Peropsin, a novel visual pigment-like protein located in the apical microvilli of the retinal pigment epithelium

Abstract

A visual pigment-like protein, referred to as peropsin, has been identified by large-scale sequencing of cDNAs derived from human ocular tissues. The corresponding mRNA was found only in the eye, where it is localized to the retinal pigment epithelium (RPE). Peropsin immunoreactivity, visualized by light and electron microscopy, localizes the protein to the apical face of the RPE, and most prominently to the microvilli that surround the photoreceptor outer segments. These observations suggest that peropsin may play a role in RPE physiology either by detecting light directly or by monitoring the concentration of retinoids or other photoreceptor-derived compounds.

Figure 1

Amino acid sequences of human and mouse peropsin aligned with human rhodopsin, octopus rhodopsin, bovine RGR opsin, and squid retinochrome. The positions of the retinylidene Schiff base (Lys-296 in rhodopsin) and its counterion (Glu-113 in rhodopsin) are indicated by arrowheads. The region encompassing the seventh putative transmembrane segment of squid retinochrome has been manually aligned to bring Lys-275, the site of the retinylidene Schiff base (26), into register with Lys-296 in human rhodopsin. h, human; m, mouse; o, octopus; b, bovine; s, squid.

Figure 2

(Left) Quantitative analysis of homologies between protein sequences in GenBank (May 1997 release) and human peropsin, bovine RGR, and squid retinochrome as determined by the blast algorithm (27). The P value is the probability of finding a comparable match by chance in a random sequence database of equivalent size. Horizontal bars indicate the range of P values for the indicated classes of sequences. Redundant database entries have not been eliminated for this analysis. (Right) Dendrogram of visual pigments and visual pigment-related proteins calculated using geneworks software with gap penalties set at 5 for each gap and 25 for lengthening a gap. In the dendrogram, the length of each line is proportional to the degree of amino acid sequence divergence. The rat 5HT-7 and human alpha 1B adrenergic receptors are two of the five nonvisual pigment G protein-coupled receptors in the GenBank database that are most homologous to peropsin.

Figure 3

RNase protection, Northern blot analysis, and in situ hybridization localize peropsin transcripts to the RPE. (A) Total RNA from the indicated adult mouse tissues was used for RNase protection with a mouse peropsin probe. Peropsin transcripts are detected only in the eye. A control reaction with an RNA polymerase II probe is shown at bottom. (B) Northern blot of total RNA from the indicated bovine tissues was hybridized to a probe encompassing the human peropsin coding region. Peropsin transcripts are detected only in the RPE. Arrowheads indicate the mobilities of the ribosomal RNAs. (C) In situ hybridization using a digoxigenin-labeled mouse peropsin probe localizes peropsin transcripts in the adult rat to the RPE (arrowhead). (Left) Antisense probe. (Right) Sense probe. Albino rats were used for this experiment so that RPE melanin would not obscure the signal. At long substrate incubation times, a faint hybridization signal is seen in the inner nuclear layer; the origin of this signal is not known. Arrowhead, nucleus of RPE cell. OS, outer segments; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer.

Figure 4

Affinity-purified anti-peropsin antibodies recognize a single protein with an apparent molecular mass of approximately 38 kDa. Fifteen micrograms of total protein from mouse brain or mouse eye from which the lens was removed were loaded on each lane of a 10% polyacrylamide/SDS gel and either stained with Coomassie blue (Left) or immunoblotted using affinity-purified rabbit antibody prepared against a synthetic 20 amino acid peptide corresponding to the carboxyl-terminus of mouse peropsin (Right). The molecular masses of protein size standards, indicated at the left, are, from top to bottom, 192, 127, 73, 43, 32, and 17 kDa.

Figure 5

Histochemical localization of peropsin in the adult mouse eye. Albino (CD1) mice were used in all experiments so that RPE melanin would not obscure the signal. (A) Immunoperoxidase and (B) immunofluorescent staining with affinity-purified anti-peropsin antibodies localizes peropsin to the apical face of the RPE. (B) Nuclei are visualized with 4′,6-diamidino-2-phenylindole. (C) Immunogold staining with anti-peropsin antibodies of a 1 μm section of unicryl-embedded tissue localizes peropsin to the apical microvilli of the RPE. Gold particles were silver-enhanced and the section was counterstained with methylene blue. Photoreceptor outer segments are visible in the lower half of the micrograph. (D) Confocal image of anti-peropsin immunostaining of the RPE. Peropsin is localized to the dense network of microvilli that protrude from the apical face of the RPE; occasional larger structures (double arrowhead) protrude from the RPE and contain peropsin immunoreactivity. Single arrowhead, nucleus of RPE cell. CH, choroid; OS, outer segments; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer; AV, apical microvilli.

Figure 6

Immunoelectron microscopic localization of peropsin in the mouse eye. Immunogold labeling with anti-peropsin antibodies is localized to the apical face of the RPE and to the microvilli that surround the rod outer segments (ROS). (A) At low magnification, the full thickness of the RPE is seen. Numerous large phagosomes are present within the RPE, and the infoldings at the basal face of the RPE are seen immediately adjacent to Bruch’s membrane at the top of the photomicrograph. (B) At higher magnification, individual immunolabeled microvilli (arrows) are seen between unlabeled rod outer segments. (Bars = 0.5 μm.)

Figure 7

Rrh maps in the distal region of mouse chromosome 3. Rrh was placed on mouse chromosome 3 by interspecific backcross analysis. Segregation patterns of Rrh and flanking genes in 180 backcross animals that were typed for all loci are shown at the top. For individual pairs of loci, more than 180 animals were typed (see text). Each column represents the chromosome identified in the backcross progeny that was inherited from the (C57BL/6J × M. spretus) F1 parent. Solid boxes represent the presence of a C57BL/6J allele and open boxes represent the presence of a M. spretus allele. The number of offspring inheriting each type of chromosome is listed at the bottom of each column. A partial chromosome 3 linkage map showing the location of Rrh in relation to linked genes is shown at right. Recombination distances between loci, in centimorgans, are shown to the left of the chromosome and the positions of loci in human chromosomes, where known, are shown to the right. References for the human map positions of loci cited in this study can be obtained from the Genome Data Base, a computerized database of human linkage information maintained by The William H. Welch Medical Library of The Johns Hopkins University (Baltimore).