Molecular and Biochemical Parasitology 100 (1999) 135–140

Short communication

The Theileria annulata sporozoite and macroschizontpolypeptide encoded by the spm1 gene shares

phenylalanine-glycine motifs with nuclear pore proteins�

Pamela Knight a,b,*, Susanna Williamson a, Duncan Brown a, Jane Kinnaird b,Mark Fox b, Khalid Hussain b, Roger Hall c, Andy Tait d

a Centre for Tropical Veterinary Medicine, Uni6ersity of Edinburgh, Easter Bush, Roslin, Midlothian, Edinburgh EH25 9RG, UKb Department of Veterinary Parasitology, Uni6ersity of Glasgow, Bearsden Rd., Glasgow G61 1QH, UK

c Department of Biology, Uni6ersity of York, York YO1 5DD, UKd Wellcome Unit of Molecular Parasitology, Anderson College, 56 Dunbarton Road, Uni6ersity of Glasgow, Glasgow G11 6NU, UK

Received 4 August 1998; received in revised form 20 January 1999; accepted 23 January 1999

Keywords: Theileria ; Sporozoite; Nucleoporin; Nucleocytoplasmic transport; Homology; Repetetive motifs

Nuclear pore proteins (nucleoporins) are com-ponents of the nuclear pore complex in eukaryoticcells which form sites for export of mRNA andassociated proteins from the nucleus to the cyto-plasm and import of proteins into the nucleus[1,2]. The structure and functional organisation ofthe nuclear pore complex is not fully understood,but recent evidence has established a role for

nucleoporins in transport of macromolecules toand from the nucleus [1,3–5]. Many nucleoporinshave now been sequenced and characterised inboth yeast and higher eukaryotes [6,7]. Groups ofnucleoporin proteins are characterised by con-served repeated ‘signature’ motifs; phenylalanine-glycine (FG), glycine-leucine-phenylalanine-glycine (GLFG) and x-phenylalanine-x-phenylala-nine-glycine (XFXFG), where ‘x’ can be any sin-gle amino-acid residue [6]. However, nucleoporinshave not been identified in the protozoa to date.Here we describe the isolation and sequence anal-ysis of a gene (spm1, sporozoite andmacroschizont protein 1) encoding a nucleoporin-like protein from the protozoan parasite Theileriaannulata.

* Corresponding author. Present address: Department ofVeterinary Clinical Studies, Veterinary Field Station, Univer-sity of Edinburgh, Easter Bush, Roslin, Midlothian, Edin-burgh, EH25 9RG, UK. Tel.: +44-131-6506273; fax:+44-131-6506588.

E-mail address: pam.knight@ed.ac.uk (P. Knight)� Note: The sequence data reported herein has been submit-

ted to Genbank™ and assigned the accession number Y15794.

0166-6851/99/$ - see front matter © 1999 Elsevier Science B.V. All rights reserved.

PII: S 0166 -6851 (99 )00023 -7

P. Knight et al. / Molecular and Biochemical Parasitology 100 (1999) 135–140136

Part of this gene was isolated initially, possiblythrough a fortuitous cross-reaction, using a mon-oclonal antibody (4B11) which was one of a panelraised against the sporozoite stage of T. annulata(S. Williamson. Ph.D. thesis. Edinburgh 1988)with a view to identifying antibodies which couldblock sporozoite invasion of the host cell. How-ever, Mab 4B11 detected several polypeptides onwestern blots of parasite extracts, in addition to acandidate ligand for host cell invasion [8]. One ofthe clones isolated from an immunoscreen of a T.annulata genomic expression library in lgt11 [9]with Mab 4B11 contained an 800 bp genomicfragment (KP6), which was subcloned intopGEM7ZF-SK+ and fully sequenced on bothstrands. The KP6 sequence had a complete openreading frame which showed homology to nucle-oporin sequences. Using the KP6 insert as a hy-bridisation probe, a full length cDNA clone wasthen isolated from a T. annulata library preparedfrom macroschizont-infected leukocyte cDNAcloned into Uni-ZAP XR (Ankara D7 cloned cellline grown at 37°C [9]). Approximately 3×105

plaques were screened in total, with positiveclones subjected to two further rounds of purifica-tion. Two positive clones were identified andanalysed, one (pBluescript-spm1) with a 2.9 kbinsert, was selected for further study, and a seriesof unidirectional deletions were constructed inboth directions for sequence analysis. The 2888 bpcDNA from pBluescript-spm1 encoded a pre-dicted 771 amino acid sequence from nucleotides34 to 2346. The KP6 region, which includes the4B11 epitope, is situated in the centre of thepredicted polypeptide sequence (amino acidresidues 275–540). In common with the majorityof Theileria genes which have been sequenced sofar the coding sequence is A/T rich (58%).

The spm1 predicted amino acid sequence wascompared to other sequences in the Genbankdatabase using the genetics computer groupBLAST program. The highest similarity scoreswere obtained with nucleoporin sequences rich inGLFG motifs, namely NUP100 (NSP100),NUP116 (NSP116) and NUP 49 (NSP 49),[10,11], a related group of GLFG nucleoporinsidentified in yeast, NUP214 (human CANprotein) a putative oncogene product associated

with myeloid leukomyogenesis [12] andCaenorhabditus elegans nucleoporin-like sequence(gene F53F10.5) [13]. The homologous regionsbetween spm1 and these protein sequences werelargely confined to the GLFG and XFG nucle-oporin signature motifs. These motifs occur 10and 23 times, respectively, in the predicted spm1sequence, predominantly in the central/C-terminalregion, where many have a similar spatial ar-rangement to those of the well-characterised yeastnucleoporin sequences NUP 100, NUP116 andNUP49 (Fig. 1). The spm1 sequence is rich in thesmall, polar amino acids serine and threonine,which comprise 34% of the entire coding region,especially in the regions between the FG andGLFG motifs. This is also a feature of many ofthe nucleoporins which have been sequenced (forreview see [6]). The predicted molecular mass ofthe spm1 encoded polypeptide is 81-kDa; themolecular mass of nucleoporin sequences canrange from 49 to 214-kDa [6,10–13].

The central GLFG rich region of spm1 pre-dicted amino acid sequence (a.a. 289–671 (Fig. 1))was also compared to the expressed sequencetagged databases using BLAST searches. In addi-tion to the yeast nucleoporin sequences describedabove, high similarity scores (50–58%) were ob-tained with predicted amino acid sequences ofseveral C. elegans cDNA clones (accession nos.C47094, C40541 and C45548; Kohara Y, Moto-hashi T, Tabara H, Watanabe H, Sugimoto A,Sano M, Miyata A and Nishigaki A. Expressionmap of the C. elegans genome. Unpublished.).These sequences all contained GLFG motifs andso could possibly encode nucleoporins. However,no predicted amino acid sequences containingGLFG motifs were identified from the Plasmod-ium falciporum and Toxoplasma gondii EST data-bases. The spm1 predicted amino acid sequencealso has two consensus sequences for the ATP/GTP binding motif A [14], which were identifiednear the C-terminus (amino acid positions 612–619 and 639–646). These motifs are not generallya feature of nucleoporins, and may imply anadditional function for spm1.

Southern blot analysis of T. annulata genomicDNA confirmed we had identified a T. annulatagene. The 800 bp KP6 insert from pGEM7ZF-

P. Knight et al. / Molecular and Biochemical Parasitology 100 (1999) 135–140 137

KP6, representing the central part of the spm1gene, hybridised to EcoRI restriction fragments inDNA from geographically isolated T. annulatapiroplasm stocks (Ankara, Gharb and Hissar) ofapproximately 12 kb at high stringency, while no

hybridisation was observed to bovine BL20 cellline DNA (Fig. 2A). No EcoRI sites are presentwithin the probe sequence, but a single site existsin the spm1 gene. The presence of spm1 RNAtranscripts in different life-cycle stages of T. annu-

Fig. 1. Amino acid alignment of spm1 with yeast nucleoporins. A comparison of the central region (a.a. residues 289–671) of thespm1 predicted amino acid sequence, containing the GLFG repeat motifs, is aligned with the amino acid sequences of the yeastnucleoporins NUP100 (959 a.a.), NUP116 (1113 a.a.) and NUP49 (472 a.a.) [10,11] using the genetics computer group PILEUPprogram. The GLFG and FG motifs in the spm1 sequence, and any corresponding motifs in the nucleoporin sequences, are shownin bold (FG) and matching sequences are boxed. Dots indicate gaps inserted by the program. The two regions homologous toATP/GTP binding motif A [14] are overlined (……). Numbers refer to amino acid positions in each sequence. Sequencing was carriedout with vector-derived and sequence specific primers, and all sequence analysis performed using programs available in the geneticscomputer group package [18] at the SEQNET facility, Daresbury, UK and the Genbank WWW site.

P. Knight et al. / Molecular and Biochemical Parasitology 100 (1999) 135–140138

Fig. 2.

of the spm1 cDNA (2888 bp). Since spm1 appearsto be expressed in all the T. annulata life-cyclestages investigated we would expect a basic‘housekeeping’ role for this gene product. Thehomologies between the spm1 predicted aminoacid sequence and well-characterised nucleoporinsequences indicate that we have identified a nucle-oporin-like sequence from T. annulata. The pres-ence of GLFG and FG motifs in the predictedspm1 sequence is interesting, since evidence isaccumulating that these motifs in nucleoporinsmediate protein–protein interactions and RNAexport. Many nucleoporins in yeast and metazoahave been shown to be necessary for import ofproteins into the nucleus and/or export ofmRNA, in the form of ribonucleoprotein, fromthe nucleus to the cytoplasm [1,7]. FG repeats

Fig. 2. Southern and northern blots probed with the 800 bpEcoRI insert from pGEM7ZF-KP6, part of the spm1 gene,labelled with 32 Pa dCTP by random priming.(A) Southernblot analysis to show the presence of the spm1 sequence in T.annulata but not bovine host DNA. Genomic DNA from threeuncloned geographically-separated isolates of T. annulata weredigested with EcoRI, separated on 0.6% agarose gels andhybridised at high stringency with the spm1 derived KP6 insertfrom pGEM7ZF-KP6. Tracks were loaded with 2 mg totalDNA derived from T. annulata piroplasms from (A) Ankara[19], (G) Gharb (H. Ouhelli. Ph.D. thesis. Toulouse 1985) and(H) Hissar [20] stocks, and (Bv) uninfected bovine leucocytes(BL20 cell line [21]). The sizes of the restriction fragments areshown in kb.(B) & (C) Northern blot analysis demonstratingthe 3 kb spm1 transcript in different life-cycle stages of T.annulata. Total RNA prepared from different T. annulatalife-cycle stages was electrophoresed through 0.8% formalde-hyde agarose gels and blotted onto nylon membranes. Trackscontain approximately 10 mg total RNA prepared from unin-fected (U) and T. annulata (Ankara) sporozoite infected (I)tick salivary glands and undifferentiated macroschizont-in-fected leucocytes (M) (Fig. 2B), and from T. annulata (AnkaraD7) macroschizont-infected leucocyte cultures at day 0 (M0),day 2 (M2), day 4 (M4) and day 6 (M6) after heat-induceddifferentiation [9], and piroplasms derived from infected blood(P) (Fig. 2C). Sporozoites were prepared from 3 day fedinfected adult Hyalomma anatolicum anatolicum ticks and T.annulata macroschizont-infected lymphoblastoid cell lines weremaintained in tissue culture as described [22]. Differentiatingmacroschizont-infected leucocytes were prepared from heat-in-duced cultures [9]. Piroplasms were prepared from T. annulatainfected bovine blood [23]. The positions of the RNA ladderare shown in kb (Fig. 2B); and the position of the 3 kb signalin later life-cycle stages indicated (Fig. 2C).

lata was assessed on northern blots. The KP6probe hybridised predominantly to a transcript of3 kb in tracks containing RNA from sporozoiteinfected tick salivary glands, but there was nohybridisation to RNA from uninfected tick sali-vary glands (Fig. 2B). Hybridisation to transcriptsof 3 kb could also be observed in RNA fromundifferentiated T. annulata-infected leukocytes,heat-induced macroschizont-infected leukocytecultures at various stages of differentiation intomerozoites [9], and in piroplasms (Fig. 2B, C).Thus the spm1 gene appears to be transcribed inall bovine host stages of T. annulata examined.

In summary, here we have described the genefor a newly identified T. annulata molecule spm1,which is transcribed in sporozoites and all thelife-cycle stages within the bovine host (Fig. 2B,C). The size of the RNA transcript in all stageswas approximately 3 kb, correlating with the size

P. Knight et al. / Molecular and Biochemical Parasitology 100 (1999) 135–140 139

rather than GLFG or XFXFG repeats are afeature of some recently identified nucleoporinsessential for ribonucleprotein export, such asXenopus NUP98 [5], where they act as proteinligand docking sites [15], and human hRIP, inwhich the interaction with the HIV Rev protein inRNA export [16] is mediated by the FG motifs[17]. The yeast nucleoporins NUP100, NUP116and NUP49 have all been implicated in nucleocy-toplasmic transport of RNA [10,11] involving theGLFG region [4] and contain FG/GLFG motifswith a similar spatial arrangement to those inspm1 (Fig. 1). It would be interesting to knowwhether the FG motifs in spm1 also have a role inprotein–protein interactions and/or RNA export,and whether the mode of transport of macro-molecules to and from the nucleus is conservedbetween protozoan parasites and higher eukary-otes. It is hoped that the identification and furthercharacterisation of such sequences will increaseour understanding of the molecular interactionswithin Theileria parasites.

Acknowledgements

Thanks to Erol Kirvar, Gwen Wilkie and MaryThomas at CTVM for their assistance with para-site material, Brian Shiels, Sue McKellar andeveryone at WUMP for assistance and discussionin the early stages of this work, Bob Munro atVCS for photography in this manuscript andMichael Rout at The Rockefeller University, NewYork for helpful recommendations. Some of thesequencing was carried out by Julia Bartley andthe University of Durham sequencing service.This work was supported by funding from theWellcome Trust, the BBSRC and the OverseasDevelopment Association.

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