Defective intracellular transport and processing of OA1 is a major cause of ocular albinism type 1

Human Molecular Genetics, Dec 2000

Ocular albinism type 1 (OA1) is an X-linked disorder mainly characterized by a severe reduction of visual acuity, hypopigmentation of the retina and the presence of macromelanosomes in the skin and eyes. Various types of mutation have been identified within the OA1 gene in patients with the disorder, including several missense mutations of unknown functional significance. In order to shed light into the molecular pathogenesis of ocular albinism and possibly define critical functional domains within the OA1 protein, we characterized 19 independent missense mutations with respect to processing and subcellular distribution on expression in COS-7 cells. Our analysis indicates the presence of at least two distinct biochemical defects associated with the different missense mutations. Eleven of the nineteen OA1 mutants (∼60%) were retained in the endoplasmic reticulum, showing defecNStive intracellular transport and glycosylation, consistent with protein misfolding. The remaining eight of the nineteen OA1 mutants (∼40%) displayed sorting and processing behaviours indistinguishable from those of the wild-type protein. Consistent with our recent findings that OA1 represents a novel type of intracellular G protein-coupled receptor (GPCR), we found that most of these latter mutations cluster within the second and third cytosolic loops, two regions that in canonical GPCRs are known to be critical for their downstream signaling, including G protein-coupling and effector activation. The biochemical analysis of OA1 mutations performed in this study provides important insights into the structure–function relationships of the OA1 protein and implies protein misfolding as a major pathogenic mechanism in OA1.

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Defective intracellular transport and processing of OA1 is a major cause of ocular albinism type 1

Marilena d'Addio 2 3 Alessandro Pizzigoni 2 3 Maria Teresa Bassi 2 3 Cinzia Baschirotto 2 3 Caterina Valetti 1 2 Barbara Incerti 2 3 Maurizio Clementi 0 2 Michele De Luca 2 5 Andrea Ballabio 2 3 4 Maria Vittoria Schiaffino 2 3 0 Medical Genetics, Department of Pediatrics, University 1 Department of Experimental Medicine, Anatomy Section, University of Genoa , 16132 Genoa , Italy 2 of Padua , 35128 Padua , Italy 3 Telethon Institute of Genetics and Medicine (TIGEM) , 20132 Milan , Italy 4 Universita Vita-Salute San Raffaele , 20132 Milan , Italy 5 IDI IRCCS, Istituto Dermopatico dell'Immacolata , 00167 Rome , Italy Ocular albinism type 1 (OA1) is an X-linked disorder mainly characterized by a severe reduction of visual acuity, hypopigmentation of the retina and the presence of macromelanosomes in the skin and eyes. Various types of mutation have been identified within the OA1 gene in patients with the disorder, including several missense mutations of unknown functional significance. In order to shed light into the molecular pathogenesis of ocular albinism and possibly define critical functional domains within the OA1 protein, we characterized 19 independent missense mutations with respect to processing and subcellular distribution on expression in COS-7 cells. Our analysis indicates the presence of at least two distinct biochemical defects associated with the different missense mutations. Eleven of the nineteen OA1 mutants (60%) were retained in the endoplasmic reticulum, showing defecNStive intracellular transport and glycosylation, consistent with protein misfolding. The remaining eight of the nineteen OA1 mutants (40%) displayed sorting and processing behaviours indistinguishable from those of the wild-type protein. Consistent with our recent findings that OA1 represents a novel type of intracellular G protein-coupled receptor (GPCR), we found that most of these latter mutations cluster within the second and third cytosolic loops, two regions that in canonical GPCRs are known to be critical for their downstream signaling, including G protein-coupling and effector activation. The biochemical analysis of OA1 mutations performed in this study provides important insights into the structure-function relationships of the OA1 protein and implies protein misfolding as a major pathogenic mechanism in OA1. - Ocular albinism type 1 (OA1; X-linked ocular albinism of the NettleshipFalls type; MIM 300500) is the most common form of ocular albinism, with an estimated prevalence of about 1:50 000 (1). This disorder is transmitted as an X-linked trait with affected males showing the complete phenotype and heterozygous females showing only minor retinal and cutaneous signs of the disease. Affected males with ocular albinism have substantial reduction of visual acuity and manifest horizontal and occasionally rotary nystagmus, strabismus and marked photophobia. Ophthalmologic examination reveals iris translucency, foveal hypoplasia and hypopigmentation of the retina. As with other types of albinism, patients with ocular albinism have misrouting of the optic tracts, resulting in loss of stereoscopic vision (1). Cutaneous changes are mild in ocular albinism: hypopigmentation of the skin may be noted only by comparison with unaffected siblings (1). Ultrastructural examination of skin melanocytes and of the retinal pigment epithelium typically reveals the presence of structural abnormalities of melanosomes, mainly represented by macromelanosomes, suggesting an underlying defect in the biogenesis of these organelles (2). Consistently, the recent data that we obtained with an Oa1 mouse knockout strongly support the idea that the protein product of the OA1 gene is required for proper maturation of melanosomes (3). We previously isolated the human and mouse OA1 genes and showed that they encode predicted proteins of 404 and 405 amino acids, respectively, display several putative transmembrane domains and are exclusively expressed in the pigment cells of the skin and eye (melanocytes and retinal pigment epithelium) (4,5). By using polyclonal antibodies we identified the endogenous OA1 protein in normal human melanocytes as a 60 kDa glycoprotein and a 4845 kDa doublet, corresponding to unglycosylated precursor polypeptides. Immunofluorescence and immunogold analyses in human melanocytes demonstrated that the endogenous OA1 is localized to the melanosomal membrane, consistent with its possible involvement in melanosome biogenesis (6). More recently, by using several complementary approaches, we found that OA1 shares significant structural and functional similarities with the G proteincoupled receptor (GPCR) superfamily (7). However, in contrast to all other GPCRs identified so far, OA1 is not localized to the plasma membrane, but is instead targeted to intracellular organelles, namely the melanosomes in pigment cells and the lysosomes in transfected non-melanocytic cells. These data indicate that the OA1 protein represents the fi (...truncated)


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Marilena d’Addio, Alessandro Pizzigoni, Maria Teresa Bassi, Cinzia Baschirotto, Caterina Valetti, Barbara Incerti, Maurizio Clementi, Michele De Luca, Andrea Ballabio, Maria Vittoria Schiaffino. Defective intracellular transport and processing of OA1 is a major cause of ocular albinism type 1, Human Molecular Genetics, 2000, pp. 3011-3018, 9/20, DOI: 10.1093/hmg/9.20.3011