Peripherin/rds is an integral membrane glycoprotein found in the rim regions of vertebrate photoreceptor cell discs. Natural mutations of the encoding gene result in degenerative retinal disorders, such as retinitis pigmentosa. The retinal degeneration slow (rds) phenotype, observed in mice, is considered to be an appropriate model for peripherin/rds-mediated retinitis pigmentosa. Associated abnormalities in the outer segment of photoreceptor cells have implicated peripherin/rds in some aspect of disc morphology, yet it remains unclear whether such morphological effects are the cause or the result of this condition. Here we present the first direct evidence to support a role for peripherin/rds in maintaining the flattened vesicle morphology characteristic of photoreceptor outer segments. In vitro expression yields a 36-kDa immunoreactive species, which is inserted into membranes and undergoes N-glycosylation, inter-and intramolecular disulfide bonding, and dimerization. Electron microscopy reveals that peripherin/rds flattens microsomal vesicles. This effect appears to be dependent on disulfide bond formation but not N-glycosylation. The inability of two pathogenic peripherin/rds mutants (P216L and C165Y) to flatten membrane vesicles implicates such mutations as the primary cause of the retinal degeneration observed in retinitis pigmentosa.The outer segment of the vertebrate rod photoreceptor cell exhibits a highly specialized structure, comprised of a stack of 1000 or more flattened vesicles or discs (1). This organization is vital to the visual process, as it maximizes the area available for photoreception and allows efficient renewal of the photoreceptor outer segments. Any elements involved in the formation and maintenance of this structure are, therefore, of vital importance to the visual process. One protein that has been implicated to have such an involvement is peripherin/rds. Peripherin/rds is a 39-kDa integral membrane glycoprotein localized exclusively to the rim regions of photoreceptor cell discs (2, 3). A topological model has been proposed (4) in which the protein possesses four transmembrane helices and cytosolically oriented N and C termini. The protein has been shown to form disulfide-linked homodimers (2). These peripherin/rds dimers and homodimers of the related disc rim protein ROM-1 (5) are believed to non-covalently associate to form a functional heterotetrameric complex (6, 7).The clinical significance of the human peripherin/rds gene is illustrated by its involvement in a wide range of degenerative retinal disorders, including retinitis pigmentosa (8). To date, over 50 pathogenic mutations within the human gene have been identified. In addition there is a mutation in the murine gene that causes the related disorder, retinal degeneration slow (rds) (9). Phenotypically rds mice exhibit distinctive photoreceptor degeneration (10, 11), and hence, murine rds is considered an important animal model for the inherited human retinal degenerative condition. Mice homozygous for the rds condition ha...
We introduced a targeted single base deletion at codon 307 of the rds-peripherin gene in mice, similar mutations being known to cause autosomal dominant retinitis pigmentosa (RP) in man. Histopathological and electroretinographic analysis indicate that the retinopathy in mice homozygous for the codon 307 mutation appears more rapid than that in the naturally occurring null mutant, the rds(-/-) mouse, suggesting that the rds-307 mutation displays a dominant negative phenotype in combination with that due to haplosufficiency. RP is the most prevalent cause of registered visual handicap in those of working age in developed countries, the 50 or so mutations so far identified within the RDS-peripherin gene accounting for up to 10% of dominant cases of the disease. Given the sequence homologies that exist between the murine rds-peripherin and the human RDS-peripherin gene, this disease model, the first to be generated for peripherin-based RP using gene targeting techniques, should in principle be of value in the work-up in mice of therapeutics capable of targeting transcripts derived from the human gene.
We report the protein isolation, cloning and characterization of members of an unusual protein family, which comprise the most abundant proteins present in the squid eye. The proteins in this family have a range of molecular weights from 32 to 36 kDa. Electron microscopy and detergent solubilization demonstrate that these proteins are tightly associated with membrane structures where they may form tetramers. Despite this, these proteins have no stretches of hydrophobic residues that could form typical transmembrane domains. They share an unusual protein sequence rich in methionine, and contain multiple repeating motifs. We have therefore named these proteins Methionine-Rich Repeat Proteins (MRRPs). The use of structure prediction algorithms suggest very little recognized secondary structure elements. At the time of cloning no sequence or structural homologues have been found in any database. We have isolated three closely related cDNA clones from the MRRP family. Coupled in vitro transcription/translation of the MRRP clones shows that they encode proteins with molecular masses similar to components of native MRRPs. Immunoblot analysis of these proteins reveals that they are also present in squid brain, optic lobe, and heart, and also indicate that MRRP-like protein motifs may also exist in mammalian tissues. We propose that MRRPs define a family of important proteins that have an unusual mode of attachment or insertion into cell membranes and are found in evolutionarily diverse organisms.
Bee venom PLA2 possesses a binding site for long-chain fatty acids that can be acylated by long-chain fatty acid imidazolides [Drainas, D. and Lawrence, A.J. (1978) Eur. J. Biochem. 91, 131-138]. Occupation of the site either by oleic acid or the oleoyl residue enhances the catalytic activity by 45.7-fold in the presence of 20% 1-propanol and occupation of the site by the oleoyl residue increases the lytic activity against rabbit erythrocytes by 60-fold. Treatment of the enzyme with oleic acid and glutaraldehyde is known to produce irreversible activation [Lawrence, A.J. and Moores, G.R. (1975) FEBS Lett. 49, 287-291]. Here we show that reduction of the glutaraldehyde-treated enzyme with borohydride stabilizes the activated state and enables the fatty acid to be removed, revealing that a large proportion of the induced activation does not require the presence of oleic acid and indicating that activation is due to a change in the conformation rather than the hydrophobicity of the protein. A kinetic study of enzyme activated by oleoyl imidazolide showed that this modification stabilizes the protein against reversible inactivation by 1-propanol. Comparison of the CD spectra of native and oleoyl imidazolide-activated enzyme shows a change in secondary structure with apparent increase in both alpha-helix and beta-sheet content. During reaction of the enzyme with oleoyl imidazolide, the protein fluorescence shows a biphasic response with an initial fall attributed to occupation of the binding site followed by a progressive decrease with a shift of the emission maximum from 341 to 348 nm. The rate of the second phase closely matched the rate of increase in catalytic activity of the enzyme. Free oleic acid caused a rapid fall in fluorescence emission without the subsequent slow change. These results support the proposal that oleic acid or the oleoyl residue occupy a very similar site on the protein and that occupation of this site increases the exposure of one or both of the Trp residues to the aqueous environment. Binding studies show that activation by oleoyl imidazolide does not increase the affinity of the enzyme for the erythrocyte membrane. It is proposed that occupation of a long-chain fatty acid binding site on the enzyme enhances catalytic activity by changing the conformation of the protein rather than acting as a hydrophobic anchor to the substrate surface.
The acidic isoform of phospholipase A(2) from Naja mossambica mossambica was activated by treatment with a molar equivalent of oleoyl imidazolide. Modification of the protein was accompanied by 50% quenching of tryptophan fluorescence and a significant red shift. The (3)H(9,10) labeled oleoyl residue was co-eluted with the enzyme during gel filtration in the presence of 20% 1-propanol or excess albumin, both of which remove free oleic acid from the enzyme. In contrast, the adduct was labile as to electrophoresis on SDS-PAGE and acid or alkali urea PAGE. The formation of a covalently linked adduct was demonstrated by electrospray mass spectrometry in the presence of 2% formic acid. No such adduct was formed by the phospholipase A(2) isoform from Naja naja atra, which differs in sequence from the N. mossambica mossambica isoform by seven residues including 2 histidine residues and 1 lysine residue. We conclude that oleoyl imidazolide activates the N. mossambica mossambica enzyme by forming an acyl adduct which is unstable as to protein denaturation. The magnitude of tryptophan fluorescence quenching indicates that the site of acylation lies in the sequence WWHF.
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