cDNA cloning and chromosomal mapping of a predicted coiled-coil proline-rich protein immunogenic in meningioma patients
cDNA cloning and chromosomal mapping of a predicted coiled-coil proline-rich protein immunogenic in meningioma patientsDirk Heckel1, Nicole Brass1, Ulrike Fischer1, Nikolaus Blin5, Ingo Steudel2, Özlem Türeci3, Oliver Fackler4, Klaus D. Zang1 and Eckart Meese1,*
1Institut für Humangenetik, Theoretische Medizin, Universität des Saarlandes, 2Neurochirugie, 3Innere Medizin I and 4Abteilung für Virologie, Universitätskliniken, Universität des Saarlandes, 66421 Homburg/Saar, Germany and 5Institut für Humangenetik und Anthropologie der Universität Tübingen, Wilhelmstrasse 27, 72074 Tübingen, Germany
Received April 28, 1997;Revised and Accepted August 26, 1997
There is increasing evidence that tumor expressed genes induce immune responses in cancer patients. To identify meningioma expressed antigens, we established a meningioma expression library which was screened with autologous serum. Out of 20 positive cDNA clones eight share high sequence homologies as determined by sequence analysis. These eight clones can be grouped into three classes which differ in length and which are characterized by specific sequence variations. The longest open reading frame was found to be 2412 bp encoding an immunoreactive antigen termed meningioma expressed antigen 6 (MEA6). Using five sequence specific primer pairs, somatic hybrid panel mapping revealed locations of the three classes on several human chromosomes including chromosomes 2, 3, 6, 7, 9, 13 and 14. The mapping results were confirmed by fluorescence in situ hybridization. RT-PCR showed consistent expression of all classes in several meningiomas and additional tissues using the same set of primer pairs as for chromosomal mapping. The expression data were confirmed by northern blot analysis. For the predicted amino acid sequence BLASTX revealed a homology to a human C219-reactive peptide which was previously isolated by an antibody directed against p-glycoprotein. Sequence properties of the MEA protein include an acidic activation domain, a proline-rich region and two coiled-coil domains indicating protein binding and activation functions.
Meningioma are among the most common tumors of the human nervous system (1 ). Cytogenetic and molecular analysis revealed several specific chromosomal alterations with the loss of human chromosome 22 sequences as the most frequent change in the meningioma karyotype (2 -4 ). Recently, inactivation of the NF2 gene which maps at 22q12.2 has been found in the majority of meningioma (5 ). However, 40% of meningioma retain both copies of chromosome 22 and do not show mutations within the NF2 gene clearly indicating that additional genes are involved in the tumorigenesis of meningioma. A variety of approaches have been employed to identify further genes associated with meningioma development including subtractive cDNA library screening and deletion mapping on chromosome 22 (5 -7 ). As yet, studies have failed to identify a commonly deleted region on chromosome 22 which could serve as a starting point for positional cloning. To circumvent these limitations an immunological approach has been employed to identify genes which are involved in the tumorigenesis of meningioma. A variety of tumors are known to express antigens which elicit an immune response in patients including colon cancer, lung cancer and breast cancer (8 -10 ). The majority of the studies reported antibodies against known proteins as, for example, the products of the genes p53 and c-myc (11 ,12 ). To identify novel antigens expressed in human tumors, expression libraries derived from various tumors have been screened with autologous sera. This approach has led to the identification of several novel antigens expressed in human tumors including eIF4[gamma] in lung carcinoma (9 ), Hom-RCC3.1.3 in renal cancer and HOM-Glio-30.3.1 in a glioma (13 ,14 ). However, immunoreactive antigens have been reported only in malignant tumors, not in benign tumors such as meningioma.
In this study, we established an expression library from a meningioma and screened the library with the autologous patient serum. Positive clones were sequenced, analyzed for expression in meningioma and localized to human chromosomes. We identified a group of clones representing a novel gene expressed in meningioma and other tissues.
A cDNA expression library was established from a meningioma with normal karyotype and expressed fusion proteins were screened with autologous serum. In detail, poly(A) mRNA was reverse transcribed into cDNA and inserted in the ZAP ExpressTM Expression vector (Stratagene) in sense orientation with respect to the lacZ promoter. Recombinant proteins were expressed in Escherichia coli and screened with preabsorbed patient serum. Antigen-antibody complexes were detected by a secondary antibody binding to the constant region of the human IgG-heavy chain. Positive clones were isolated and subjected to a second round of screening with the autologous serum (Fig. 1 A and B). In addition, positive clones were screened with sera from unrelated individuals including sera from patients with glioblastoma, pilocytic astrocytoma, neurinoma and lung cancer, respectively. As demonstrated in Figure 1 C there were no antigen-antibody complexes found with any of the control sera. As an additional positive control mea was subcloned into a pQE expression vector, expressed in E.coli and identified by Western blotting using autologous serum as probe (Fig. 1 D).
In this study we identified a novel immunoreactive antigen termed MEA by screening a meningioma specific expression library with autologous patient serum. Importantly, screening with a heterologous serum of a second meningioma patient also indicated an antibody response for the MEA protein (data not shown). However, we did not obtain an antibody response against MEA protein using sera of patients with other tumors including glioblastoma, pilocytic astrocytoma, neurinoma, and squamous cell lung carcinoma. These results indicated that the MEA proteins act as antigens expressed in meningioma. As for the mechanism of the expression of an immunoreactive antigen our data do not provide evidence for major variations of the MEA mRNA expression level in meningioma and control tissues. However, the identified nucleotide exchanges or, alternatively, posttranslational modifications of the MEA protein may account for MEA antibodies. Further light will be shed on the role of MEA proteins in meningioma by studies analyzing the activity of antibodies against MEA proteins in meningioma.
Forty percent of the positive cDNA clones share high sequence homologies indicating an overrepresentation of the corresponding transcripts and a possible role of the antigen in the development of meningioma. While several studies report the identification of autoantigens in different malignant tumor types, our investigation provides first evidence for the expression of autoantigens in benign tumors.
As for the function of the new autoantigen, the nucleotide sequence did not show any homology to known genes. The predicted amino acid sequence, however, revealed a homology to a recently reported peptide encoded by `human mRNA for KIAA0268 gene' (16 ) which itself was reported to be similar to a smaller peptide isolated with a monoclonal antibody C219, allegedly specific for p-glycoprotein. Surprisingly, neither the C219 reactive peptide nor the corresponding nucleotide sequence showed homology to p-glycoprotein or the MDR gene (17 ).
The deduced amino acid sequence MEA apparently contains two coiled-coil domains. The first of the two predicted coiled-coil structures is characterized by a heptat repeat of four leucine residues which possibly indicates a leucine zipper motive. The second coiled-coil domain includes the area with the highest sequence homology between the predicted amino acid sequence of MEA and the peptide encoded by the `human mRNA for KIAA0268 gene'. It has recently been demonstrated that the prediction of coiled-coil domains is in agreement with experimental data obtained by circular dichroism analysis and electron microscopy (18 ). The high percentage of acidic amino acids at the N-terminal end of the predicted protein most likely indicates an acidic activation domain which has previously been reported in numerous eukaryotic transcription factors (19 ). The C-terminal portion of the protein is highly enriched for proline residues which have been reported to interact with specific protein domains including the scr-homology-domain type 3 (SH3) and WW domains found in proteins of signal transduction pathways (20 ,21 ). Together, the predicted coiled-coil domains, the high percentage of acidic amino acids in the N-terminal part and the proline stretches in the C-terminal portion of the protein strongly indicate that the MEA antigen is involved in protein-protein interaction.
To gain further insight into the role of MEA we determined the chromosomal localization of the MEA encoding gene. Somatic hybrid panel mapping and fluorescent in situ hybridization indicated corresponding sequences on several chromosomes including chromosomes 2, 3, 6, 7, 9, 13 and 14. There were no signals on chromosome 22 which is affected by loss of heterozygosity in the majority of meningioma (2 -4 ). In addition to chromosome 22 changes, previous cytogenetic and molecular genetic studies show that chromosomes 1, 6, 11, 13, 14, 18, 19, X and Y were also involved in structural and numerical alterations in meningioma (22 ). It remains to be seen whether the alterations of chromosomes 6, 13 and 14 can be specifically related to possible antigen encoding genes on these chromosomes.
There is no easy explanation for the chromosomal mapping data which indicate different locations for the 5'- and 3'-ends of the mea sequences. Primer pairs MEAaltA and MEA3' localized at the 3'-end map on chromosome 14 while primer pair MEA5' localized at the 5'-end has been excluded from chromosome 14. On the other hand the data are consistent with open reading frames on chromosomes 6, 7, 13 and 14. The RT-PCR data indicate that several MEA encoding sequences are transcribed from various chromosomal loci. Provided the majority of the mea sequences represent intronless pseudogenes it is conceivable that chromosome 14 harbors the active MEA encoding gene. Chromosome 14 was also found to give the strongest hybridization signals in the in situ hybridization experiment. However, based on our data it cannot be ruled out that chromosomes 6, 7 and 13 contain coding mea sequences. This question cannot be finally answered until the complete intron-exon structure of the MEA encoding genes has been analyzed.
In summary, the implications of our study are five-fold. First, we identified a novel immunoreactive antigen expressed in meningioma. This is the first autoantigen reported in benign tumors. Second, chromosomal mapping indicated locations of closely related sequences on several human chromosomes, possibly indicating a group of evolutionary conserved genes. Third, the novel gene appears to be widely expressed in different tissues including meningioma. Fourth, the predicted amino acid sequence shows homology to a human protein similar to a C219-reactive peptide which was isolated by an antibody directed against p-glycoprotein. Fifth, the predicted coiled-coil domains and the identification of two regions bearing a high percentage of acidic amino acids and proline residues, respectively, strongly indicate involvement of the MEA protein in protein-protein interaction.
Genomic DNA was isolated from tumor tissue and blood lymphocytes according to standard protocols (22 ). Following proteinase K digestion, proteins were extracted with chloroform and high molecular weight DNA was precipitated with isopropanol. RNA isolation was according to the manufacturer's instructions (Stratagene). Frozen tissue was homogenized, proteins were phenol-chloroform extracted and RNA was precipitated twice with isopropanol and finally resuspended in DEPC treated H2O. Integrity and concentration of RNA was evaluated using formaldehyde gels.
Clone DNA (10 ng) was labeled with biotin-16-dUTP by nick translation according to the manufacturer's instruction (Gibco BRL, Nick Translation System). Biotinylated DNA was hybridized against metaphase chromosome spreads of a normal karyotype and visualized using avidin conjugated to fluorescein isothiocyanate. After three rounds of amplification using goat anti-avidin antibodies fluorescent signals were analyzed in a Zeiss microscope and documented with the program ISIS3 of MetaSystems.
Total RNA was applied to oligo(dT) cellulose push columns and poly(A) mRNA was eluted according to the Poly(A) Quick® Kit (Stratagene). cDNA synthesis was performed with the ZAP ExpressTM cDNA synthesis kit (Stratagene). In brief, 4.5 [mu]g of poly(A) mRNA was reverse transcribed by MMLV-reverse transcriptase using a oligo(dT) primer with a 5' XhoI restriction site. The cDNA was ligated to EcoRI adapters, XhoI digested, size fractionated with Sephacryl S-500 columns and cloned into ZAP ExpressTM vector arms. The vector was packaged using the Gigapack® III Gold Packaging Extract (Stratagene). Transfection was with E.coli XL1blue MRF' host strain grown in LB-medium supplemented with 0.2% (w/v) maltose and 10 mM MgSO4. One round of amplification was performed and the phage titer determined to be 1.3 * 1010 p.f.u./ml.
Blood serum was isolated from 10 ml samples using serum gel monovettes and was stored at -75oC. Prior to use the serum samples were diluted 1:10 in 1* TBS, 0.5% (w/v) dry milk and 0.01% thimerosal. Preabsorption columns were assembled by incubating sonificated E.coli XL1blue MRF'cells in 1* TBS with Affinity Adsorbent (Glutaraldehyde-activated; Boehringer Mannheim) in BioRad Polyprep chromatography columns overnight. `Lytical' columns are prepared by using bacteria lysed by non-recombinant ZAP express phages. The serum was preabsorbed by gravity flow using each column type five times. The preabsorbed serum was diluted to a final concentration of 1:100 in 1* TBS, 0.5%(w/v) dry milk and 0.01% thimerosal.
Escherichia coli XL1blueMRF' cells were transfected with the cDNA expression library and plated to an approximate density of 10 000 p.f.u./plate on NZCYM agar plates in the presence of 12.5 [mu]g/ml tetracycline. After 4 h of incubation at 42oC, fusion protein expression was induced by applying Duralose UVTM-membranes (Stratagene) which were soaked in 2 M IPTG. Subsequent to a second incubation for 4 h at 37oC the plates with the filters were stored overnight at 4oC. Membranes were removed, washed twice for 15 min in 1* TBST and blocked with 5% (w/v) dry milk in 1* TBS for 1 h. Following three additional wash steps of 10 min in 1* TBS the membranes were incubated for 3.5-4 h in diluted autologous serum. Membranes were washed three times for 10 min in 1* TBS and incubated with goat anti-human IgG antibody conjugated to alkaline phosphatase for 1 h. Antigen-antibody complexes were detected by 0.005%(w/v) BCIP prediluted in 100%(v/v) DMF and 0.01%(w/v) NBT prediluted in 70%(v/v) DMF in 1* color developing solution.
PCR was carried out in a thermal cycler (PTC100 MJ Research Inc.) for 26-28 cycles. Initial denaturation was at 94oC for 5 min, each cycle consisted of incubation for 1 min at 94oC, annealing for 45 s at 58oC and extension for 45 s at 72oC. Final extension was carried out for 10 min at 72oC. Annealing for primer pair MEAINS was at 60oC for 45 s followed by an extension step of 1 min at 72oC. The sequences of the different primer pairs are listed in Table 1 .
RNA was treated with 20 U DNaseI per 2 [mu]g RNA for 15 min. Absence of DNA was evaluated by Alu-PCR using A1S-primer. Reverse transcription was performed twice using oligo-dT and random primers for 1 h at 37oC using MMLV-reverse transcriptase (Stratagene). PCR was performed as described above.
Sequencing was performed according to the manufacturer's instructions using the PERKIN ELMER ABIPrism Cycle sequencing kit. Clone inserts were sequenced with an automated sequencer (373A DNA sequencer, Applied Biosystems). Sequence alignment was done with BLASTN and BLASTX algorithms. Protein pattern searches were carried out with the Baylor College of Medicine search launcher using the COILS 2.2 program and the algorithms PROSITE, BEAUTY and SAPS.
The N-terminal portion of the mea sequence (nucleotides 52-1580) were subcloned into the BamHI- and SalI-sites of pQE30 expression vector (Qiagen) using PCR-primers for insert preparation. Escherichia coli M15 cells were made competent for transformation according to the manufacturer's instructions (Qiagen) and were transformed with the pQE30-mea11-N constructs. Transformants were screened for the presence of inserts of the correct size by PCR analysis utilizing vector-specific primers. Single positive colonies were grown in liquid culture overnight at 37oC, subsequently diluted 1:60 with LB broth to a final volume of 20 ml and regrown to a OD600 of 0.6. A 2 ml aliquot was saved as control. For expression induction of the recombinant protein, IPTG was added to a final concentration of 1 mM. Expression was allowed to proceed for 4 h before the cells were harvested by centrifugation at 4000 r.p.m. for 10 min. As a control, transformants containing the pQE30 vector without insert were treated accordingly. Cell pellets were resuspended in 2-fold sample buffer [6% SDS; 125 mM Tris pH 6.8; 10%(v/v) mercaptopropanediol; 10% (v/v) glycerol] and extracted by sonication (3 * 10 s at 500 W). Cell extracts were separated by electrophoresis on a 10% SDS-polyacrylamide gel at 25 mA for3 h. The proteins were transferred to a nitrocellulose filter (Amersham) by electroblotting at 330 mA for 1 h. The filters were prehybridized for 30 min in 1* PBS and 5% dry-milk and subsequentlysealed for overnight incubation at 4oC with patient serum diluted 1:100 in 1* TBS and 0.5% dry-milk. After washing 3 * 10 min in 1* PBS the filter was hybridized with a secondary goat anti-human IgG, Fc[gamma] specific antibody conjugated to peroxidase (Dianova) diluted 1: 5000 in 1* PBS 5% dry-milk for 1 h at 4oC. Detection was carried out using the ECL detection reagents (Amersham) according to the manufacturer's instructions.
This work was supported by a grant from the Deutsche Forschungsgemeinschaft (SFB 399, A4). The excellent technical help of Mrs Evi Vollmar is acknowledged. The E.coli host strain M15 was kindly provided by Norbert Schuster.
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*To whom correspondence should be addressed. Tel: +49 6841 166038; Fax: +49 6841 166186; Email: hgemee@med-rz.uni-sb.de
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