Parasitol Res (2003) 90: 84–86 DOI 10.1007/s00436-002-0600-0
SH O RT CO MM U N IC A T IO N
Harald Klein Æ Stefanie Mueller Æ Bettina Loeschner Ralf R. Toenjes Æ Gundula Braun Æ Eva-Christina Mueller Albrecht Otto Æ Thomas Montag
Cloning, sequencing and recombinant expression of the open reading frame encoding a novel member of the Sarcocystis muris (Apicomplexa) microneme lectin family Received: 8 December 2001 / Accepted: 3 January 2002 / Published online: 14 February 2003 Springer-Verlag 2003
Abstract Micronemes are characteristic secretory organelles located within the apical cell region of apicomplexan parasites. The protein contents are exocytosed during an early phase of host cell invasion and contribute to parasite motility and the invasion of target cells. We report here on the cloning and heterologous expression of a novel member of the Sarcocystis muris microneme lectin family. The deduced amino acid sequence is in total agreement with that obtained after sequencing the native protein and is characterized by two copies of the apple domain motif. The recombinant polypeptide is expressed in a biologically active conformation as demonstrated by its galactose binding properties.
Micronemes of the cyst merozoites of Sarcocystis muris contain at least two major proteins with apparent molecular masses of approximately 15 kDa. Both are galactose specific lectins (S. muris lectins, SML) functionally related to the Sarcocystis gigantea lectins previously described by Montag et al. (1987). They can be purified using a combination of different biochemical methods including lactose affinity chromatography (unpublished data). Using confocal laser scan microscopy in combination with a cross-reactive monoclonal
H. Klein Æ S. Mueller Æ B. Loeschner Æ T. Montag (&) Paul-Ehrlich-Institute, Division Parasitology/Diagnostics, Paul-Ehrlich-Strasse 51-59, 63225 Langen, Germany E-mail:
[email protected] Tel.: +49-6103-772665 Fax: +49-6103-771250 R.R. Toenjes Æ G. Braun Paul-Ehrlich-Institute, Department of Medical Biotechnology, Langen, Germany E.-C. Mueller Æ A. Otto Max Delbrueck Center for Molecular Medicine, Division Protein Chemistry, Berlin, Germany
antibody (mAb) which is directed to the two SML polypeptides mentioned above, Entzeroth et al. (1992) demonstrated the secretion of these proteins during the invasion of host cells, with an increased amount of lectin localized at the moving junction. Taking advantage of the same antibody, we have isolated a full-length cDNA clone encoding one of the microneme proteins from a S. muris cyst merozoite expression library (SML1, previously named pSM/1.6; Eschenbacher et al. 1993). Subsequent screening of a subgenomic S. muris library gave rise to an additional reading frame, showing 86.7% identity to the cDNA clone based on the deduced amino acid (aa) sequences (SML3, previously named GEN/4; Klein et al. 1996). Either polypeptide precursor consists of a typical N-terminal secretory signal sequence followed by a hydrophilic propeptide of unknown function and the mature protein. The derived sequences of the mature molecules (138 aa) showed 79.7% identity and their theoretical molecular masses are 15,019 Da and 15,112 Da, respectively (Klein et al. 1996). However, complete sequencing of the two native lectins isolated from S. muris cyst merozoites gave an unexpected result. Whereas one sequence is in total agreement with the deduced aa sequence of the cDNA clone encoding mature SML1, the second did not match with the suggested sequences of the two clones mentioned above (Mueller et al. 2001). This implies that the Sarcocystis genome harbours a third gene encoding a microneme protein (SML2) of cyst merozoites. The present study was designed to isolate the corresponding open reading frame (ORF) and to express the recombinant lectin in a biologically active conformation in a secretory baculovirus expression system (BVES). Based on the known nucleotide sequences of the two clones referred to earlier, degenerate primers (forward: 5¢-GCA-GGN-CCN-CAA-CTC-GAC-GTC-AG C-3¢; reverse: 5¢-GGC-ACG-YTT-RCT-AGA-NGT-YT G-GCT-GTA-GAG-3¢) spanning the entire ORF were synthesized. The oligonucleotides were deduced from aa residues 1–8 of the N-terminal sequence and aa residues 129–138 from the C-terminal sequence of the novel
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15 kDa microneme protein. A polymerase chain reaction was performed using these primers and total DNA isolated from S. muris cyst merozoites. A specific fragment of about 414 bp was obtained and ligated into the plasmid pBAD-TOPO (Invitrogen). At least four randomly picked clones containing inserts were sequenced by the dideoxy chain termination method (Sanger et al. 1977). Figure 1 shows the nucleotide and derived aa sequence. As expected, the cloned DNA fragment represents a single ORF of 414 bp encoding a protein of 138 aa. The calculated molecular mass is 15,078 Da. The deduced aa sequence is in perfect agreement with the sequence obtained for the native protein. The encoded polypeptide is predominantly hydrophilic [mean hydrophobicity index: –0.45 (Kyte and Doolittle 1982)] and rich in cysteine (8.7%). Due to the frequently occurring Asp and Glu residues, the calculated pI of the protein is slightly acidic (6.54). A single potential asparaginelinked glycosylation site could be detected at aa position 81 (Fig. 1). However, attempts to detect glycosylation of the native protein failed (data not shown), leading to the assumption that this site is not used in vivo. The aa sequence alignment of SML2 with those of SML1 and SML3 (Fig. 2) reveals 72.5% and 65.2% identity, respectively. Large blocks of conserved aa sequences are scattered throughout the ORF, although the vast majority of identity is located within the first half of the sequences. The presence and spacing of the 12 cysteine residues are rigorously conserved in all SML molecules, indicating their importance for the structure and/or function of the microneme proteins via disulfide bond formation. Furthermore, all polypeptides possess the duplicated apple domain-like motif C-X3-C-X5-CX2-F/Y-T-Y/F, which was also found in six copies in
a microneme protein (MIC4) of the closely related coccidian parasite Toxoplasma gondii (Brecht et al. 2001). In addition, multiple copies of this motif were identified in a microneme protein (EtMIC 5) of Eimeria tenella (Brown et al. 2000). Based on the conserved nature of this domain in apicomplexan parasites, one could speculate that polypeptides containing apple domainlike motifs may represent a new family of microneme proteins with adhesive properties. For heterologous expression of the novel SML in a soluble and biologically active conformation in the BVES, the corresponding SML2 ORF was subcloned into the baculovirus transfer vector pMelBacA (Invitrogen) which is designed to directly express recombinant proteins through the secretory pathway to the extracellular medium. Recombinant baculoviruses were generated by homologous recombination after cotransfection of Sf9 insect cells (Invitrogen) with genetically engineered plasmid DNA and Bac-N-Blue linear virus DNA (Invitrogen). For the expression of secreted SML2, High Five insect cells (Invitrogen) were infected with recombinant viruses harbouring the SML2 ORF. The supernatant (30 ml) of a 3-day culture was passed through a lactose column using the SMART-HPLC system (Amersham Pharmacia Biotech). As for the native protein, galactose-binding activity of the recombinant lectin was monitored by desorption of bound material with a galactose gradient. As shown in Fig. 3, the recombinant protein was purified to near homogeneity and migrates at approximately 15 kDa (lane 3), which is in the expected range. Additionally, it could be assumed that the lectin was expressed in its native structure since sugar binding activity strongly depends on the correct conformation of the saccharide recognition site. The identity of purified recombinant SML2 was further established by its reaction with a polyclonal antiserum raised against an Escherichia coli-derived version of SML1 (Fig. 4, lanes 1 and 2, respectively). The observed band-doublet could be the result of different N-terminal signal peptide cleavage sites used
Fig. 1 Nucleotide and deduced amino acid (aa) sequence of the open reading frame encoding a novel Sarcocystis muris major microneme protein (SML2). Numbering of the aa sequence starts at the N-terminus of the mature protein. The consensus sequence N-X-S/T (X=any aa except proline) of a potential asparaginelinked glycosylation site is underlined and the cysteine residues are marked by asterisks
Fig. 2 Multiple sequence analysis of the three open reading frames encoding microneme proteins of S. muris. Identical aa residues are boxed (black for all sequences, and grey for two sequences). Conserved cysteine residues are marked by asterisks and the duplicated apple domain-like motif C-X3-C-X5-C-X2-F/Y-T-Y/F (X=any aa) is indicated by plus signs. Sequence numbering is given in brackets and starts at the N-terminus of the mature proteins
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Fig. 3 Silver-stained SDS-PAGE analysis of recombinant SML2 (8–25%, reducing conditions). Lane 1 shows the supernatant of an insect cell culture 3 days after infection with recombinant baculoviruses harbouring the open reading frame encoding SML2. Lane 2 contains the flow-through of the lactose column. Lane 3 represents recombinant SML2 after elution with a galactose gradient. Molecular size standards are given on the left (M)
lectin-sugar interactions are involved in the multistep invasion process of host cells by apicomplexan parasites. At least for S. muris, sequence variations in distinct regions of the aa sequence may fine tune the specificity and/or affinity of the lectins towards complex oligosaccharides, which possibly provide alternative pathways or different functions for the successful invasion of host cells. At present it is unclear whether the ORF encoding SML3 that was isolated from a subgenomic library is expressed in the cyst merozoite as well. Experiments to purify the corresponding native protein by simple lactose affinity chromatography failed, although a recombinant form of SML3 clearly possesses galactose binding activity (unpublished data). Therefore, it is possible that SML3 is expressed in one of the other developmental invasive stages in the life cycle of S. muris. Acknowledgements We gratefully acknowledge Nadja Zyto for critically reading of the manuscript and helpful discussions. Note: the nucleotide sequence data reported in this paper are available in the GenBank, EMBL and DDBJ databases under the accession numbers L13471 (SML1), AF130977 (SML2), and U21964 (SML3).
References
Fig. 4 Western blot analysis of recombinant SML2, probed with a polyclonal antiserum directed against recombinant SML1 (10–20% SDS-PAGE gel, reducing conditions). Lane 1 contains the supernatant of an insect cell culture 3 days after infection with recombinant viruses harbouring the open reading frame encoding SML2. Lane 2 shows the galactose eluate of the lactose column. Lane 3 represents the flow-through of the sugar affinity chromatography. Molecular size standards are given on the right (M)
by the secretory apparatus of the insect cells. The flow-through (lane 3) of the lactose column was completely free of recombinant protein, indicating the efficiency of the purification procedure. The total yield of highly purified and biologically active recombinant SML2 was approximately 100 lg per 30 ml cell culture supernatant. Since the microneme proteins we are dealing with are galactose-specific lectins, it could be assumed that
Brecht S, Carruthers VB, Ferguson DJ, Giddings OK, Wang G, Jaekle U, Harper JM, Sibley LD, Soldati D (2001) The toxoplasma micronemal protein MIC4 is an adhesin composed of six conserved apple domains. J Biol Chem 276:4119–4127 Brown PJ, Billington KJ, Bumstead JM, Clark JD, Tomley FM (2000) A microneme protein from Eimeria tenella with homology to the apple domains of coagulation factor XI and plasma pre-kallikrein. Mol Biochem Parasitol 107:91–102 Entzeroth R, Kerkhoff H, Koenig A (1992) Microneme secretion in Coccidia: confocal laser scanning and electron microscopy of Sarcocystis muris in cell culture. Eur J Cell Biol 59:405–413 Eschenbacher KH, Klein H, Sommer I, Meyer HE, Entzeroth R, Mehlhorn H, Rueger W (1993) Characterization of cDNA clones encoding a major microneme antigen of Sarcocystis muris (Apicomplexa) cyst merozoites. Mol Biochem Parasitol 62:27–36 Klein H, Mehlhorn H, Rueger W (1996) Characterization of genomic clones encoding two microneme antigens of Sarcocystis muris (Apicomplexa). Parasitol Res 82:230–237 Kyte J, Doolittle RF (1982) A simple method for displaying the hydropathic character of a protein. J Mol Biol 157:105–132 Montag T, Tietz HJ, Brose E, Liebenthal C, Mann W, Hiepe T, Hiepe F, Coupek J (1987) The mitogenicity of extracts from Sarcocystis gigantea macrocysts is due to lectin(s). Parasitol Res 74:112–115 Mueller JJ, Mueller EC, Montag T, Zyto N, Loeschner B, Klein H, Heinemann U, Otto A (2001) Characterization and crystallization of a novel Sarcocystis muris lectin, SML2. Acta Crystallogr D 57:1042–1045 Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A 74:5463–5467
