Ординатура / Офтальмология / Английские материалы / Recent Advances in Retinal Degeneration_LaVail, Hollyfield, Anderson _2008

Vasireddy V, Vijayasarathy C, Huang J, Wang XF, Jablonski MM, Petty HR, Sieving PA, Ayyagari R (2005) Stargardt-like macular dystrophy protein ELOVL4 exerts a dominant negative effect by recruiting wild-type protein into aggresomes. Mol Vis 11:665–676

Westerberg R, Tvrdik P, Unden AB, Mansson JE, Norlen L, Jakobsson A, Holleran WH, Elias PM, Asadi A, Flodby P, Toftgard R, Capecchi MR, Jacobsson A (2004) Role for ELOVL3 and fatty acid chain length in development of hair and skin function. J Biol Chem 279:5621–5629

Zhang K, Kniazeva M, Han M, Li W, Yu Z, Yang Z, Li Y, Metzker ML, Allikmets R, Zack DJ, Kakuk LE, Lagali PS, Wong PW, MacDonald IM, Sieving PA, Figueroa DJ, Austin CP, Gould RJ, Ayyagari R, Petrukhin K (2001) A 5-bp deletion in ELOVL4 is associated with two related forms of autosomal dominant macular dystrophy. Nat Genet 27:89–93

Organization and Molecular Interactions

of Retinoschisin in Photoreceptors

Camasamudram Vijayasarathy, Yuichiro Takada, Yong Zeng, Ronald A. Bush, and Paul A. Sieving

1 Introduction

Retinoschisin (RS), the product of the RS1 gene on the X-chromosome (Xp22), is a 24 kDa secreted protein which is expressed exclusively in the retina (Reid et al., 1999; Sauer et al., 1997) and pineal gland (Takada et al., 2006). In the retina, photoreceptors, bipolar cells, and ganglion cells synthesize RS (Takada et al., 2004). The structural features and functional implications of the 224 amino acid RS sequence include a N-terminus signal peptide (amino acids 1–23) and a 157 amino acid (amino acids 68–217) discoidin domain (Reid et al., 1999; Sauer et al., 1997). The signal peptide guides the secretion of mature RS across the plasma membrane and is cleaved off during the translocation process (Molday, 2007). Discoidin domains, which are highly conserved across species and found on the extracellular cell surface, are known to be involved in cell adhesion and signaling (Reid et al., 1999). Based on this structural feature, RS is believed to function as an adhesive protein in preserving the structural and functional integrity of the retina (Weber et al., 2002). A spectrum of missense mutations occurring throughout the RS1gene was found to interfere with one of the several steps in RS biosynthesis and secretion pathway (Molday, 2007; Wang et al., 2006). The loss of function mutations lead to a form of macular disruption and degeneration in males known as X-linked retinoschisis (XLRS) (Sikkink et al., 2006). XLRS patients exhibit a split or schisis and cystic-like spaces within the retinal layers that lead to early and progressive loss of vision. While there is no established treatment for XLRS, a recent study reported a reduction in foveal cysts and improvement in vision in XLRS patients who were treated with a topical form of dorzolamide, a carbonic anhydrase inhibitor (Apushkin and Fishman, 2006). Further, the feasibility of gene therapy as an approach to treating retinoschisis was also demonstrated in mouse model of XLRS (Molday, 2007; Zeng et al., 2004). Although these studies have advanced our

Paul A. Sieving

National Eye Institute, National Institutes of Health, Bethesda, MD 20892, USA, Tel: 301-496-2234, Fax: 301-496-9972

e-mail: pas@nei.nih.gov

present in the retina displayed no evidence of interaction with RS (Vijayasarathy et al., 2007). Furthermore, the Col2a1 mutant mouse in which retinoschisis was reported (Donahue et al., 2003) revealed normal retinal localization and distribution of RS (Fig. 2C). In summary these results do not suggest an interaction between RS and retinal cell collagens that have been tested in this study.

Blue native gels, resolve multi-protein complexes in accordance to their molecular masses (Schagger and von Jagow, 1991). On a blue native gel, RS from retinal cell lysates migrated with electrophoretic mobilties that corresponded to 200 kDa octamer and a >700 kDa diffuse complex (Fig. 3). Subfractionation of the gel slice corresponding to 700 kDa region, followed by in gel tryptic digestion and MS/MS peptide sequence analysis, identified RS in two of the three fractions. In both cases, the coverage was 15% by amino acid count in the discoidin domain region of RS. The RS sequence information did not indicate post-translational modification of amino acid residues. None of the extracellular matrix (ECM) protein sequences were found in this diffuse RS band to indicate RS interaction with ECM. The proteins identified in this diffuse band included NCAM (neural cell adhesion molecule), Na+ K+ ATPase, crystallins and other cytoskeletal and mitochondrial membrane proteins. Although a recent study (Steiner-Champliaud et al., 2006) suggested RScrystallin interaction, further investigation is needed to determine the significance of intracellular protein–RS interactions as well as the authenticity of the multi-protein complex in terms of RS interacting proteins.

RS is a peripheral membrane protein bound by ionic forces to the outer leaflet of the photoreceptor inner segment plasma membrane. Most importantly, the high affinity of RS for membrane surfaces would restrict its diffusion into the extracellular matrix. The results suggest that RS may act locally to maintain photoreceptor inner segment stability and architecture, and it is not secreted only for export to the inner retina as previously proposed (Reid and Farber, 2005). We propose that

Fig. 3 RS-multiprotein complex. Whole cell lysates (40 μg protein) from mouse retina solubilized in Triton X-100 were adjusted to 0.5 M Bis-Tris pH 7.0. The smples were electrophoressed on 4–13% acrylamide gels under native conditions at 4◦ C as described by Schagger and von Jagow (1991). RS was localized by immunoblotting. The Serva-Blue G dye stained molecular weight markers: thyroglobulin (669 kDa) and amylase(200 kDa)

disruption of the inner segment architecture due to the loss of RS on inner segments might be the basic mechanism that underlies the displacement and disorganization of photoreceptors that we have seen in RS deficient mice retinas. RS bound by ionic forces or phospholipids on the membrane surface can participate in cell-matrix and cell-cell interactions which will influence cytoskeletal organization. Future studies will be needed to address the molecular mechanisms and the role of RS in such events.

Acknowledgments This study was supported by the NIH Intramural Research Program through National Institute on Deafness and Other Communication Disorders and National Eye Institute, Bethesda, MD 20892, USA.

References

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Donahue, L. R., Chang, B., Mohan, S., Miyakoshi, N., Wergedal, J. E., Baylink, D. J., Hawes, N. L., Rosen, C. J., Ward-Bailey, P., Zheng, Q. Y., et al. (2003). A missense mutation in the mouse Col2a1 gene causes spondyloepiphyseal dysplasia congenita, hearing loss, and retinoschisis. J Bone Miner Res18, 1612–1621.

Fraternali, F., Cavallo, L., and Musco, G. (2003). Effects of pathological mutations on the stability of a conserved amino acid triad in retinoschisin. FEBS Lett544, 21–26.

Gilbert, G. E., and Drinkwater, D. (1993). Specific membrane binding of factor VIII is mediated by O-phospho-L-serine, a moiety of phosphatidylserine. Biochemistry32, 9577–9585.

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Takada, Y., Fariss, R. N., Møller, M., Bush, R. A., Rushing, E. J., Sieving, P. A., and Bush, R. A. (2006). Retinoschisin expression and localization in rodent and human pineal and consequences of mouse RS1 gene knockout. Mol Vis12, 1108–1116.

Takada, Y., Fariss, R. N., Tanikawa, A., Zeng, Y., Carper, D., Bush, R., and Sieving, P. A. (2004). A retinal neuronal developmental wave of retinoschisin expression begins in ganglion cells during layer formation. Invest Ophthalmol Vis Sci45, 3302–3312.

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Vijayasarathy, C., Gawinowicz, M. A., Zeng, Y., Takada, Y., Bush, R. A., and Sieving, P. A. (2006). Identification and characterization of two mature isoforms of retinoschisin in murine retina. Biochem Biophys Res Commun349, 99–105.

Vijayasarathy, C., Takada, Y., Zeng, Y., Bush, R. A., and Sieving, P. A. (2007). Retinoschisin is a peripheral membrane protein with affinity for anionic phospholipids and affected by divalent cations. Invest Ophthalmol Vis Sci48, 991–1000.

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Part VII

Basic Science Underlying Retinal

Degeneration

Proteomics Profiling of the Cone Photoreceptor

Cell Line, 661W

Muayyad R. Al-Ubaidi, Hiroyuki Matsumoto, Sadamu Kurono,

and Anil Singh

1 Introduction

Cultured retinal cells are convenient experimental systems offering great advantages in the assessment of numerous retinal processes. The two most important advantages are the ease of evaluation of an isolated cellular function without the effects of other retinal cell types and an avoidance of use of the more costly animal research. The Two obvious disadvantages are the loss of the architecture of the native tissue, and lack of functional influence of other retinal cell types. However, for most of the research applications, the advantages of use of in vitro systems offset the potential limitations.

Retinal cell culture can be used to determine effectiveness of promoter fragments from retina-specific genes, the functional role of domains on a retinal protein and how mutations would effect that function and to express retinal proteins for biochemical analyses. Last but not least, retinal cell culture can be used to determine the toxicity of pharmacologic agents, and for studies of cell death and differentiation.

Except for Müller cells, all other retinal cells are terminally differentiated, specialized neuronal cells with a limited, if any, capacity for cell division. However, immortalized cell lines currently exist for several retinal cell types including Müller cells (Roque et al., 1997). A cell line expressing retina-specific genes, including the photoreceptor proteins IRBP and cone transducin, has also been isolated from a mouse ocular tumor (Bernstin, Kutty, Wiggert, Albert, & Nickerson, 1994). Furthermore, Y-79 (Reid et al., 1974) and WERI-Rb (McFall, Sery, & Makadon, 1977) are immortalized cell lines derived from human retinoblastoma tumors. It was initially believed that the Y-79 cells had originated from a cone cell lineage (Bogenmann, Lochrie, & Simon, 1988), but later these cells were shown to express rod opsin, rod transducin, rod phosphodiesterase, and recoverin (Di Polo & Farber, 1995;

M.R. Al-Ubaidi

Department of Cell Biology, University of Oklahoma Health Sciences Center, 940 Stanton L. Young Blvd. (BMSB781), Oklahoma City, OK 73104, USA; IBERICA Holdings Co., Ltd, Kurume University Translational Research Center, Kurume, Fukuoka 830-0011, Japan, Tel: 405-271-2382, Fax: 405-271-3548

e-mail: muayyad-Al-ubaidi@ouhsc.edu