Toshimichi Shinohara

LEDGF, a survival factor, activates stress-related genes

LEDGF is a survival factor and it enhances survival of various cell types against stress. LEDGF is also a transcriptional activator and it binds to promoter elements of heat shock and stress-related genes to activate expression of these genes. The elevated levels of the stress-related family of proteins, such as heat shock proteins, antioxidant proteins, and detoxication enzymes might suppress apoptosis induced by stress. The protective mechanisms against stress in mammalian cells and in yeast are surprisingly similar.

Both PCE-1/RX and OTX/CRX Interactions Are Necessary for Photoreceptor-specific Gene Expression

RX, a homeodomain-containing protein essential for proper eye development (Mathers, P. H. Grinberg, A., Mahon, K. A., and Jamrich, M. (1997) Nature 387, 603–607), binds to the photoreceptor conserved element-1 (PCE-1/Ret 1) in the photoreceptor cell-specific arrestin promoter and stimulates gene expression. RX is found in many retinal cell types including photoreceptor cells. Another homeodomain-containing protein, CRX, which binds to the OTX element to stimulate promoter activity, is found exclusively in photoreceptor cells (Chen, S., Wang, Q. L., Nie, Z., Sun, H., Lennon, G., Copeland, N. G., Gillbert, D. J. Jenkins, N. A., and Zack, D. J. (1997) Neuron 19, 1017–1030; Furukawa, T., Morrow, E. M., and Cepko, C. L. (1997) Cell 91, 531–541). Binding assay and cell culture studies indicate that both PCE-1 and OTX elements and at least two different regulatory factors RX and CRX are necessary for high level, photoreceptor cell-restricted gene expression. Thus, photoreceptor specificity can be achieved by multiple promoter elements interacting with a combination of both photoreceptor-specific regulatory factors and factors present in closely related cell lineages.

Aldose reductase and ϱ-crystallin belong to the same protein superfamily as aldehyde reductase

Aldose reductase (EC 1.1.1.21) has been implicated in a variety of diabetic complications. Here we present the first primary sequence data for the rat lens enzyme, obtained by amino acid and cDNA analysis. We have found structural similarities with another NADPH‐dependent oxidoreductase: human liver aldehyde reductase (EC 1.1.1.2). The identity between these two enzymes is 50%. Both enzymes share approx. 40–50% homology with ϱ‐crystallin, a major lens protein present only in the frog, Rana pipiens. We propose that aldose reductase, aldehyde reductase and ϱ‐crystallin are members of a superfamily of related proteins.

The proximal promoter of the mouse arrestin gene directs gene expression in photoreceptor cells and contains an evolutionarily conserved retinal factor-binding site.

Regulatory sequences and nuclear factors governing tissue-restricted expression of the mouse arrestin gene were investigated. The results showed that while proximal promoter sequence positions -38 to +304 are sufficient to direct low levels of retina-specific gene expression, sequences extending upstream to position -209 support higher levels of expression in the retina, as well as detectable expression in the lens, pineal gland, and brain. Within the interval between positions -209 and -38, a broadly expressed nuclear factor, Bd, binds to sequences centered between positions -205 and -185, a region which contains two direct repeats of the hexamer, TGACCT. The proximal promoter binds three apparently retina-specific nuclear factors, Bp1, Bp2, and Bp3, through overlapping sequences centered between positions -25 and -15. Bp1 and Bp3 also recognize a closely related sequence found in the promoter regions of several other vertebrate photoreceptor-specific genes. Moreover, the consensus binding site for Bp1, designated PCE I, is identical to RCS I, an element known to play a critical role eliciting photoreceptor-specific gene expression in Drosophila melanogaster. The results suggest that PCE I and RCS I are functionally as well as structurally similar and that, despite marked differences in the fly and vertebrate visual systems, the transcriptional machinery involved in photoreceptor-specific gene expression has been strongly evolutionarily conserved.

Retinal photoreceptor neurons and pinealocytes accumulate mRNA for interphotoreceptor retinoid-binding protein (IRBP)

We have utilized cDNA probes and in situ hybridization techniques to define the subcellular localization of interphotoreceptor retinoid‐binding protein (IRBP) mRNA in bovine and monkey retinas. Results suggest that the mRNA is mainly localized in rod photoreceptor neurons within the outer nuclear layer of the retina. IRBP mRNA is also abundant in cells of the pineal gland, strengthening the analogy between rod photoreceptor cells and pinealocytes.

Lens epithelium-derived growth factor (LEDGF/p75) and p52 are derived from a single gene by alternative splicing.

A human gene that encodes lens epithelium-derived growth factor (LEDGF) was isolated, and the DNA sequence and the exon/intron organization was determined. The gene contains at least 15 exons and 14 introns and encodes LEDGF mRNA and p52 mRNA. Exons 1-15 encode LEDGF mRNA, and exons 1-9, and a part of the ninth intron encode a splice variant (p52). Sequences of the exon/intron junctions of the gene have the highly conserved GT/AG rule. Most intron/exon junctions correspond to junctions of individual protein motifs. Almost equal amounts of LEDGF and p52 are expressed in lens epithelial cells in culture. The LEDGF gene is assigned to chromosome 9p22.2, which is adjacent to the major cell malignancy locus.

The sequence of human retinal S-antigen reveals similarities with α-transducin

The complete amino acid sequence of human retinal S‐antigen (48 kDa protein), a retinal protein involved in the visual process has been determined by cDNA sequencing. The largest cDNA was 1590 base pairs (bp) and it contained an entire coding sequence. The similarity of nucleotide sequence between the human and bovine is approximately 80%. The predicted amino acid sequence indicates that human S‐antigen has 405 residues and its molecular mass is 45050 Da. The amino acid sequence homology between human and bovine is 81%. There is no overall sequence similarity between S‐antigen and other proteins listed in the National Biomedical Research Foundation (NBRF) protein data base. However, local regions of sequence homology with α‐transducin (Tα) are apparent including the putative rhodopsin binding and phosphoryl binding sites. In addition, human S‐antigen has sequences identical to bovine uveitopathogenic sites, indicating that some types of human uveitis may in part be related to the animal model of experimental autoimmune uveitis (EAU).

S-antigen: Characterization of a pathogenic epitope which mediates experimental autoimmune uveitis and pinealitis in Lewis rats

S-antigen (48K protein) is a photoreceptor cell protein highly pathogenic for the induction of experimental autoimmune uveitis (EAU) and intimately involved in the visual process. EAU is characterized, in part, as a T-cell mediated autoimmune disease which results in a severe inflammation of the uveal tract, and pineal gland. In order to determine specific sites in S-antigen responsible for its pathogenicity we synthesized twenty-three peptides, corresponding to the entire 404 amino acid sequence, and tested each peptide for its ability to induce EAU in Lewis rats. One peptide, peptide M (18 amino acids in length), was found to be highly pathogenic and consistently induced an EAU that was identical to the disease caused by native S-antigen. Clinically, the disease that developed in the eye was characterized by iris and pericorneal hyperemia, followed by inflammatory exudates in the anterior and vitreous chambers. Histopathologically a severe inflammatory response was observed which resulted in the complete destruction of the photoreceptor cell layer of the retina. In order to more fully characterize this pathogenic site, 14 additional smaller peptides (eight to eighteen amino acids in length) corresponding to the left and right portions of peptide M were synthesized. Of these peptides, peptide M16L, M15L, and M12L induced EAU, further localizing this pathogenic site to a small well-characterized region of S-antigen consisting of twelve amino acids. In addition, animals with ocular inflammatory disease had an associated pinealitis characterized by a lymphocytic infiltration of the subcapsular and central area of the pineal gland. The significance of these findings and the relationship of S-antigen in the pathogenesis of EAU and other autoimmune diseases is discussed.

S-antigen: identification of the MAbA9-C6 monoclonal antibody binding site and the uveitopathogenic sites

The location of the monoclonal antibody, MAbA9-C6, binding site and two uveitopathogenic sites in S-antigen have been determined. Using cyanogen bromide, S-antigen was cleaved into nine peptides, designated C1 to C9. MAbA9-C6 bound selectively to one large peptide designated C5, consisting of 122 amino acids. Six peptides (20 to 22 amino acids in length) designated 2,3,K,L,N and M, corresponding to the entire length of peptide C5, were synthesized chemically. In a radioimmunoassay and a dot-binding immunoassay, MAbA9-C6 bound selectively to one of the six peptides, peptide 3, indicating that this region of peptide C5 contains the MAbA9-C6 binding site. Twelve smaller peptides (ten amino acids in length), corresponding to the amino acid sequence of peptide 3, were synthesized and used in a competitive inhibition binding assay. These studies localized the MAbA9-C6 binding site to a small region within peptide 3. In addition, peptide K and peptide M were highly pathogenic for the induction of experimental auto-immune uveitis (EAU). Clinical and histological evidence of a severe uveo-retinitis, indistinguishable from that seen with native S-antigen, was documented in Lewis rats immunized with the synthetic peptides (50 micrograms), 11-12 days following immunization. Our results show that the MAbA9-C6 binding site and the two uveitopathogenic sites lie in close proximity to each other within the region of S-antigen corresponding to peptide C5. Furthermore, microcomputer analysis of the average hydrophilicity/hydrophobicity values of the amino acid sequence corresponding to peptide C5 shows that the MAbA9-C6 binding site and one uveitopathogenic site (peptide K) lie on the adjacent peaks. The significance of these findings and their relationship to the role of S-antigen in the pathogenesis of EAU and the phototransduction of vision is discussed.

Sequence analysis of bovine retinal S-antigen: Relationships with α-transducin and G-proteins

S‐Antigen is a major soluble protein of the retina and pineal. It is capable of inducing experimental autoimmune uveitis (EAU) in laboratory animals and also seems to play an important role in the visual cycle. The results of partial cDNA sequence analysis reveal interesting homologies with a‐transducin, a GTP‐binding protein of retina and other purine nucleotide‐binding proteins. In particular S‐antigen shows over 50% identity to the proposed pertussis toxin ADP‐ribosylation site of α‐transducin. It also contains the Gly‐X‐X‐X‐X‐Gly‐Lys pattern common to phosphoryl binding sites. A possible relationship between S‐antigen and purine nucleotide‐binding proteins is discussed. There is also evidence for a repetitious β‐structure in the C‐terminal half of S‐antigen, with a monoclonal antibody epitope in a helical region at the C‐terminus.