Robert S. Molday

Immunospecific ferromagnetic iron-dextran reagents for the labeling and magnetic separation of cells

Ferromagnetic iron dextran particles were prepared by reacting a mixture of ferrous chloride and ferric chloride with dextran polymers under alkaline conditions. Particles purified by gel filtration chromatography were in the size range of 30-40 nm, had an electron dense core of about 15 nm, were stable against aggregation in physiological buffer, showed little non-specific binding to cells and had a magnetic moment. Protein A from Staphylococcus aureus was covalently coupled to periodate-oxidized ferromagnetic iron-dextran particles. These conjugates were used to indirectly label antigen sites on human red blood cells and thymocytes for visualization by scanning and transmission electron microscopy. Cells labeled with these immunospecific ferromagnetic particles are were quantitatively retained by a simple permanent magnet and could be separated from unlabeled cells. Applications of these novel reagents in the separation of cells, cell membranes and receptors in drug targeting studies are discussed.

Transgenic mice with a rhodopsin mutation (Pro23His): A mouse model of autosomal dominant retinitis pigmentosa

We inserted into the germline of mice either a mutant or wild-type allele from a patient with retinitis pigmentosa and a missense mutation (P23H) in the rhodopsin gene. All three lines of transgenic mice with the mutant allele developed photoreceptor degeneration; the one with the least severe retinal photoreceptor degeneration had the lowest transgene expression, which was one-sixth the level of endogenous murine rod opsin. Of two lines of mice with the wild-type allele, one expressed approximately equal amounts of transgenic and murine opsin and maintained normal retinal function and structure. The other expressed approximately 5 times more transgenic than murine opsin and developed a retinal degeneration similar to that found in mice carrying a mutant allele, presumably due to the overexpression of this protein. Our findings help to establish the pathogenicity of mutant human P23H rod opsin and suggest that overexpression of wild-type human rod opsin leads to a remarkably similar photoreceptor degeneration.

Cloning of the CDNA for a novel photoreceptor membrane protein (rom-1) identifies a disk rim protein family implicated in human retinopathies

The molecules essential to the continual morphogenesis and shedding of the opsin-containing disks of vertebrate photoreceptors are largely unknown. We describe a 37 kd protein, rom-1, which is 35% identical and structurally similar to peripherin/retinal degeneration slow (rds). Like peripherin, rom-1 is a retina-specific integral membrane protein localized to the photoreceptor disk rim. The two proteins are similarly oriented in the membrane, and each has a highly conserved (15/16 residues) cysteine- and proline-rich domain in the disk lumen. Although both rom-1 and peripherin form disulfide-linked dimers, they do not form heterodimers with each other, but appear to associate noncovalently. These results suggest both that rom-1 and peripherin are functionally related members of a new photoreceptor-specific protein family and that rom-1, like peripherin, is likely to be important to outer segment morphogenesis. The association of mutations in RDS with retinitis pigmentosa indicates that ROM1 is a strong candidate gene for human retinopathies.

Rod and cone photoreceptor cells express distinct genes for cGMP-gated channels

Signal transduction in vertebrate rod and cone photoreceptor cells involves ion channels that are directly gated by the internal messenger cGMP. Rods and each type of cones express genetically related yet different forms of photopigments. Enzymes that control the light-stimulated hydrolysis of cGMP in rods and cones are also the product of distinct genes. Two different cDNA clones encoding cGMP-gated channels have been characterized from the chicken retina. Expression of cDNAs in Xenopus oocytes gives rise to cGMP-stimulated channel activity. Antibodies against a synthetic peptide specific for the C-terminal amino acid sequence derived from one clone stain outer segments of cone but not rod photoreceptors. Therefore chicken rod and cone cells each express different forms of cGMP-gated channels that are genetically related to each other. Expression in COS-1 cells produces the complete form of both channel polypeptides, whereas Western blot analysis indicates that channels in outer segment membranes are present in a processed form that is significantly shorter than the full-length polypeptide.

ABCR expression in foveal cone photoreceptors and its role in stargardt macular dystrophy

Mutations in the gene encoding ABCR are responsible for Stargardt macular dystrophy. Here we show by immunofluorescence microscopy and western-blot analysis that ABCR is present in foveal and peripheral cone, as well as rod, photoreceptors. Our results suggest that the loss in central vision experienced by Stargardt patients arises directly from ABCR-mediated foveal cone degeneration.

The heteromeric cyclic nucleotide-gated channel adopts a 3A:1B stoichiometry

Cyclic nucleotide-gated (CNG) channels are crucial for visual and olfactory transductions. These channels are tetramers and in their native forms are composed of A and B subunits, with a stoichiometry thought to be 2A:2B (refs 6, 7). Here we report the identification of a leucine-zipper-homology domain named CLZ (for carboxy-terminal leucine zipper). This domain is present in the distal C terminus of CNG channel A subunits but is absent from B subunits, and mediates an inter-subunit interaction. With cross-linking, non-denaturing gel electrophoresis and analytical centrifugation, this CLZ domain was found to mediate a trimeric interaction. In addition, a mutant cone CNG channel A subunit with its CLZ domain replaced by a generic trimeric leucine zipper produced channels that behaved much like the wild type, but less so if replaced by a dimeric or tetrameric leucine zipper. This A-subunit-only, trimeric interaction suggests that heteromeric CNG channels actually adopt a 3A:1B stoichiometry. Biochemical analysis of the purified bovine rod CNG channel confirmed this conclusion. This revised stoichiometry provides a new foundation for understanding the structure and function of the CNG channel family.

Inactivation of the murine X-linked juvenile retinoschisis gene, Rs1h, suggests a role of retinoschisin in retinal cell layer organization and synaptic structure

Deleterious mutations in RS1 encoding retinoschisin are associated with X-linked juvenile retinoschisis (RS), a common form of macular degeneration in males. The disorder is characterized by a negative electroretinogram pattern and by a splitting of the inner retina. To gain further insight into the function of the retinoschisin protein and its role in the cellular pathology of RS, we have generated knockout mice deficient in Rs1h, the murine ortholog of the human RS1 gene. We show that pathologic changes in hemizygous Rs1h−/Y male mice are evenly distributed across the retina, apparently contrasting with the macula-dominated features in human. Similar functional anomalies in human and Rs1h−/Y mice, however, suggest that both conditions are a disease of the entire retina affecting the organization of the retinal cell layers as well as structural properties of the retinal synapse.

The cGMP-gated channel and related glutamic acid-rich proteins interact with peripherin-2 at the rim region of rod photoreceptor disc membranes.

The rod cGMP-gated channel is localized in the plasma membrane of rod photoreceptor outer segments, where it plays a central role in phototransduction. It consists of α- and β-subunits that assemble into a heterotetrameric protein. Each subunit contains structural features characteristic of nucleotide-gated channels, including a cGMP-binding domain, multiple membrane-spanning segments, and a pore region. In addition, the β-subunit has a large glutamic acid- and proline-rich region called GARP that is also expressed as two soluble protein variants. Using monoclonal antibodies in conjunction with immunoprecipitation, cross-linking, and electrophoretic techniques, we show that the cGMP-gated channel associates with the Na/Ca-K exchanger in the rod outer segment plasma membrane. This complex and soluble GARP proteins also interact with peripherin-2 oligomers in the rim region of outer segment disc membranes. These results suggest that channel/peripherin protein interactions mediated by the GARP part of the channel β-subunit play a role in connecting the rim region of discs to the plasma membrane and in anchoring the channel·exchanger complex in the rod outer segment plasma membrane.

Rom-1 is required for rod photoreceptor viability and the regulation of disk morphogenesis.

The homologous membrane proteins Rom-1 and peripherin-2 are localized to the disk rims of photoreceptor outer segments (OSs), where they associate as tetramers and larger oligomers. Disk rims are thought to be critical for disk morphogenesis, OS renewal and the maintenance of OS structure, but the molecules which regulate these processes are unknown. Although peripherin-2 is known to be required for OS formation (because Prph2−/− mice do not form OSs; ref. 6), and mutations in RDS (the human homologue of Prph2) cause retinal degeneration, the relationship of Rom-1 to these processes is uncertain. Here we show that Rom1−/− mice form OSs in which peripherin-2 homotetramers are localized to the disk rims, indicating that peripherin-2 alone is sufficient for both disk and OS morphogenesis. The disks produced in Rom1−/− mice were large, rod OSs were highly disorganized (a phenotype which largely normalized with age) and rod photoreceptors died slowly by apoptosis. Furthermore, the maximal photoresponse of Rom1−/− rod photoreceptors was lower than that of controls. We conclude that Rom-1 is required for the regulation of disk morphogenesis and the viability of mammalian rod photoreceptors, and that mutations in human ROM1 may cause recessive photoreceptor degeneration.

Membrane Topology of the ATP Binding Cassette Transporter ABCR and Its Relationship to ABC1 and Related ABCA Transporters IDENTIFICATION OF N-LINKED GLYCOSYLATION SITES

ABCR is a member of the ABCA subclass of ATP binding cassette transporters that is responsible for Stargardt macular disease and implicated in retinal transport across photoreceptor disc membranes. It consists of a single polypeptide chain arranged in two tandem halves, each having a multi-spanning membrane domain followed by a nucleotide binding domain. To delineate between several proposed membrane topological models, we have identified the exocytoplasmic (extracellular/lumen) N-linked glycosylation sites on ABCR. Using trypsin digestion, site-directed mutagenesis, concanavalin A binding, and endoglycosidase digestion, we show that ABCR contains eight glycosylation sites. Four sites reside in a 600-amino acid exocytoplasmic domain of the N-terminal half between the first transmembrane segment H1 and the first multi-spanning membrane domain, and four sites are in a 275-amino acid domain of the C half between transmembrane segment H7 and the second multi-spanning membrane domain. This leads to a model in which each half has a transmembrane segment followed by a large exocytoplasmic domain, a multi-spanning membrane domain, and a nucleotide binding domain. Other ABCA transporters, including ABC1 linked to Tangier disease, are proposed to have a similar membrane topology based on sequence similarity to ABCR. Studies also suggest that the N and C halves of ABCR are linked through disulfide bonds.