C C T G C C ~ A G A A A G TTC T A G A A C C T C T A A A C C C A G T C C C C T A G T A A C C C T A G A A T C G G C G G T A A F V S G A D K M P A G E S S R T S K P S P L V T L E S A V T CAC AACC TTCAAAGGAC C C ATTCAAGAC AATTAGTGCC TTGTCAAAAGCAACAAAAGTATGGAAGTCAGCGGTA TCAG TATCAGGTGAC T Q P S K D P F K T I S A L S K A T K V W K S A V S V S G D S CTAAGAC TGTACCTACTCCAGTTTCGGAACCAATGATCACTCGATCTTTTCAAGAAC CAGTATCTCAAGAACTTGAATTCCAATCAGATA K T V P T P V S E P M I T R S F Q E P V S Q E L E F Q S D T
270
361
CTGAAATTAATGAGTCAGGATCCGGTTCAGATGAGGATGAGGATGACGATGACGATGAGGAGGAAGAAGAAGACGATAAATCTACCTCAT
450
451
E I NLE S G S G CTAA~CGGAAAAGGCAGCCCAAAAGC K N G K G S P K
540
541
TATCACAAACTGGATTC-C~ACCAAGTGGTTCCCACGCTCAACAAGATCCCGGTGTAGGTGTTCCAGGAGT~TGTTCCAGGAGTAGGTG
91 181 271
631 721 811 901 991 1081 1171 1261 1351 1441 1531 1621 1711 1801 1891 1981 2071 2161 2251 2341 2431 2521 2611 2701 2791
D E D E D D D D D E E E E E D D K S T S S TCAGCCTC-GAGTATCTTCAAGCAGTACATCCTCAGCAAGTC CAACATCTCCAAC TACAACAT A Q P G V S S S S T S S A S P T S P T T T L
180
360
S
630
S Q T G L G P S G S H A Q Q DIP G V G V P G V G V P G V G V l TTC CAGGAGTAGGTGTTCC AGGAG TAGGTGTTCCAGGTGTAGGTGTGC CAGGTGTAGGAGGTGTTCC CGGAGTTGGCGTTGCACCAGGGG 720 IP G V G V P G V GV P G V G V P G V G G V PG V G V A P G V J TAGGTGTTCCAGGAGTTC-G TGTTGCAC C AGGTGTAGGTGTTGGAGC TGATAGTAGTGGATTGCCTGGAAGTC~TGGTC TTGGAGCAGGAG 810 IG V P G V G V A P G V G V GIA D S S G L P G S G G L G A G A C A A A G G C T G G G A A A G G T C A A G G A T C T G G T C T A C A G G G A C C A G G A G G T G T T G G A G T A G TAC C T G G T G T A G G T G T A G C A G C T T C TTC T T C T T 900 K A G K G Q G S G L Q G P G G V G V V IP G V G V IA A S S S S CAC CAGGAAAACCTC CAGGAGTAGGAGCAGGAGTTATGC C TGGAGTTGGTGTA CGAGCACAAGGAGGAGTAATAATTGGTGCGCCAGGAG 990 P G K P P G V G A G V M . IP G V G V IRA Q G G V I I G A P G V T A G C A G G T G T G C C A G G A G G A A A G C C A G G A C A A C C A G TATC T C A A G A A C T T G A A C T G A A A T C A G A C A C T G A A A T T A A T G A G T C A G G T TC C A 1080 A G V P G G K P G Q P V S Q E L E L K S D T E I NLE S G S S G T T C A G A A G G G G A A G A C G A T G A C G A T G A A G A A G A G G A A G A A G A A A A T A A A T C T A C C T CATC T A A A G G A G C A G G A G G A A A G G C T G G A A A A G 1170 S E G E D D D D E E E E E E N K S T S S K G A G G K A G K G GTCAAGGATCTGTATCACCAGGAGGAGGATCC TCAGCAAGTCAAACATCTCCAACTACAACACCACAATCTGGC TTGGCATCAAGTGGTT 1260 Q G S V S P G G G S S A S Q T S P T T T P Q S G L A S S G S CTCATGCTCAACAAAGTCC TCAACAAGATCCAGCGC CTAGTAAACCTAGTGGAGGAGGTGTGCCAGGAGT'I~AGTTCCTC, GTGTTGGCG 1350 H A Q Q S P Q Q D P A P S K P S G G G VIP G V G V P G V G V I TTC C CGGTGTTGGAG TAC CAGGAG TAGGAGTTGCGC CGGGAGTTGGTGTTGTAC CTGGAGTAC~TG C A A C A A C T T C TTC A T C A T C A A 1440 IP G V G V P G V G V A P G V G V V P G V G G IA T T S S S S T CAACTTC AACTTCAACTTCAACTACTACTACTACTACAAC TTCATCAGGAAAA CCTTCAGACCAAGGAAGCCATGGTACTTCTCCAAGAA 1530 T S T S T S T T T T T T T S S G K P S D Q G S H G T S P R N ATGCAGTAACCAGACAAACTGACTCAATATCAGGAC C C A T A C C A T C A C C A G G A G A T C C A A G A G C A A T T A C TC-GA C A A A T G G G T G A A G G A G 1620 A V T R Q T D S I S G P I P S P G D P R A I T G Q M G E G E AAAC,G T T T G C T G T A C A G T T C C T G G G A G A T T T T A A A C C A A A A C C A A G G A G A T A T G A A G G A C A A G G A A C A G A T G C A G T A A A A C TAAAA CAAT 1710 R F A V Q F L G D F K P K P R R Y E G Q G T D A V K L K Q F T C A T T T T CGAAGAC, G T C A A A T C G C T G G T G C A A A C C T T A A T A A A C C T T A A A T T A G C A A T T G C A A A C G A C T T T G T T G A A A T C A G T G A A A A G T 1800 I F E E V K S L V Q T L I N L K L A I A N D F V E I S E K L T G A A A A A G A A A A A T C A A A A T T A C G TA C C G A A A T T A A A G T T G T T A A A A G G A G A A C A A T T T G A CAC C A A A C A G A A G G T A G C C A A C G T A C T A A 1890 K K K N Q N Y V P K L K L L K G E Q F D T K Q K V A N V L K AAGC43TT C A A T T C T C T G T A C T T C G T A T T T T T T A T G A A C C T T A A C CTAGC G A A A G A A G T T A A C A A A C C G G A A G A A T T G G C A G A A T T T C T T T 1980 G F N S L Y F V F F M N L N L A K E V N K P E E L A E F L W GGAAACTAAA TACAA TCCC AGATAAAGTAGGAAGAGAATTTGAGTTAGCAATAGAAAAAAC TAAAGGTTCAGAGAAAAAGAAGGAA TTAG 2070 K L N T I P D K V G R E F E L A I E K T K G S E K K K E L E
AAGAAGCATTTAATTCAATAC~TTAGGTTTCAAAATA@CACAGTACGCAACAAATGACATCCTCTCAAGTATAACAAATTCAGTCTACT E A F N S I G L G F K I A Q Y A T N D I L S S I T N S V Y S CCC TGATAAAACTAAAGAATTTTGGAGATGATTTTGTTACCGAAGTAAGAAAGTCAC TGCAAATGGTTC CACAC CAAAAGAACCTAAACG L I K L K N F G D D F V T E V R K S L Q M V P H Q K N L NL G GAT CAGC ATTTATAGTCAAAATCT CAGAAATAATCAACAAAAAAGGAACAGAAGATC AGGATCAAACAT CAGGAAGTGGGTCAAAAGGAA S A F I V K I S E I I N K K G T E D Q D Q T S G S G S K G T CAGAAGGAGGA TCAC TAAGGG~ AAGATTTGACAGAAGAAGAAGTTTTGAAAGTTC TGGATGAA C TAG TGAAGGATGTAAG CGAAGAAC E G G S L R G Q D L T E E E V L K V L D E L V K D V S E E H ATGTTGGAATAGGAGATTTAAGTGACCCAAGTAGCAGAACACCAAATGCAAAACCAGCCGAACTTGGAC CTTCACTAGTGATACAAAATG V G I G D L S D P S S R T P N A K P A E L G P S L V I Q N V TACCGTCAGACCCCTCAAAAGTGACACCAACACAGCCTTCAAATTTGC~ACAAGTACCAACAACAGGGCCGGGGAALCGGGACGGATGGAA P S D P S K V T P T Q P s N L P Q V P T T G P G N G T D G T CAA CAAC AGGA C CAGGTGGAAACGGGGAAGGAC.GCAAAGA TTTGAAGGAAGGAGAAAAGAAAGAAGGATTATTT CAAAAGAT CAAAAACA T T G ~ ~ G N G ~ q G K D U ~< ~ G E K K E $ ~ ~ Q K ~ K ;~ A A C T C T T G G G C T C A G G A T T C G A A G T C G C A A G T A T T A T T A T A C C A A T G A C A A C A A T C A T A T T CAGC A T A G TC C A C T A A A A C T A A A A A C A C A L L G S G F E V A S I I I P M T T I I F S I V H * ACTAACCACAC TAAT TTATAATATACAAAAAAAAA 2825
2160 2250 2340 2430 2520 2610 2700 2790
Fig. 1 c D N A sequence of 7". annulata sporozoite surface antigen (SPAG-I). The predicted amino acid sequence is shown below. The PGVGV repeat regions showing homology to elastin are boxed. The C-terminal region encoded by the insert of 2gtl I-SRI, used to isolate this clone, is underlined. Putative N-linked glycosylation sites are marked with arrowheads.
108 {])
-~
2
I
2
3
mmmmm
i
"
I'
1: D/E regions: amino acids 92-144 and 323-364 2: [PGVGV]n : amino acids 179-236 and 424-456 2: T/S region : amino acids 458-478 4:1A7 epitope : between amino acids 778-891
(ii) a s
179
PGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGGVPGVGVAPGVGVPGVGVAPGVGV
,,rlllllllJllllllllillil E
335
S
588
E
i010 648 1070 708 1130
S
424 335
I I illllll
236
lllltll,;l
IIIII
PGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGV.GVPGVGV.PGVGVPGVGV.PGVGV
389
CCCGGTGTAGGTGTTCCAGGAGTTGGTGTTCCAGGAGTAGGTGTTCCAGGAGTAGGTGT'? II II II il II II II II II II I. I, II II I I ] I I I I I :I CCAGGAGTTGGAGTCCCTGGTGTCGGGGTCCCTGGTGTCGGGG'F'FCCAGGTGTCGGGGTC
647 II
II 1069
C CAGGAGTAGGTGTTCCAGGTGTAGGTGTGCCAGGTGTAGGAGGTGTTCCCGGA,<;TTGGC I II II If I I I I I I I I I I I I! II I, IIIII II II II 'I IIII: CCT(;GTGTCC~;GGTTCCAGGTGTCGGAGTCCCAGGTGTT. . .G G A G T C C C A G G T G T T G G A GTTGCACCAGGGGTAGGTGTTCCAGGAGTTGGTGTTGCACCAGGTGTAGGq~q"I' II 'I II II rl II ~I li I!II II II !IIII GTC...CCTGGTGTTGGGGTCCCTGGTGLFI'G'GCGTC...CCTGGTGTTGGAGTT
PGVGVPGVGVPGVGVPGVGVA
IIIIIIIIIIIIIIIIII E
I II
.... P G V G V .... V P G V G G A
!:
P G V G V P G k iG V P G V G V P G V G V P G k
IIIII
707 ] 129
761 II
:II 1183
456
lilll.:
IGV.m G V G V P G V G V P G V G V P
375
{iii) 92
PVSQELEFQSDTEINESGSGSDEDEDDDDDEEEEEDDKSTSSK
IIIIII1:.111111
324
:::111111111::11111} (c PVSQELELKSDTEINESGSSS--EGEDDDDEEEEEENKSTS~K
]~4
I,Izl
264
Fig. 2. Structural features of SPAG-I. (i) Cartoon of the polypeptide SPAG-I. The modules marked with a I represent a directly repeated peptide sequence whose precise composition is shown in (iii) below and which contain the D/E regions. Module 2 represents the regions containing the PGVGV and VGVAPG repeats. Box 3 represents the T/S region. Box 4 represents the region containing the IA7 epitope. (ii) Alignment of the PGVGV repeat regions encoded by SPAG-1 with the homologous region in bovine elastin. In all cases the top line (S) represents the Theileria sequence and the lower line (E) the bovine sequence. (a) Comparison of the amino-terminal PGVGV repeat. (b) Comparison of the DNA sequence encoding the N-terminal PGVGV repeat. (c) Comparison of the more C-terminal PGVGV repeat. The VGVAPG motif is underlined. (iii) Comparison of the internal repeat sequence designated as module 1 in (i) above. The D/E regions are underlined. For more details see text. The alignments were made using the BESTFIT programme on the U W G C G package [15].
units 8 and 10. It is interesting that this remarkable degree of homology at the protein level is not reflected to the same extent at the nucleotide level, since over this region there is a high degree of third base wobble (Fig. 2(ii)b). Over the region compared in Fig. 2(ii)b there are 43 silent third base differences of which 18
show A/T for G/C substitutions whilst only 3 show the reverse. This is consistent with the known A/T richness of the Theileria genome [25]. The second region of PGVGV repeats (residues 424--456, Fig. 2(ii)c) consists of only 6 units, the first 3 being perfect and the last 3 being slightly degenerate. The fourth unit,
109
of threonine (13 residues) and serine (9 residues) and thus called the T/S region (designated as module 3 in Fig. 2(i)). At the amino terminus there is a stretch of 18 mainly hydrophobic amino acids (residues 1-18), which is predicted by the S I G N A L P E P programme [15] to be a signal peptide. At the C-terminus there is a 24-residue hydrophobic domain (residues 883-907) which could be a membrane anchor. There are also 5 potential N-linked glycosylation sites marked with an arrowhead (Fig. 1). Overall the protein is relatively rich in glycine (15.1%), serine (11.8%), proline (8.3%) and valine (8.9%). There are no cysteine residues and hence no disulphide bonds. Codon usage also shows marked bias in favour of codons with an A in the third position (analysis not shown).
containing a terminal alanine residue, is identical to units 8 and 10 in the first repeat region. These units, with an additional alanine residue, may be highly significant since in all 3 cases a hexapeptide of the form V G V A P G can be generated (underlined in Fig.2 (ii) a). This hexapeptide also occurs in elastin, outside the repeat region, and has been demonstrated to be chemotactic and a ligand for the elastin receptor identified on a range of cells including fibroblasts and monocytes [26-28], (see Discussion). A number of other features may be noted from this sequence (Fig. 2). There appears to have been an internal duplication event such that the sequences designated as modules 1 and 2 occur as tandem repeats towards the amino terminal half of the molecule. Module 2 contains the PGVGV repeats just described. The 2 versions of module 1 are almost perfect matches over 41-43 amino acids and the alignments are shown in Fig. 2(iii). Within each of the versions of module 1 is a region of extreme negative charge consisting entirely of glutamic and aspartic acid residues (shown underlined in Fig. 2(iii)). The more amino terminal of these contains 9 aspartates and 7 glutamates; the other consists of 4 aspartates and 7 glutamates. These modules are thus designated as D/E regions. Immediately downstream of the second block of PGVGV repeats is a region of 22 polar amino acids consisting
A
Monoclonal antibody to the VGVAPG elastin motif recognises sporozoites. To extend the findings from our sequencing data, and confirm that the hexapeptide V G V A P G was present, as predicted, on sporozoites, an immunofluorescence assay was undertaken using a monoclonal antibody, BA4, which specifically recognises this peptide [18]. The results are shown in Fig 3A. As can be seen, the sporozoites are clearly labelled and thus express this determinant. Positive (anti-sporozoite monoclonal I A7) and negative (antimerozoite monoclonal 5El) controls are
B
C
Fig. 3. Indirect immunofluorescence assay on sporozoites. (A) BA-4 anti VGVAPG monoclonal [18]; (B) IA7 anti-sporozoite monoclonal (positive control); Panel C. 5E 1 anti-merozoite and piroplasm monoclonal (negative control). The sporozoites in the positive panels (A and B) are identified as fluorescent specks. The photographs were taken at x 400 magnification.
I10
1
2
-
115
Cell-free protein synthesis demonstrates that S P A G - I is synthesisised as a precursor. In our previous characterisation of this antigen we demonstrated that it was complex and that at least 4 peptides (85, 70, 63 and 54 kDa) carried the epitope defined by mAb I A7, in extracts of purified sporozoites [8]. We were unclear whether these represented independent gene products or were the result of processing from a common precursor. To resolve this issue we translated sporozoite m R N A in a reticulocytelysate cell-free system and immunoprecipitated the product with a rabbit antiserum against thc original 2gtll-SRl fusion protein. These results are illustrated in Fig. 4 and clearly show that the primary translation product is a single major polypeptide with an apparent Mr of 115 000. This coincides with the size of the largest band revealed by Western blotting using material containing immature sporozoites [8]. We interpret this to mean that the products observed on maturc sporozoites are derived by proteolytic processing from this common precursor (see discussion). We note that there is a size discrepancy between the primary translation product (115 kDa) and the predicted size, 91.9 kDa. A similar anomaly was present in the 2gtll-SR1 fusion protein and we assume that these variations are due to the unusual structural features and amino acid composition of the molecule [8].
Discussion
Fig. 4. Cell-free translation and immunoprecipitation of SPAGI. Lane I, immunoprecipitation with pre-immune rabbit serum; lane 2, immunoprecipitation with rabbit antiserum to the ).gtl I-SRI fusion protein [8]. Arrow, size in kDa.
shown in Figs 3B and 3C respectively and react as expected. Specificity controls performed on slides of merozoites (an extra-cellular stage in the blood) using the same dilutions as in the figure demonstrated that BA4 did not react whilst 5El did stain as expected (data not shown).
In this paper we describe the gene for a surface antigen expressed on the infective (sporozoite) stage of T. annulata. The most remarkable feature of this sequence is the presence of 2 regions mimicking the elastin repetitive structure consisting of tandem arrays of the pentapeptide PGVGV [9,23,24]. In addition the hexapeptide VGVAPG, which occurs twice in bovine elastin, occurs 3 times in the repetitive domains in SPAG-I. Molecular mimicry by parasites is a fascinating phenomenon for which there are essentially 2 explanations which are not mutually exclusive (extensively reviewed in
I11
ref. 29). In the first place one can imagine that the parasite mimics the host in order to escape the immune response. On the other hand mimicry of functional molecules which the parasite can exploit to its own advantage is a particularly compelling idea. Both these explanations can be advanced for the mimicry we observe in the SPAG-1 antigen of T. annulata. In particular, we can speculate that SPAG-1 functions as a ligand for host cell recognition. The VGVAPG peptide has been shown to be chemotactic for monocytes and most significantly binds to the elastin receptor identified in these cells [26-28], which are a major class of target cells for T. annulata. It is interesting to note that in culture the sporozoite can infect fibroblasts [30] which also carry high affinity elastin receptors [27]. Use of the elastin receptor by T. annulata sporozoites could explain to some extent the host cell specificity of this parasite. In this regard it is noteworthy that Theileria parva does not significantly invade monocytes, but rather prefers T-cells [3,4] and we would predict that this parasite will not carry ligands for the elastin receptor on its surface. If SPAG-1 is a ligand for host target cells, then it becomes tempting to suggest that functional ligand domains are revealed during processing of this antigen from a l l5-kDa precursor into the 4 polypeptides detected by Western blotting with mAb 1A7 on the mature sporozoite [8]. Obvious candidates as functional sequences are the 3 VGVAPG elastin ligand peptides. It is clear that the processing must involve cleavages towards the aminoterminal end of the molecule, since the observed mature products all retain the 1A7 epitope known to reside near the C-terminus [8]. We have not resolved the fate of the Nterminal fragments, but since many, if not all of them would be likely to carry at least one of the elastin repeats we predict that they will be retained on the surface exposing the proposed ligand sequences. The extensive mimicry we describe here is on a previously unobserved scale, although there are many interesting examples of mimicry of host components by viruses and parasites
[29,31]. For example, in the malaria system there is similarity between the extracellular matrix glycoprotein thrombospondin and 2 Plasmodium proteins, the circumsporozoite antigen [32] and TRAP, a blood stage protein [33]. The exact significance of this similarity is not clear but it has been noted that the liver cells possess a thrombospondin receptor and it is tempting to speculate that this is a receptorligand system for the entry of malaria parasites into the liver parenchyma. The degree of similarity is not so extensive as for the elastin mimicry we have observed. However it may be that mimicry of host matrix proteins is a more common feature in host-parasite interactions than has previously been recognised. Finally, in the design of sub-unit vaccines it is important to bear these considerations in mind since vaccination with determinants that mimic the host could have unfortunate autoimmune consequences.
Acknowledgements This work was supported by the Agricultural and Food Research Council, the Wellcome Trust and the Overseas Development Administration. We thank Duncan Brown, Pamela Knight, Howard Baylis and Brian Shiels for helpful discussion. For provision of parasite material we are indebted to Duncan Brown, Alan Walker, June Fletcher and all the staff of the Protozoology Division, Centre For Tropical Veterinary Medicine, University Of Edinburgh. For excellent technical assistance we are grateful to Anne Megson and Susan McKellar.
References 1 Tait, A. and Sacks, D.L. (1988) The cell biology of parasite invasion and survival. Parasitol. Today 4, 228234. 2 Perkins, M.E. (1989) Erythrocyte invasion by the malarial merozoite: recent advances. Exp. Parasitol. 69, 94-99. 3 Spooner, R.L., Innes, E.A., Glass E.J. and Brown, C.G.D. (1989) Theileria annulata and T. parva infect and transform different bovine mononuclear cells. Immunology 66, 284-288.
112 4 Glass, E.J., Innes. I-.A.. Spooner, R.I,. and Brown. C G . I ) . (1989) Infection of bovine monocyte.:macrophage populations with Theileria annulata and Theih,ria parva. Vet. lmmunol. Immunopathol. 22, 355 368 5 Jura. W.G.Z.O.. Brown, ( ' . G D . , and Kelly. B. (1983) Fine structure and invasivc behaviour of the early developmental stages of Theileria annuhtta in vitro. Vet. Parasitol. 12, 31 -44. 6 Jura, W.G.Z.O. (1984) Factors affecting the capacity of lTwih,ria atmulala sporozoites to invade bovine peripheral blood lymphocytes. Vet. Parasitol. 16. 215 223. 7 Shaw, M.K., Tilney, I,.G. and Mousoke. A.J. (1991) The entry of Theileria parva sporozoites into bovine lymphocytes: evidence for MH(" class 1 involvement. J. Cell Biol. 113, 87 101. 8 Williamson, S., Tait. A., Brown, I).. Walker, A., Beck, P., Shiels, B., Fletcher. J. and Hall, R. (1989) Theih,ria annulata sporozoite surface antigen expressed in Fscherichia coli elicits neutralizing antibody. Proc. Natl. Acad. Sci. USA 86, 4639 4643. 9 Raju, K. and Anwar, R.A. Primary structures of bovine elastin a.b. and c deduced from the sequences o f c l ) N A clones. (1987) J.Biol. Chem. 262, 5755 5762. 10 (Jill, BS.. Bhattacharyula. Y. and Kaur, D. (1976) I m m u n i z a t i o n against bovine tropical thcilcriosis (7heileria annulata infections). Res. Vet. Sci. 21. 146 149. II Gubler. U. and Hoffman, B.J. (1983) A simple and very efficient method for generating eDNA libraries. C~cne 25, 263 269. 12 Seed, B. (1987) An LFA-3 eDNA encodes a phospholipid-linked m e m b r a n e protein homologous to its receptor CD2. Nature 329. 840 842. 13 Sangcr, F.. Nicklcn. S. and Coulson, A.R. (1977) DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74, 5463 5467. 14 Bankier, A.T.. Weston. K.M and Barrell. B.G. (1987) Random cloning and sequencing by the MI3/dideoxynucleotide chain termination method. Methods Enzymol. 155. 51 93. 15 Dcvereux, J. (1989) Genetics computer group sequence analysis software package. Nucleic Acids Res. 12. 387 395. 16 Williamson. S.M. (1988) PhD Thesis. University of F,dinburgh. 17 Walker, A.R. and McKcllar. S.B. (1983) Observations on the separation of Theileria sporozoites from ticks. Int. J. Parasitol. 13, 313-318. 18 Wrenn. D.S, Griffin, G.L., Senior, R.M and Mecham, R.P. (1986) Characterization of biologically active domains on elastin: identification of a monoclonal antibody to a cell recognition site. Biochemistry. 25. 5172 5176. 19 Glascodine. J.. Tetley, I,., Tait, A., Brown. C.G.D. and Shiels. B. (1990) Developmental expression of a
7"heileria annulata mcrozoite antigen. Mol. Biochen~. Parasitol. 40. 105 112. 20 Pelham, It. and Jackson. R. (1976) An efficient mRNA dependent translation system from reticulocyte lysates. Fur. J. Biochem. 67, 247 256. 21 HarIow. E. and Lane, I). (1988) Antibodies. A l,aboratory Manual, Chapter I I. Cold Spring Harbor Laboratory, ('old Spring Harbor, NY. 22 l,aemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680 682. 23 Bressan. G.M., Argos, P. and Stanley, K.K. (1987) Repeating structure of chick tropoelastin revealed by complementary cl)NA cloning. Biochemistry 26, 1497 15O3. 24 Fazio, M.J., Olsen, D.R., Kauh. E.A., Baldwin. C.T.. Indik. Z.. Ornstein-Goldstein, N., Yeh, H.. Rosenbloom. J. and Uitto, J. (1988) Cloning of full-length elastin cl)NAs from a human skin fibroblast recombimint eDNA library: further elucidation of ahernative splicing utilizing exon-specific oligonucleotides. J. Invest. Dermatol. 91, 458464. 25 AIIsop, B. and Allsop, M.T.E.P. (1988) Theileria parva genomic I)NA studies reveal intra-spccific sequence diversity. Mol. Biochem. Parasitol. 28. 77 84. 26 Mecham, R.P., Hinek, A., Entwistle, R., Wrenn, I)S., Griffin, G. and Senior, R.M. (1989) Biochemistry 28, 3716 3722. 27 Blood, C.tI., Sasse, J.. Brodt, P.A. and Zetter, B.R. (1988) Identification of a tumour cell receptor for VGVAPG, an elastin derived chemotactic peptide. J. ('ell. Biol. 107, 1987 1993. 28 Robert. 1,.. Jacob, M.P., Fulop. T.. Timar, J and Ilornebeck, W. (1989) Elastonectin and the elastin receptor. Pathol. Biol. 37, 736 741. 29 Damian, R.T. (1989) Molecular mimicry: parasite evasion and host defense. Curr. Top. Microbiol. lmmunol. 145. 101 115. 30 Brown, C.G.D. and Gray. M.A. (1981) Infection of bovine fibroblastic cell lines with 7heileria parva and 7annulata. Trans. R. Soc. Trop. Med. Hyg. 75, 323 324. 31 Oldstone. M.B.A. (1989) Molecular mimicry as a mechanism and as a probe uncovering etiologic agent(s) of autoimmune disease. Curr. Top. Microbiol. lmmunol. 145, 127 135. 32 Goundis. D. and Reid, K.B.M. (1988) Properdin, the terminal complement components, thrombospondin and the circumsporozoite protein of malaria parasites contain similar sequence motifs. Nature 335, 82 85. 33 Robson. K.J.H., Hall, J.R.S., Jennings, M.W.. Harris, T.JR.. Marsh. K.. Newbold, C.I., Tate, V.E. and Weatherall, D.J. (1988) A highly conserved amino-acid sequence in thrombospondin, properdin and in proteins from sporozoites and blood stages of a human malaria parasite. Nature 335. 79 82.