Research report Ophthalmic Genetics 1381-6810/04/ US$ 22.00 Ophthalmic Genetics 2004, Vol. 25 No. 2, pp. 101 110 DOI: 10.1080/13816810490514333 2004 Taylor & Francis Ltd. Accepted 23 January 2004 Rab escort protein 1 (REP1) in intracellular traffic: a functional and pathophysiological overview Markus N. Preising 1 Carmen Ayuso 2 1 Department of Pediatric Ophthalmology, Strabismology and Ophthalmogenetics, Klinikum, University of Regensburg, Germany 2 Genetics Department, Fundación Jiménez Díaz, Madrid, Spain Abstract The intracellular distribution of proteins, compartments, substrates, and products is an active process called intracellular traffic. Control of intracellular traffic is established by small GTP-binding proteins (Rab proteins). Rab proteins are modified by geranyl-geranyl moieties necessary for membrane association and target-protein recognition. Geranyl-geranyl groups are transferred to Rab proteins by geranyl-geranyl transferase 2 (GGTase2). GGTase2 requires Rab escort protein 1 (REP1) to bind Rab proteins. REP1 null mutations underlie an X-linked retinal degeneration called choroideremia (CHM). This review summarizes the current biochemical and clinical knowledge on REP1 and CHM. Key words Rab escort protein 1 (REP1); choroideremia (CHM) Correspondence and reprint requests to: Dr. rer. medic. Markus N. Preising, Dipl.Biol. (PhD) Department of Pediatric Ophthalmology, Strabismology and Ophthalmogenetics Klinikum, University of Regensburg 93042 Regensburg, Germany Tel: +49-941-944-9276 Fax: +49-941-944-9258 E-mail: markus.preising@klinik. uni-regensburg.de Biochemical properties of Rab escort protein 1 (REP1) Cells are sectioned into compartments to separate cellular functions like digestion, excretion, protein production, and protein processing. The cellular compartments are bordered by intracellular membranes from which vesicles bud in order to transport products and substrates between different cellular compartments and the plasma membrane. The movement between cellular compartments is called intracellular traffic (for an overview on intracellular traffic, see Olkkonen & Ikonen 1 ). Transport vesicles as well as movable compartments like melanosomes, endosomes, and lysosomes are guided by small GTPbinding proteins (Rab proteins) (Fig. 1(2 6)) to identify their target compartment. 2 Rab proteins are linked to compartment/vesicle membranes by a gerany-geranyl moiety.the geranyl-geranyl moiety is transferred to the Rab protein by geranyl-geranyl transferase 2 (GGTase2). 3 GGTase2 is not able to interact with Rab proteins directly. A mediator of interaction is necessary. Therefore, nascent Rab proteins are taken Rab escort protein 1 (REP 1) in intracellular traffic 101
Fig. 1. Geranylation of Rab proteins (1) using REP1 as co-factor for GGTase2. Geranylated Rab proteins are accompanied by REP1 while being directed to their target membranes. REP1 acts as GTP dissociation inhibitor (GDI) in the first cycle of Rab activity. For further cycles of activity, GDIs are bound to Rabs for activation by controlled GTP hydrolysis. Rabs are used to target ER-microsomes to the Golgi apparatus (2), controlled budding and fusion of Golgi vesicles (3), budding of secretory vesicles (4), fusion of lysosomes and endosomes (5) as well as endosome budding (6). After delivery of the Rab protein to its acceptor on the target membrane, REP1 re-enters another cycle of Rab geranylation. ER, endoplasmatic reticulum; M, microsomes; GA, Golgi apparatus; sv, secretory vesicle; L, lysosome; E, endosome; PP-GG, geranylphosphate; -GG, geranyl moiety; GDI, GTP dissociation inhibitor. from the endoplasmic reticulum (ER) (after translation) and presented to GGTase2 for geranyl-geranyl modification by Rab escort protein 1 (REP1) 4,5 and REP2, 6 a protein that closely resembles REP1 (Fig. 1(1)). Both proteins are expressed throughout the body. Their substrate specificity is overlaps, thus REP1 and REP2 are able to substitute each other in a substrate-specific extent. 6 After geranylation, Rab proteins are less soluble in the cytoplasm. In addition, they become activated by GTP binding. Activated Rab proteins tend to lose the bound GTP which is crucial for their activity.thus, REP1 acts as a GTP dissociation inhibitor (GDI) until the Rab protein is guided to its target compartment and membrane acceptor to start the first cycle of activity. 1 It prevents GTP dissociation and keeps the Rab protein solubilized. After delivering the Rab proteins to their first 102 M.N. Preising and C. Ayuso
target, REP1 dissociates and re-enters another cycle of RAB geranylation and targeting. GDI function is established afterwards by a RABspecific GDI which is not able to fulfill the REP1 escort function on nascent RAB. 4 REP1 is an X-linked gene (Xq21.1) of 15 exons predicting a protein of 653 amino acids. 7 REP2 is assigned to chromosome 1q42-ter and, although it contains three additional amino acids, it is encoded by a single exon. 6,8 Both proteins share several regions of conserved sequence with each other and with a bovine GDI. 8 Although REP1 is an X-linked gene, inactivation by lyonization 9 is incomplete as recently shown. 10,11 On the attempt to create a REP1 knock-out mouse, the loss of REP1 caused embryonic death to hemizygous males and heterozygous females when the gene was transmitted from heterozygous females. This indicates different functional properties of REP1 in mice compared to humans. 12 Clinical features of patients with mutations in REP1 Mutations in REP1 cause a disease called choroideremia (CHM). CHM is an X-linked eye disease, characterized by progressive degeneration of the photoreceptors, retinal pigment epithelium, and choriocapillaris. 13 Typically, affected hemizygous males complain of night blindness in the first or second decade of life, followed by progressive visual field restriction with progression from annular scotomas leading to tunnel vision and often blindness. 14 Several studies have shown that in the majority of the cases analyzed patients with CHM present good central vision until age 40 50 years. Nevertheless, impairment of visual acuity (VA) is eventually noticed in late age, and only a few patients retain any central vision beyond 60 years of age. 15 17 Fundus changes vary along with the progression of the disease. The diffuse, progressive atrophy of the choriocapillaris and retinal pigment epithelium (RPE) begins peripherally and spreads centrally (Fig. 2A). At the onset, there are pigmentary stippling and focal areas of choroidal atrophy in the equatorial fundus that eventually encompass the more peripheral retina and posterior pole. As the disease progresses, degenerative changes of the retinal pigment epithelium (RPE) and loss of choriocapillaris occur with the subsequent formation of scalloped lesions through which large choroidal vessels can be seen (Fig. 2A). In the final stage, the choroidal vessels within the macula may also atrophy, especially after the age of 40 and, in advanced stages, the sclera becomes visible on fundus examination in the areas of total choroidal and RPE atrophy. 16 Angiographically, these areas of choriocapillaris loss are represented by areas of hypofluorescence with linear hyperfluorescent streaks that represent the choroidal vessels running through. In contrast, the areas of preserved choriocapillaris are hyperfluorescent (Fig. 2B). The ERG is markedly reduced from childhood onwards with delays in the b-wave implicit time, increased dark-adaptation thresholds, and complete absence of rod responses. Both rod- and cone-mediated central function decline with age. 18 Expression and progression are variable among the affected men, even within the same family. Rab escort protein 1 (REP 1) in intracellular traffic 103
B A C Fig. 2. (A) Fundus photography of a CHM patient at the age of 17 years. Note the preserved macular region compared to the atrophic peripheral retina. (B) Autofluorescence of the same patient at age 17 years showing areas of loss of RPE and choriocapillaris indicated by visible choroidal vessels. (C) Female carrier of CHM exhibiting spots for atrophic retina with loss of RPE (with kind permission from Prof. Birgit Lorenz, Department of Pediatric Ophthalmology, Strabismology and Ophthalmogenetics, University of Regensburg, Germany). Clinical findings in carriers Heterozygous female carriers are mostly asymptomatic with no serious visual impairment and normal visual fields. They do, however, show characteristic pigment changes in the midperiphery fundus resembling the fine mottling observed in the initial stage of the disease in males and patchy areas of chorioretinal degeneration (Fig. 2C). 19,20 They often show fundus abnormalities alone, in the absence of demonstrable photoreceptor cell dysfunction. 21 Photoreceptor function is measured by Ganzfeld ERG in CHM patients and carriers. Usually, no variations from normal amplitude and implicit time are found in the carriers. In addition, all stages of reduced rod-mediated function report without correlation to age and type of REP1 mutations, with more preserved central and peripheral conemediated function and with abnormalities only in elder patients. 15 In a recent paper, we showed that carriers may exhibit obvious reductions in photopic Ganzfeld ERG and multifocal ERG (mferg) either as teens or as adults. 22 30-Hz flicker stimulation showed significantly reduced amplitudes. Scotopic loss of Ganzfeld ERG was evaluated as subnormal in these carriers. This is in accordance with the clinical find- 104 M.N. Preising and C. Ayuso
ings in elder carriers who develop degeneration of the macula, which develops together with the peripheral mottling. Male patients, in contrast, will keep a functional macula over several years before this part finally degenerates. Histopathology So far, histology has been performed in 18 probands of which most are carriers (14 eyes reviewed from literature in Kärnä 15 and two eyes each in MacDonald et al. 23 and Syed et al. 24 ). Interestingly, the authors unanimously report atrophy of the RPE and photoreceptor layer. Especially in females, the macula was severely affected with complete loss of the photoreceptor layer, RPE, and finally the choroid. The heterozygous carriers revealed normal photoreceptors in the periphery, which contrasts with the findings in male patients. The RPE was abnormal, with irregular thickness and pigmentation associated with variable lipofuscin content. Photoreceptor and RPE loss appeared to be independent. The choriocapillaris was normal, except in retinal areas where it was seriously degenerated. 25 27 Finally, hyalinization and thickening of the walls of the choriodal vessels together with atrophy of the optic nerve could be observed (reviewed in Kärnä 15 ). Differential diagnosis CHM is diagnosed by the combination of ophthalmic examination, visual field testing, and ERG recordings. It is commonly misdiagnosed as X-linked retinitis pigmentosa (XLRP) or gyrate atrophy. Various choroidal degenerations can mimic the appearance of CHM. The clinical appearance also resembles diffuse choriocapillaris atrophy with autosomal dominant or recessive inheritance, or Wagner vitreoretinal degeneration. Since individual recognition of peripheral field loss occurs at advanced stages of the disease, misinterpretation of clinical data often occurs. The disease proceeds to an end-stage and appears similar to many other retinal degenerations. Abnormal plasma ornithine levels are indicative of gyrate atrophy. Additionally, careful genetic workup reveals an X-linked inheritance pattern in CHM in families with several cases, which allows differentiation from autosomal recessive disorders. 28 30 The impaired night vision, constricted peripheral field, diffuse pigmentary abnormalities, and abolished ERG seen in the early stages of CHM are similar to XLRP. The distinguishing feature is the diffuse choroidal atrophy that is uncommon in early XLRP. 31 Finally, careful examination of obligate carriers like mother and grandmother of the patient will reveal differences. CHM carriers more often present with a carrier fundus and the ERG and mferg results will be able to distinguish between the diseases before fundus changes are obvious. Genetics of choroideremia To date, no missense mutations have been reported in REP1 (for an overview on REP1 mutations, see http://www.retina-international.org/sci-news/repmut.htm). The catalogue of mutations includes gross deletions ranging from a few exons to several kilobases. These deletions cover about 50% of all mutations in REP1 (Van den Hurk et al. 32 and authors observations). Further mutations include small deletions of single to several base pairs, dis- Rab escort protein 1 (REP 1) in intracellular traffic 105
rupting the open-reading frame, splice-site mutations, and nonsense mutations. These have to be considered Null mutations. A total of 30 40% of the CHM patients cannot be correlated with a mutation in sporadic patients. In families with several affected individuals, the mutation detection rate reaches 60% and more (Van den Hurk et al. 32 and authors observations). This has been attributed to mutations in regions of REP1 not scanned by the mutation screens.the lack of hypoor hypermorphic mutations in REP1 has been explained by either prenatal lethality or by phenotypes other than CHM caused by REP1 missense mutations. 32 Prenatal lethality is in agreement with the results from the mouse models mentioned before. 12 According to our recent hypothesis, REP1 and REP2 expression gets dysregulated due to the incomplete lyonization of REP1. 22 CHM is a disorder confined to the posterior pole of the eye. This has been explained by tissue-specific REP1 substrates (Rab27a 33 ). In these tissues, complete loss of REP1 activity can be substituted by REP2 to different extents. Minimal expression of wild-type REP1 on a mutant background will reduce REP2 substitution and induce REP1 expression to reach normal activity. Normal activity cannot be established since the mutant allele is mostly expressed in these cells. Thus, a deadly spiral is started. This is in agreement with the retinal degeneration pattern of RPE in males versus carriers. 22 It would also be in agreement with prenatal lethality of pathologic missense mutations. In hemizygous males carrying missense mutations, the presence of an REP1 gene product reduces REP2 expression, thus inducing REP1 expression which does not contribute to the activity of REP1. The necessary activity of REP1 will never be reached and cells using REP1 will degenerate. Since REP1 activity is necessary from very early embryonic development on, this will cause male fetuses to die prenatally. In a recent attempt, an adeno-associated virus (AAV) construct carrying REP1 was used to successfully transform human lymphoblastoid cells. 34 Since lymphoblastoids do not present with phenotypic changes in CHM patients, further research in higher animals needs to be conducted to evaluate the effect of a functional REP1 copy on an REP1 mutation background. Genetic diagnosis of choroideremia In many CHM families, only single affected males are present and no X-linked pattern can be derived from the nuclear family. A rapid way to exclude REP-1 involvement in family cases is with linkage analysis using known intragenic markers. 7,35 As mentioned above, all REP1 mutations to date have been identified as null mutations, abolishing the REP1 gene product. Therefore, the most elegant approach to identify patients with the REP1 mutation would be to perform western blotting against an REP1 antibody. 36 Loss of immunoreactivity will show the presence of a mutation even in parts of the gene not screened by usual exon-screening methods. Those mutations producing a transient but mutant mrna of REP1 can be traced by the protein truncation test (PTT) since all known REP1 mutations predict premature translation stop signals. 37 The PTT identifies mutations by the in-vitro translation of mrna fragments of patient mrna. Unfortunately, neither mrna nor blood 106 M.N. Preising and C. Ayuso
samples are available in most cases sent from external centers to the genotyping units. Therefore, laborious screening of all 15 exons of REP1 with single-strand conformation polymorphism (SSCP) analysis, denaturing gradient high-pressure liquid chromatography (DHPLC), or direct sequencing of PCR amplimers is necessary. In some rare instances when deletions are big enough, high-resolution karyotyping might be helpful, since large genomic rearrangements (large deletions or translocations) are difficult to detect by molecular methods. 7,12,38 41 Diseases related to functional deficits in REP1 substrates REP1 is a crucial component of a protein complex in each cell of the body. This complex and its substrates and products are involved in disorders caused by functional deficits of the intracellular traffic. While mutations in Rab27a result in the Griscelli syndrome (GS), 42 GGtase2 has been found to underlie the mouse model gunmetal. 43 Gunmetal is a mouse disorder of clotting, showing prolonged bleeding times, thrombocytopenia, and a reduced platelet granule content. This syndrome is similar to the features of human Hermansky-Pudlak syndrome (HPS) although GGtase2 is not among the seven currently known genes for HPS. GS is characterized by pigmentary dilution of hair and skin and hemophagocytic syndrome. Neurological deficits occur. A mouse model of Rab27a mutations is called the ashen mouse. 44,45 Rab27a was identified as a critical gene for organelle-specific protein trafficking in melanocytes and platelets. 45 Both disorders are caused by failures in the transport of secretory vesicles, melanosomes, and vesicles in neuronal cells vital to establish neuronal function. Conclusion Choroideremia (CHM) is a disorder of intracellular traffic caused by a ubiquitously expressed gene, REP-1. The local restriction of symptoms to the retina is attributed to retina-specific genes interacting with REP-1. The phenotype of CHM can be confused with other retinal disorders like autosomal recessive gyrate atrophy or X-linked retinitis pigmentosa in older patients. Mutation screening of REP-1 is labor-intensive, but can be relieved when blood samples are provided from single cases or DNA samples from complete families. Since CHM is an X-linked disorder caused by null mutations, it provides an excellent model disease for gene therapy trials based on the substitution of the affected gene. References 1 Olkkonen VM, Ikonen E. Genetic defects of intracellular-membrane transport. N Engl J Med. 2000;343: 1095 1104. 2 Olkkonen VM, Stenmark H. Role of Rab GTPases in membrane traffic. Int Rev Cytol. 1997;176:1 85. 3 Farnsworth CC, Seabra MC, Ericsson LH, Gelb MH, Glomset JA. Rab geranylgeranyl transferase catalyzes the geranylgeranylation of adjacent cysteines in the small GTPases Rab1A, Rab3A, and Rab5A. Proc Natl Acad Sci USA. 1994;91:11963 11967. 4 Alexandrov K, Horiuchi H, Steele Mortimer O, Seabra MC, Zerial M. Rab escort protein-1 is a multifunctional protein that accompanies newly prenylated rab proteins to their target membranes. Eur Mol Biol Organ (EMBO) J. 1994;13:5262 5273. Rab escort protein 1 (REP 1) in intracellular traffic 107
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44 Haddad EK, Wu X, Hammer III JA, Henkart PA. Defective granule exocytosis in Rab27a-deficient lymphocytes from ashen mice. J Cell Biol. 2001;152:835 842. 45 Wilson SM, Yip R, Swing DA, et al. A mutation in Rab27a causes the vesicle transport defects observed in ashen mice. Proc Natl Acad Sci USA. 2000;97:7933 7938. 110 M.N. Preising and C. Ayuso