Human Molecular Genetics, 2001, Vol. 10, No. 11 1177-1183
© 2001 Oxford University Press
Mutations in the X-linked RP2 gene cause intracellular misrouting and loss of the protein
Max-Planck-Institut für Molekulare Genetik, Ihnestraße 73, 14195 Berlin, Germany
Received 22 January 2001; Revised and Accepted 29 March 2001.
DDBJ/EMBL/GenBank accession no. AJ303371.
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ABSTRACT |
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Mutations in RP2 cause the second most frequent form of X-linked retinitis pigmentosa, a severe retinal degeneration that leads to loss of visual acuity and blindness. The RP2 gene encodes a protein with homology to cofactor C, a tubulin-folding chaperone. By searching protein sequence databases, we identified a whole set of similar molecules from diverse organisms. Protein sequence alignments show that RP2 and cofactor C represent members of two distinct orthologous groups. All previously identified missense mutations affect amino acid residues which are conserved in all RP2 orthologues or both orthologous groups. Intracellular localization of the wild-type protein and mutated variants was determined by fluorescence microscopy of cells expressing RP2 with a green fluorescent protein tag. A mutation in the N-terminus of RP2 abolishes localization to the plasma membrane in HeLa cells. C-terminal protein truncation mutations, which account for 2/3 of the pathogenic RP2 variants, lead to scattered fluorescent foci in the cytoplasm of COS-7 and HeLa cells. Analysis of protein extracts from the respective cells with anti-RP2 antibodies identified truncated proteins of expected size in a low-speed centrifugation pellet while the wild-type protein appeared in the supernatant. Moreover, no protein was detected in immortalized cell lines from patients with protein truncation mutations while mRNA was still present. Thus, loss of the protein and/or aberrant intracellular distribution might be the basis for the photoreceptor cell degeneration in most RP2 cases.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Familial forms of retinitis pigmentosa (RP), a progressive degeneration of the light-sensitive photoreceptor cells in the retina, occur with a frequency of approximately 1 in 3700 individuals (1). The premature cell death primarily affects rod photoreceptors and starts in the mid-periphery of the retina. The degeneration progresses over two to three decades and can finally result in complete blindness of affected individuals. There is no effective treatment for the disease. Genetic linkage studies revealed more than 30 RP loci in the human genome and several gene defects have been identified by positional cloning or candidate gene approaches over the past years. Mutations were frequently observed in genes encoding different components of the photo-transduction cascade or structural proteins of photoreceptor cells (2–6). But genes of unknown function were also found to be mutated in patients with RP. The X-chromosomal forms of RP account for 3–20% of all familial cases (1,7–9). Four different loci were reported but most of the X-linked cases (80–90%) are due to mutations in RPGR (retinitis pigmentosa GTPase regulator) in Xp21.1 and the RP2 gene in Xp11.3. Both genes were isolated by positional cloning, show ubiquitous expression and contain domains with homology to known proteins (10–13). The N-terminus of RPGR is homologous to RCC1 (regulator of chromosome condensation), a guanine nucleotide exchange factor of the ras-like GTPase ran (14,15). RP2 codes for a polypeptide of 350 amino acids. The N-terminal portion of RP2 shows homology to human cofactor C, one of at least five chaperones (A–E) which act as GTPase-activating proteins in the tubulin-folding supercomplex (16,17). But the precise function of RP2 remains elusive. Twenty different pathogenic RP2 mutations reported so far represent two classes; missense and protein truncation mutations (13,18–22). The majority of missense mutations (five out of six) are clustered in the cofactor C homologous portion and might interfere with proper catalytic activity of RP2. The remaining mutations, that is 2/3 of all pathogenic changes, lead to truncated polypeptides by introducing a premature stop codon or frameshifts.
Here we report consequences of different mutations on protein level. We have tagged RP2 with the green fluorescent protein (GFP) and determined the intracellular localization of the fusion protein in different cell types by fluorescence microscopy. Subsequently, mutated variants were expressed in the same way. The majority of mutations leads to aberrant protein distribution in the cell. In addition, we raised anti-RP2 antibodies and analysed protein extracts from immortalized cell lines of patients with RP2 mutations.
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RESULTS |
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Protein sequence alignment
The predicted RP2 protein consists of 350 amino acids and possesses three distinct domains; a stretch of 40 hydrophilic residues at the N-terminus followed by a cofactor C homologous part (amino acid residues 42–192) and a C-terminal domain of unknown function (amino acid residues 193–350). Cofactor C is one of several proteins which are involved in the folding of
- and ß-tubulins. We have identified several RP2 and cofactor C homologous sequences by database searches and performed multiple protein sequence alignments (Fig. 1). Comparison of the amino acid sequences revealed RP2 and cofactor C orthologues in several species. Interestingly, all missense mutations in the RP2 gene reported to date affect amino acid residues which are conserved in all RP2 orthologues (Cys86Tyr, Leu253Arg) or even both orthologous groups (Pro95Leu, Arg118His). Mutations affecting completely conserved residues tend to show a more severe phenotype, although the statistical basis for this is still small. The Arg118His mutation was reported in three more families after its first description in one of our patients with severe disease manifestations (13,18,21,23). All but one of the missense mutations occur in the cofactor C homologous part of RP2. The hydrophilic N-terminal domain may contain an important signal for intracellular protein localization, as reported recently (24). The C-terminus of RP2 (amino acid residues 193–350) has no significant homology to other database entries and contains no characteristic protein motifs.
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Intracellular localization of the RP2 protein
To determine the intracellular localization of RP2, we expressed GFP-tagged fusion constructs in three different cell types, including fibroblasts, COS-7 and HeLa cells. Expression of the fusion proteins was confirmed by western blot analyses of protein extracts from transfected cells with anti-GFP and anti-RP2 antibodies. Fluorescence microscopy of transfected cells revealed striking differences in the localization of the fusion protein with respect to the cell type. Exclusive cytoplasmic staining was observed in two human fibroblast cell lines, irrespective of whether RP2 was tagged at its N- or C-terminus. In COS-7 cells, the N-terminally-tagged RP2 protein (GFP-RP2) was found in the nucleus and cytoplasm. A similar picture was obtained when GFP was expressed alone. In contrast, C-terminally-tagged RP2 (RP2-GFP) predominantly stained the cytoplasm of COS-7 cells. In HeLa cells, however, RP2-GFP localized to the plasma membrane (Fig. 2), whereas GFP-RP2 stained nucleus and cytoplasm (data not shown). Thus, subcellular distribution of the fusion protein depends on the cell type and the position of the GFP-tag. When RP2 was tagged at its N-terminus, the protein localization resembled fluorescence distribution of the empty GFP-vector, whereas a C-terminal GFP-tag revealed predominant staining of the cytoplasm in COS-7 and an intense staining of the plasma membrane in HeLa cells. Membrane localization of RP2 was also reported for a human neuroblastoma cell line and for Chinese hamster ovary (CHO) cells (24).
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Intracellular localization of mutant proteins
In order to examine the consequences of RP2 mutations on protein distribution, we expressed GFP-tagged mutated variants in HeLa and COS-7 cells. Mutations were introduced in the RP2 open reading frame (ORF) by site-directed mutagenesis. In this way a deletion of the serine residue at position 6 and the missense mutation Arg118His, both of which are found in patients with retinal degeneration, were mimicked. The deletion at position 6 clearly showed an effect on the intracellular distribution of RP2-GFP in COS-7 as well as HeLa cells (Fig. 2). Plasma membrane staining was abolished in HeLa and predominant nuclear staining was observed in COS-7. An arginine substitution at position 118 for histidine did not affect intracellular distribution of the fusion protein in HeLa and COS-7. As most of the reported RP2 mutations (>65%) result in frameshifts or lead to premature stop codons, we also expressed three truncated variants in HeLa and COS-7 cells. The three truncated forms lack 199, 150 or 55 (
exon 4) amino acid residues from the C-terminus of RP2. Each variant was GFP-tagged at the N- or C-terminus and fusion proteins were expressed in HeLa and COS-7 cells. All these fusion proteins form fluorescent foci in the cytoplasm of COS-7 as well as HeLa cells (Fig. 2), irrespective of the position of the GFP-tag. Depending on the length of the C-terminal deletion, the precipitate-like distribution can be observed in all transfected cells (Tyr151Stop) or only in part of them ( exon 4, 
10–20% of transfected cells). Expression of mutant proteins was confirmed by western blot analysis with antibodies raised against GFP and RP2. Strikingly, the amount of fusion protein was markedly reduced in the soluble protein fraction from transfected cells expressing truncated variants (Fig. 3). In contrast, the wild-type and two other mutant variants with a deletion of a single amino acid at position 6 (Ser6) and the missense mutation Arg118His revealed a band of the expected size with both antibodies. Subsequently, protein extracts were fractionized in a soluble and pellet fraction by low-speed centrifugation and analysed on western blots (Fig. 3). All RP2 variants with C-terminal deletions were detected in the pellet fraction with the anti-GFP as well as the anti-RP2 antibody, indicating that these proteins are either insoluble or associated with cell organelles.
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Loss of the RP2 protein in lymphoblastoid cell lines from patients
To investigate the effect of pathogenic mutations in vivo, we examined RP2 expression on RNA and protein level in immortalized lymphoblastoid cell lines from six patients with mutations in RP2. The spectrum includes one missense mutation (Arg118His), two nonsense (Gln26Stop, Tyr151Stop), two frameshifts (Tyr151FS, deletion of exon 4) and a LINE1 retro-transposition in intron 1 of RP2 (13). Northern blot analysis showed that all of the patient cell lines express the RP2 mRNA except the cells with the LINE1 retro-transposition (Fig. 4). Expression levels are variable but do not show a significant correlation with the type of mutation. Marginal transcription was observed in two patient cell lines with mutations at codon position 151 (Tyr151Stop and Tyr151FS). In contrast, mRNA level was normal in two other patients with protein truncation mutations (
exon 4 and Gln26Stop). In order to examine protein levels in these cell lines we generated antibodies against the RP2 protein. To this end we expressed the N- (amino acid residues 1–194) and C-terminal (amino acid residues 205–350) parts of RP2 as histidine-tagged polypeptides in Escherichia coli, purified them by affinity chromatography and immunized rabbits with these protein preparations. High titer antisera were affinity-purified and used for western blotting. These antibodies detected a protein of the expected size on western blots of several lymphoblastoid cell lines and HeLa cells and also in protein extracts from peripheral blood cells (data not shown). By using protein extracts of COS-7 cells transfected with the GFP-RP2 fusion constructs we could show that the N-terminal antibody recognizes epitopes located in the first 40 amino acids of the RP2 protein. When this part of RP2 is deleted, the fusion protein is no longer detected by the N-terminal anti-RP2 antibody but can be visualized with an anti-GFP antibody (Fig. 3). Protein preparations of the patient cell lines were probed with the antibody directed against the N-terminus of RP2 but no protein was detected except for the Arg118His mutation and a control (Fig. 4). Probing the insoluble fractions of these protein preparations did not yield an RP2 signal either, nor did the C-terminal antibody detect RP2 protein in any of the mutant cell lines except for the Arg118His mutation (data not shown).
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DISCUSSION |
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We made use of the GFP of the jellyfish Aequorea victoria to determine the intracellular localization of the ubiquitously expressed RP2 protein and mutated variants. For this purpose, several cell lines were used and expression constructs were cloned with a GFP-tag at the C- or N-terminal end of RP2, designated RP2-GFP and GFP-RP2, respectively. In HeLa cells, RP2-GFP localized to the plasma membrane. This specific localization was abolished when the GFP-tag was added to the N-terminus of RP2 (GFP-RP2). These results can be explained by a masking mechanism in which the GFP-tag interferes with a functional domain in the N-terminus of RP2 that triggers proper intracellular targeting. This is consistent with previous findings which identified a potential N-myristoyl transferase (NMT) recognition site at the N-terminus of RP2 and showed that the 15 N-terminal-most amino acids are sufficient to direct GFP to the plasma membrane in CHO cells (24). Inspection of the N-terminal sequence of the mouse Rp2 protein reveals the presence of an NMT recognition site also in this species (25). Additionally, we observed that subcellular protein distribution varied in different cell types that were used for transient expression of fusion proteins. In COS-7 cells, RP2-GFP predominantly stains the cytoplasm but not the plasma membrane as it does in HeLa cells. Cell type-dependent protein trafficking to the plasma membrane has been shown previously, e.g. for the cystic fibrosis transmembrane conductance regulator (CFTR), and may be attributed to cell type-specific expression of trafficking factors (26). Therefore it remains to be determined which retinal cell type contains RP2 and in which compartment.
We were also interested in finding out whether or not pathogenic RP2 mutations have an effect on intracellular protein distribution and therefore generated several mutated variants by in vitro site-directed mutagenesis. The Arg118His missense mutation in the cofactor C homologous part of RP2 has no effect on protein localization in COS-7 or HeLa cells. This fully agrees with previous findings in CHO cells which showed that membrane localization of RP2 is not disrupted by Arg118His (24). However, this and four additional missense mutations in the cofactor C homologous domain may have a deleterious effect on protein function. Indeed, this arginine residue is conserved in all members of both orthologous groups, RP2 and cofactor C, and a patient with this missense mutation showed a severe phenotype (23). Cofactor C as part of the tubulin folding supercomplex is responsible for the GAP activity of the latter and GAPs are known to share invariant Arg residues indispensible for their catalytic function (27). Arg118 of RP2 could be such a conserved Arg finger mediating GAP activity for a so far unknown target protein while the corresponding residue in cofactor C mediates GAP activity for ß-tubulin. The deletion of a single serine residue at position 6 of RP2 changes the intracellular protein distribution in COS-7 and HeLa cells. A similar observation was reported for CHO cells that stably express the Ser6 mutant variant and can be explained by the disruption of the NMT recognition site at the N-terminus of RP2 (24). A patient with this mutation shows rather mild disease symptoms (23). The less severe phenotype may be explained by a reduced amount of an otherwise functional protein in a particular compartment of the cell whereas the Arg118His mutation may completely abolish normal function.
The most striking consequences for protein localization were observed for RP2 variants with C-terminal deletions. This was independent of the cell-type and also found when the GFP-tag was added to the N-terminal end of RP2. C-terminal truncations were always associated with fluorescent foci in the cytoplasm, more pronounced in the perinuclear area. One explanation of this finding is an enhanced degradation of these incomplete proteins by the lysosomal pathway (28). The association of truncated proteins with cell organelles is supported by our observation that these proteins disappeared from the soluble protein fraction where the wild-type protein is found. This characteristic pattern was also observed when the protein was truncated by only 55 amino acids ( exon 4). An enhanced protein degradation can also explain the lack of the protein in lymphoblastoid cell lines from patients with protein truncation mutations. We could not detect RP2 protein upon western blot analysis, neither in the supernatant nor the pellet, even in patient cell lines with normal mRNA levels. Obviously, nonsense-mediated decay (NMD), a mechanism that detects and degrades mRNAs with premature translation termination signals, cannot explain the complete loss of the gene product (29,30). More likely, C-terminal protein truncations lead to misfolding and subsequent degradation of the non-functional protein (28). This is of particular interest as the vast majority of RP2 mutations introduce a frameshift or premature stop codon. Consequently, loss of the protein is the underlying mechanism for most of the RP2 cases.
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MATERIALS AND METHODS |
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Database searching and sequence alignment
Using the RP2 protein sequence we searched GenBank, SWISS-PROT and TrEMBL (31) for homologous entries with the BLAST algorithm (32). Protein sequences taken directly from the database or deduced from nucleotide records were aligned and a consensus calculated with the PILEUP and PRETTY programs (Wisconsin Package Version 10.1, Genetics Computer Group).
Cloning of GFP expression constructs
For the constructs expressing the wild-type, 1–40, Tyr151Stop, 201–350 and the exon 4 RP2 variants the corresponding parts of the RP2 cDNA were PCR-amplified from the human cDNA clone (RZPD no. DKFZp577C16138Q3) with Pfu polymerase (Stratagene) and suitable primers including restriction sites:
wild-type primer 1, 5'-CTAGATAAGCTTCCATGGGCTGCTTCTTCT-3'; wild-type primer 2, 5'-ACCGTCGACTGCAGATATTCCCATCTGTATATCAGC-3'; 1–40 primer 1, 5'-CTAGATAAGCTTCCATGTTCAGTGGACTGAAGG-3'; 1–40 primer 2, 5'-ACCGTCGACTGCAGATATTCCCATCTGTATATCAGC-3'; Tyr151Stop primer 1, 5'-CTAGATAAGCTTCCATGGGCTGCTTCTTCT-3'; Tyr151Stop primer 2, 5'-ACCGTCGACTGCAGACCATTGAAAACATCCAAATTTGA-3'; 201–350 primer 1, 5'-CTAGATAAGCTTCCATGGGCTGCTTCTTCT-3'; 201–350 primer 2, 5'-ACCGTCGACTGCAGGAGGAACATAGTCCTGAACCAC-3'; exon 4 primer 1, 5'-CTAGATAAGCTTCCATGGGCTGCTTCTTCT-3'; exon 4 primer 2, 5'-CCGTCGACTGCAGAGATACAAACATCTTTGTTC-3'.
The exon 4 construct mimicks exactly the RP2 mRNA of a patient described previously, including four new amino acids upstream of the premature stop codon (13). PCR products were digested and cloned into the PstI and HindIII sites of the pEGFPC1 and pEGFPN3 vectors (Clontech Laboratories). The Ser6 and Arg118His mutants were generated from the wild-type constructs by site directed mutagenesis (QuickChange Site-Directed Mutagenesis Kit). PCR primers were as follows: Ser6 primer 1, 5'-ATGGGCTGCTTCTTCAAGAGACGGAAGGCTGACAAGGAG-3'; Ser6 primer 2, 5'-CTCCTTGTCAGCCTTCCGTCTCTTGAAGAAGCAGCCCAT-3'; Arg118His primer 1, 5'-CCTGCCAACAATTTCATGTGCGAGATTGTAG-3'; Arg118His primer 2, 5'-CTACAATCTCGCACATGAAATTGTTGGCAGG-3'. All constructs were verified by overlapping sequencing.
Transfection, expression in cell culture and microscopic analysis
For transfection of COS-7, HeLa cells and fibroblasts, 1 µg of DNA and 5 µl of lipofectace (Gibco BRL, Life Technologies) diluted in OPTIMEM medium (Gibco BRL) were used according to the manufacturer’s recommendations. 5 x 104 cells were seeded in 6-well plates containing microscope coverslips and grown overnight prior to transfection. Preparations were observed in a ZEISS Axioscop 2 after 48–72 h either alive or after fixation for 2 min in 4% PFA, 1x PBS.
Western blotting
For immunochemical detection approximately 106 transiently transfected COS-7 or HeLa cells (grown for 72 h) were lysed in 200 µl of lysis buffer for 5 min at 4°C (2% Nonidet P40, 300 mM NaCl, 50 mM Tris, 0.01% SDS pH8, including protease inhibitor cocktail from Boehringer Mannheim). Insoluble material was sedimented at 3000 g and 4°C for 5 min and the supernatant was precipitated with 4-fold excess of acetone at 20 000 g and 4°C for 15 min. Both pellets were dissolved in 200 µl of Laemmli buffer, boiled for 5 min and 20 µg protein was loaded on a 10% SDS–PAGE. Protein from lymphoblastoid cells (2 x 107) was prepared in 1.8 ml of lysis buffer in the same manner as above and 60 µg was analysed by 12% SDS–PAGE. Protein was transferred to PVDF membrane by electroblotting and detected in 1x PBS, 0.05% Tween with either an anti-RP2 (1:100 dilution, 1 h) or an anti-GFP antibody [1:500 dilution, 1 h, GFP(FL); sc8334, Santa Cruz Biotechnology] after blocking the membrane for 1 h in 1x PBS with 5% skim-milk powder (Fluka Chemie AG). Primary antibodies were detected with an anti-rabbit Ig F(ab’)2 horseradish peroxidase-conjugated secondary antibody and chemiluminescence reaction (NEN Life Science Products).
Northern blotting
Total and poly(A)+-RNA from lymphoblastoid cell lines were isolated using the RNeasy Midi and Oligotex mRNA Midi Kit (Qiagen GmbH) according to the manufacturer’s recommendations. Three micrograms of poly(A)+-RNA were separated on a 6.7% formaldehyde gel, transferred and UV crosslinked to a nylon membrane. Hybridization was carried out with the radioactively labeled RP2 ORF for 1 h at 68°C in QuickHyb hybridization solution (Stratagene) according to accompanying protocols. The absence of any detectable RP2 transcript in the LINE1 insertion cell line was shown previously by RT–PCR (13).
Production of antibodies
RP2 amino acids 1–194 and 205–350 were expressed as histidine-tagged polypeptides in E.coli using the Qiagen Expressionist Kit (Qiagen GmbH). The expression constructs contained PCR-amplified human cDNA fragments which were directionally cloned into the SstI and PstI sites of the pQE30 vector. Inserts were confirmed by sequence analysis. Expression and affinity-purification on Nickel columns in denaturing 8 M urea buffer yielded 5 mg/l bacterial culture of the N-terminal and 16.5 mg/l culture of the C-terminal protein. These preparations were further purified by 13% SDS–PAGE to immunize rabbits according to standard protocols with 100 µg of protein per immunization/boost (BioGenes GmbH). High titer antisera were affinity-purified first on a protein G-column (Amersham Pharmacia Biotech), subsequently on an antigen-loaded Ni-NTA column (Qiagen GmbH) and used in 1:100 dilution.
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ACKNOWLEDGEMENTS |
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The authors thank the patients and their families who contributed to this study. We are grateful to Susann Schweiger for helpful discussions, and to Silke Feil, Susanne Freier and Hannelore Madle for technical assistance. This work was supported by the Foundation Fighting Blindness, USA (W.B.).
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FOOTNOTES |
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+ To whom correspondence should be addressed. Tel: +49 30 84131253; Fax: +49 30 84131383; Email: berger@molgen.mpg.de
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