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(Nyctalopin on Chromosome X), the Gene Mutated in Congenital Stationary Night Blindness, Encodes a Cell Surface Protein

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(Nyctalopin on Chromosome X), the Gene Mutated in Congenital Stationary Night Blindness, Encodes a Cell Surface Protein NYX (Nyctalopin on Chromosome X), the Gene Mutated in Congenital Stationary Night Blindness, Encodes a Cell Surface Protein Christina Zeitz,1,2,3,4 Harry Scherthan,2,4 Susanne Freier,2 Silke Feil,1,2 Vanessa Suckow,2 Susann Schweiger,2 and Wolfgang Berger1,2 PURPOSE. The complete type of X-linked congenital stationary and are functionally conserved. (Invest Ophthalmol Vis Sci. night blindness (CSNB1) in human and mouse is caused by 2003;44:4184–4191) DOI:10.1167/iovs.03-0251 mutations in the NYX gene. The human NYX protein has been predicted to contain an N-terminal endoplasmic reticulum (ER) -linked congenital stationary night blindness (XLCSNB) is a signaling sequence and a C-terminal glycosylphosphatidylinosi- Xnonprogressive retinal disorder characterized by impaired tol (GPI) anchor. In the current study, these computer predic- night vision and other ocular symptoms such as myopia, hy- tions were verified experimentally by expression of domain- peropia, nystagmus, and reduced visual acuity.1 According to specific cDNA constructs in COS-7 and HeLa cells. Moreover, the clinical phenotype, XLCSNB can be divided into two types: computer-based analysis of the orthologue mouse amino acid complete (CSNB1) and incomplete (CSNB2). These two sub- sequence did not reveal a GPI anchor, which may result in a types can be distinguished on the basis of electroretinogram different protein localization compared with human NYX. (ERG) and by genetic means.2,3 CSNB2 is associated with a Therefore, the cellular localization for the mouse Nyx protein reduced rod b-wave and substantially reduced cone response was also examined. and is due to mutations in CACNA1F, an X chromosomal gene METHODS. A new method was established that differentially encoding the ␣1-subunit of an L-type calcium channel.4,5 visualizes both the protein at the surface of the living cell and CSNB1 is characterized by the complete absence of the rod subsequently in intracellular compartments. The localization of b-wave, but largely normal cone amplitudes. By positional the human and mouse V5-tagged wild-type and mutant NYX cloning we and others have isolated a gene in Xp11.4 (NYX, protein were studied. nyctalopin on chromosome X) that is mutated in patients with 6,7 RESULTS. Human and mouse V5-NYX proteins were dispersed in CSNB1. The gene consists of three exons and codes for a the form of speckles over the entire cell surface. Subsequent novel protein belonging to the leucine-rich repeat (LRR) super- staining of the same cells after detergent extraction revealed family. The predicted polypeptide contains an N-terminal ER that V5-NYX located to the ER and Golgi apparatus. Deletion of signal peptide, a C-terminal glycosylphosphatidylinositol (GPI) the GPI anchor domain of NYX resulted in a time-dependent membrane anchor, and 11 leucine-rich repeats (LRR), flanked loss of V5-NYX from the surface of living cells and accumula- by cysteine-rich LRRs. LRRs are short-sequence motifs present tion of this truncated protein in the ER and Golgi apparatus. in a number of proteins with diverse functions and cellular Deletion of the ER signal sequence in Nyx delocalized the localization. Functionally, these repeats are suggested to medi- 8 intracellular V5-Nyx protein and caused its dispersion in the ate protein–protein interactions. Several membrane proteins cytosol. Furthermore, mutations introduced in the leucine-rich have been shown to be linked to the outer leaflet of the lipid 9,10 repeat (LRR)-region, which has been described as a pathogenic bilayer through a GPI anchor. They are synthesized and variant of NYX, had no effect on subcellular localization of the attached at the endoplasmic reticulum (ER) before modifica- protein. tion and are then transported to the cell surface through the secretory pathway.11 The mammalian Golgi apparatus is the CONCLUSIONS. These data provide evidence that human and central organelle within the secretory machinery. It plays an mouse nyctalopin are membrane-bound extracellular proteins important role in processing, maturation, and sorting of newly synthesized secretory and membrane proteins received from the ER.12,13 GPI-anchored proteins may be localized in mi- From the 1Division of Medical Molecular Genetics and Gene Di- crodomains14 and have been implicated in processes such as agnostics, Institute of Medical Genetics, University of Zurich, Schwer- protein sorting.15 T4-cell activation,16 and cell signaling.17,18 zenbach, Switzerland; the 2Max-Planck Institute for Molecular Genet- Most notably, it has been shown that connectin and chaoptin, 3 ics, Berlin, Germany; and the Institute for Chemistry/Biochemistry, two prominent GPI-anchored LRR proteins, have a pivotal Free University, Berlin, Germany. 4 role in photoreceptor cell adhesion and axon guidance pro- Contributed equally to the work and therefore should be consid- 19,20 ered equivalent first authors. cesses. Supported by Grant T-GE-0602-0222 from The Foundation Fight- The amino acid sequence identity between the mouse or- ing Blindness. thologue, previously identified by screening a genomic mouse 21 Submitted for publication March 11, 2003; revised May 7, 2003; PAC library and human NYX is much higher in the LRR core accepted May 29, 2003. (Ͼ90%) than in the N- and C-terminal sequences (62% and 52%, Disclosure: C. Zeitz, None; H. Scherthan, None; S. Freier, None; respectively). Computer-based protein motif prediction re- S. Feil, None; V. Suckow, None; S. Schweiger, None; W. Berger, vealed the ER signaling peptide, the LRRs, and the GPI anchor None in the human protein sequence. However, only the signal The publication costs of this article were defrayed in part by page sequence and the LRRs were predicted in the mouse sequence. charge payment. This article must therefore be marked “advertise- ment” in accordance with 18 U.S.C. §1734 solely to indicate this fact. To understand the function of NYX, we generated V5- Corresponding author: Christina Zeitz, Division of Medical Molec- tagged human and mouse cDNA expression constructs and ular Genetics and Gene Diagnostics, Institute of Medical Genetics, studied their subcellular localization by immunofluorescence University of Zurich, Schorenstrasse 16, CH-8603 Schwerzenbach, of tagged NYX protein on live and in fixed cells after transfec- Switzerland; [email protected]. tion into COS-7 and HeLa cells. Different protocols were estab- Investigative Ophthalmology & Visual Science, October 2003, Vol. 44, No. 10 4184 Copyright © Association for Research in Vision and Ophthalmology Downloaded from iovs.arvojournals.org on 09/24/2021 IOVS, October 2003, Vol. 44, No. 10 NYX, a Cell Surface Protein 4185 lished to identify the localization of V5-NYX. Although V5-NYX GAGCTAGAATTCTCACCGAAAGCTGTCATTCAACG-3Ј (MWG). KpnI/ showed a speckled pattern at the cell surface in living cells, it EcoRI– digested PCR products were isolated from agarose gel clearly colocalized with markers for ER and Golgi in detergent- (Qiaquick; Qiagen) and cloned in KpnI/EcoRI-digested PCR products treated cells. Identical results were obtained with mouse cDNA were isolated from agarose gels (Qiaquick; Qiagen) and cloned in a expression constructs. similarly treated pcDNA4/V5-His expression vector (Invitrogen). In addition, a decisive role of the signal sequence and the GPI anchor for the proper subcellular synthesis and localiza- Cell Culture and Transfection tion of the V5-NYX protein was demonstrated by using dele- tion constructs. COS-7 and HeLa cells were spread at 1 ϫ 105 cells per well of a six-well Our results show that the ER signaling peptide and GPI plate or at 3 ϫ 106 cells per flask in DMEM/F12 (Invitrogen), contain- anchor are of functional relevance, not only in humans but also ing3mML-glutamine, streptomycin (100 U/mL), penicillin (100 in the mouse, and that V5-NYX is a membrane bound, extra- U/mL), and 10% FCS, 1 day before transfection. Transfection was cellular protein. performed according to a commercial protocol (PolyFect; Qiagen), by using 1 or 15 ␮g plasmid DNA, 10 or 50 ␮L transfection reagent (PolyFect; Qiagen), and 100 or 600 ␮L serum-free medium (Opti-MEM; MATERIALS AND METHODS Invitrogen). The transfection efficiency for all experiments was 20%. Expression Constructs Immunofluorescence Expression constructs were generated by using a mammalian expres- sion system (Invitrogen, Karlsruhe, Germany), in which pcDNA4/V5- COS-7 and HeLa cells were transfected as just described. After 24 His is used for gene fusion. Inserts were amplified with DNA Polymer- hours, transfected cells were analyzed using different staining meth- ase in a kit (HotStarTaq with Q-solution; Qiagen, Hilden, Germany) on ods. To observe proteins that localize exclusively on the plasma mem- genomic DNA and with a second DNA polymerase (ProofStart with brane, we performed live cell staining as follows. The anti-V5 antibody Q-solution; Qiagen) on purified amplified PCR products. To avoid (Invitrogen) was diluted 1:300 in an appropriate volume of cell culture overlong primers, wild-type and mutant inserts were amplified by medium containing 2 ␮L of a crude, heat-denatured lysate prepared using two forward primers. They contained the NYX signal sequence, from nontransfected HeLa or COS-7 cells. After this 10-minute absorp- a V5 recognition site (GGTAAGCCTATCCCTAACCCTCTCCTCG- tion step at 37°C, which removes nonspecific binding sites among the GTCTCGATTCTACG), and 20 nucleotides of the corresponding cDNA. anti-V5 antibodies, the antibody solution was added to the living The reverse primer, used in both PCRs, contained the NYX stop codon transfected cells in the culture dish. After 30 minutes of incubation at to avoid the C-terminal V5- and His-tags positioned downstream in the 37°C, cells were washed three times with medium to remove unbound vector. antibody. The live cells were then incubated for a further 10 minutes After the first PCR reaction, amplified products were purified by gel at 37°C with FITC-conjugated rabbit anti-mouse antibody (Dako, extraction (Qiaquick; Qiagen) and used as a template for the second Glostrup, Denmark) serum diluted 1:500 in culture medium.
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