Retina International's Scientific Newsletter

	Retina International's Scientific Newsletter
Editor's Notes from literature on RPE65

Recent update from: 20.02.2003

RPE65,

Notes

RPE65 can be extracted from Membranes by 1 M KCl at 90% efficiancy and together with CHAPS at 95% efficiancy
Extraction has almost no effect on in vitro LRAT or IMH activities
Thus RPE65 does not play a role in processing atRol into 11cRol
In RPE65 microsomal membranes
Not integral only associated

Species: Bos taurus

See also:

Chromosomal localisation	in man:
	in mouse:

Gene Data

Exons:

Primers:

Protein Data

Amino acids:

Molecular weight	on SDS-PAGE:
	calculated:

Reference: (3)

RPE65,

Notes

11cRol to 11cRAl formation is reduced on preincubation with anti-RPE65 antibody
Possible involvement of RPE65 in 11cRAl formation

Species: Bos taurus

See also:

Chromosomal localisation	in man:
	in mouse:

Gene Data

Exons:

Primers:

Protein Data

Amino acids:

Molecular weight	on SDS-PAGE:
	calculated:

Reference: (4)

RPE65,

Notes

pI = 6.41
RPE-specific
Related to function in the photoreceptor outer segment metabolism
Posttranscriptionally regulated in vivo
Not glycosylated
Associated with microsomal membranes of RPE cells
Phospholipid-binding might be necessary for function
Complete membrane embedding is unlikely
mRNA in RPE cells but not in retina

Species: Bos taurus

See also:

Chromosomal localisation	in man:
	in mouse:

Gene Data

mRNA: 3.15 kb

Exons:

Primers:

Protein Data

Amino acids: 533 AA

Molecular weight	on SDS-PAGE: 65 kDa
	calculated: 60940

Reference: (7)

RPE65,

Notes

Microsomal protein specific to RPE
5' and 3' UTR do not affect translational efficiency
TIE within 170 nt of sequence downstream of stop codon
TIE does not form base pairing to other parts of the mRNA to block elongation in WGE and RRL
Expression is posttranscriptionally regulated in primary cultured RPE

Species: Bos taurus

See also:

Chromosomal localisation	in man:
	in mouse:

Gene Data

cDNA: 3.1 kb

Exons:

Primers:

Protein Data

Amino acids:

Molecular weight	on SDS-PAGE:
	calculated:

Reference: (8)

RPE65,

Notes

Lacks TMD or signal peptide
Detergents or 0.75 - 1 M KCl required for solubilization
Peripheral membrane association
Co-sediments with PC/PS/PI as well as other liposomes
Binding is Ca²⁺ indipendent
Associated with RPE smooth ER
Localized to cytoplasm and intracellular membranes
NH-terminal amphipatic a-helices (AA 5 - 22 and 108 - 125)
Major protein associated with the microsomal membrane fraction
Major physical effect of RPE65 liposome phospholipid association is vesicle aggregation

Species: Bos taurus

See also:

Chromosomal localisation	in man:
	in mouse:

Gene Data

Exons:

Primers:

Protein Data

Amino acids:

Molecular weight	on SDS-PAGE:
	calculated:

Reference: (21)

rpe65,

Notes

IR in cones
No IR in rods

Species: Bos taurus

See also:

Chromosomal localisation	in man:
	in mouse:

Gene Data

Exons:

Primers:

Protein Data

Amino acids:

Molecular weight	on SDS-PAGE:
	calculated:

Reference: (23)

RPE65,

Notes

cDNA library from miniature poodle
487AAGA deletion in Swedish briard dogs resulting in retinal dystrophy
Identity: 98% human, 97% bovine, 93% rat

Species: Canis familiaris

See also:

Chromosomal localisation	in man:
	in mouse:

Gene Data

cDNA: 1724 bp

Exons:

Primers: 3' End, library screening primer

Protein Data

Amino acids: 533

Molecular weight	on SDS-PAGE: 61 kDa
	calculated:

Reference: (1)

rpe65,

Notes

Highly conserved throughout mammalian evolution
4 bp deletion 485delAAGA (residue 153 onward) in exon 5 caused Briard retinal dystrophy
pI: 6.3
AA similarity: 97.9% bovine, 98.5% human
DNA similarity: 91.3% human, 92.2% bovine,
2 non-consensus polyadenylation signals (ATTAAA at nt 1906 - 1911 and 2339 - 2344
Several AU-rich motifs
Polymorphisms: 459T/C (Y144), 541G/A (V171I), 1797delA (3'-UTR), IVS11+112C/T, IVS11+225delT
Exon 11 can be amplified by homologous human primers

Species: Canis familiaris

See also:

Chromosomal localisation	in man:
	in mouse:

Gene Data

cDNA: 2390 bp

Exons:

Primers: complete

Protein Data

Amino acids: 533

Molecular weight	on SDS-PAGE:
	calculated: 60.89 kDa

Reference: (22)

RPE65,

Notes

Present at high levels in vivo
Possible role in vit. A cycle suggested
Associates with serum Rol-binding protein and 11cRol dehydrogenase

Species: Homo sapiens

See also:

Chromosomal localisation	in man: 1p31
	in mouse:

Gene Data

Exons:

Primers: Polymorphism + complete

Protein Data

Amino acids:

Molecular weight	on SDS-PAGE: 61 kDa
	calculated:

Reference: (5)

RPE65,

Notes

Species: Homo sapiens

See also:

Chromosomal localisation	in man: 1p31
	in mouse: 3, Amy-2, Pxmp-1, Egf

Gene Data

Exons:

Primers:

Protein Data

Amino acids:

Molecular weight	on SDS-PAGE:
	calculated:

Reference: (6)

RPE65,

Notes

Species: Homo sapiens

See also:

Chromosomal localisation	in man: 1p31
	in mouse:

Gene Data

Exons: 14

Primers: complete

Protein Data

Amino acids:

Molecular weight	on SDS-PAGE:
	calculated:

Reference: (12)

RPE65,

Notes

Species: Homo sapiens

See also:

Chromosomal localisation	in man:
	in mouse:

Gene Data

Exons: 14

Primers: complete exons

Protein Data

Amino acids:

Molecular weight	on SDS-PAGE:
	calculated:

Reference: (13)

RPE65,

Notes

Species: Homo sapiens

See also:

Chromosomal localisation	in man:
	in mouse:

Gene Data

Exons:

Primers:

Protein Data

Amino acids:

Molecular weight	on SDS-PAGE: 61 kDa
	calculated:

Reference: (16)

RPE65,

Notes

In HeLa-cells
Basal promoter -83 bp
Regulatory elements: TATA, NF-1, SH2
PCE-1 at -137 = NRE

Species: Homo sapiens

See also:

Chromosomal localisation	in man:
	in mouse:

Gene Data

Exons:

Primers:

Protein Data

Amino acids:

Molecular weight	on SDS-PAGE:
	calculated:

Reference: (15)

RPE65,

Notes

Promoter includes D1S2803
Sequence homologies to CRAlBP
Basal promoter from -83 to + 39
No other control elements within 3.5 kb upstream
8 protected regions by DNAse I footprint
Predicted TATA-box at -27 (CATAAAA)
AP-1 consensus at -282 to -275
AP-4 consensus at -273 to -264 and -88 to -79
Ret/PCE1 consensus at -37 to -31

Species: Homo sapiens

See also:

Chromosomal localisation	in man: 1p31
	in mouse:

Gene Data

mRNA: 2.7 kb

Exons: 14

Primers:

Protein Data

Amino acids:

Molecular weight	on SDS-PAGE:
	calculated:

Reference: (14)

RPE65,

Notes

Voluntarily aborted embryo at 30 weeks gestation
Cell loss and thinning of ONL
Decreased IR for key phototransduction proteins
Abberant synaptic and inner retinal organisation
Cells loss in central posterior field
ONL contains many condensed cellular profiles
Mid and far periphery appeared similar to controls with equal ONL numbers
Less INL cells from central to mid-periphery
Photoreceptors revealed stunted OS and reduced rhodpsin labeling
Paenut agglutinin gave only sparse punctate staining compared to continous band in controls
RPE65 labeling failed in LCA patient

Species: Homo sapiens

See also:

Chromosomal localisation	in man:
	in mouse:

Gene Data

Exons:

Primers:

Protein Data

Amino acids:

Molecular weight	on SDS-PAGE:
	calculated:

Reference: (17)

RPE65,

Notes

Membrane associated
No signal peptide
No CAAT, TATA box
PCE like, AP4 (basic helix loop helix)
Alu like elements
Associated with smooth ER
Possible association with enzymes of visual cycle
Conserved in evolution

Species: Homo sapiens

See also:

Chromosomal localisation	in man:
	in mouse:

Gene Data

Exons: 14

Primers:

Protein Data

Amino acids: 533 AA

Molecular weight	on SDS-PAGE: 65 kDa
	calculated:

Reference: (19)

RPE65,

Notes

Biochemical association with RDH5 and SRBP
Direcly involved in atRol to 11cRol isomerisation

Species: Homo sapiens

See also:

Chromosomal localisation	in man:
	in mouse:

Gene Data

Exons:

Primers: complete

Protein Data

Amino acids:

Molecular weight	on SDS-PAGE:
	calculated:

Reference: (18)

RPE65,

Notes

Detected by PCR in transformed cell lines (HEK293, COS-1, COS-7) but not in primary kidney cells
Western blot was positive in COS-7 and HEK293
COS-7 share only 98% (cDNA) or 99% (AA) identity with retinal RPE65
HEK293 is identical at the cDNA level
Assumption: alternative retinoid metabolism in transformed kidney cells
Congenital mesoblastic nephroma and clear cell sarcoma express RPE65
Does not complex with RDH5 in COS-7 cells

Species: Homo sapiens (kidney cell lines)

See also:

Chromosomal localisation	in man:
	in mouse:

Gene Data

Exons:

Primers:

Protein Data

Amino acids:

Molecular weight	on SDS-PAGE: 61 kDa
	calculated:

Reference: (9)

RPE65,

Notes

Species: Mus musculus

See also:

Chromosomal localisation	in man:
	in mouse:

Gene Data

Exons: 14

Primers:

Protein Data

Amino acids:

Molecular weight	on SDS-PAGE:
	calculated:

Reference: (19)

RPE65,

Notes

Mouse knocknout
Histological differences to normal from 7th weeks of age on
By 15 weeks thinning of OS layer
RPE shows no major differences
Hereozygotes show standard ERG
-/- shows diminished b-wave, slow phot. Regeneration
ROS are disorganized
No effect in in RPE65 +/- and +/+ mice
Apo-opsin is not decreased but RHO is missing
Abolished rod ERG
11cRy are absent
atRy overaccumulate in RPE of -/- mice
Ry distribution suggests block in RPE visual cycle, possibly in isomerization of at-ester
Cone do not depend on RPE for regeneration (?!), possible 11cRal pool in cones?
RPE65 absence does not prevent vit. A uptake

Species: Mus musculus

See also:

Chromosomal localisation	in man:
	in mouse: distal 3

Gene Data

Exons:

Primers:

Protein Data

Amino acids:

Molecular weight	on SDS-PAGE:
	calculated:

Reference: (20)

RPE65,

Notes

No evidence of pseudogenes
27 kb genomic DNA
Transcription start is located 57 bp usptream from initiation codon
Higly conserved gene
Exon intron structure

Species: Mus musculus (domesticus)

See also:

Chromosomal localisation	in man: distal 3
	in mouse:

Gene Data

Exons: 14

Primers:

Protein Data

Amino acids:

Molecular weight	on SDS-PAGE:
	calculated:

Reference: (2)

rpe65,

Notes

IR in cones
Double staining with affinity purified anti-RPE65 and peanut agglutinin
Present in both types of mouse cones
In RPE65 knock-out mice no RPE65 IR was detected
No IR in rods
RPE65 levels in cones are substantially lower than in RPE
BALB/c mice have higher RPE65 levels than 129s and B6
RPE65 does not show isomerase activity on ist own

Species: Mus musculus (domesticus)

See also:

Chromosomal localisation	in man:
	in mouse:

Gene Data

Exons:

Primers:

Protein Data

Amino acids:

Molecular weight	on SDS-PAGE:
	calculated:

Reference: (23)

rpe65,

Notes

IR in cones
No IR in rods

Species: Oryctolagus cuniculus

See also:

Chromosomal localisation	in man:
	in mouse:

Gene Data

Exons:

Primers:

Protein Data

Amino acids:

Molecular weight	on SDS-PAGE:
	calculated:

Reference: (23)

RPE65,

Notes

Appears late during retinal development
Rate of accumulation parallels that of RHO
Involved in the RPE-specific metabolism of 11cRol
Identity: human: 94.5%, bovine 94%

Species: Rattus norvegicus

See also:

Chromosomal localisation	in man:
	in mouse:

Gene Data

cDNA: 2123

Exons:

Primers:

Protein Data

Amino acids: 533 AA

Molecular weight	on SDS-PAGE:
	calculated:

Reference: (11)

RPE65,

Notes

Sf9 expression system
Associated with cell membrane in this system
Cytosolic fraction protein weight is closely identical to the expected weight
Mmebrane fraction protein has a significantly higher weight
Native RPE65 is detected in cytosolic (61,161 Da) and microsomal (61,961 Da) fractions
Unstable protein, which is easily degraded during purification
Recombinant preotein is not myristoylated
Not bound to membranes by fatty acid acylation

Species: Recombinant

See also:

Chromosomal localisation	in man:
	in mouse:

Gene Data

Exons:

Primers:

Protein Data

Amino acids:

Molecular weight	on SDS-PAGE:
	calculated:

Reference: (10)

References:

Aguirre,G.D., Baldwin,V., Pearce Kelling,S., Narfstr�m,K., Ray,K., and Acland,G.M. Congenital stationary night blindness in the dog: common mutation in the RPE65 gene indicates founder effect. 1998; Mol.Vis. 4: 23
Link to PubMed
Free Online Source
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Boulanger,A., Liu,S., Yu,S., and Redmond,T.M. Sequence and structure of the mouse gene for RPE65. 2001; Mol.Vis. 7:283-7.: 283-287.
Link to PubMed
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Choo,D.W., Cheung,E., and Rando,R.R. Lack of effect of RPE65 removal on the enzymatic processing of all-trans-retinol into 11-cis-retinol in vitro. 1998; FEBS Lett. 440: 195-198.
Link to PubMed
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Crouch,R.K., Goletz,P., Yu,S., and Redmond,T.M. A possible role for RPE65 in retinoid processing. 1997; Invest.Ophthalmol.Vis.Sci. 38: S304
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Gu,S., Thompson,D.A., Srisailapathy Srikumari,C.R., Lorenz,B., Finckh,U., Nicoletti,A., Murthy,K.R., Rathmann,M., Kumaramanickavel,G., Denton,M.J., and Gal,A. Mutations in RPE65 cause autosomal recessive childhood-onset severe retinal dystrophy. 1997; Nat.Genet. 17: 194-197.
Link to PubMed
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Hamel,C.P., Jenkins,N.A., Gilbert,D.J., Copeland,N.G., and Redmond,T.M. The gene for the retinal pigment epithelium-specific protein RPE65 is localized to human 1p31 and mouse 3. 1994; Genomics. 20: 509-512.
Link to PubMed
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Hamel,C.P., Tsilou,E., Pfeffer,B.A., Hooks,J.J., Detrick,B., and Redmond,T.M. Molecular cloning and expression of RPE65, a novel retinal pigment epithelium-specific microsomal protein that is post- transcriptionally regulated in vitro. 1993; J.Biol.Chem. 268: 15751-15757.
Link to PubMed
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Liu,S.Y. and Redmond,T.M. Role of the 3'-untranslated region of RPE65 mRNA in the translational regulation of the RPE65 gene: identification of a specific translation inhibitory element. 1998; Arch.Biochem.Biophys. 357: 37-44.
Link to PubMed
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Ma,J.X., Zhang,D., Laser,M., Brownlee,N.A., Re,G.G., Hazen-Martin,D.J., Redmond,T.M., and Crouch,R.K. Identification of RPE65 in transformed kidney cells. 1999; FEBS Lett. 452: 199-204.
Link to PubMed
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Ma,J., Zhang,J., Othersen,K.L., Moiseyev,G., Ablonczy,Z., Redmond,T.M., Chen,Y., and Crouch,R.K. Expression, purification, and maldi analysis of rpe65. 2001; Invest Ophthalmol.Vis.Sci. 42: 1429-1435.
Link to PubMed
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Manes,G., Leducq,R., Kucharczak,J., Pages,A., Schmitt Bernard,C.F., and Hamel,C.P. Rat messenger RNA for the retinal pigment epithelium-specific protein RPE65 gradually accumulates in two weeks from late embryonic days. 1998; FEBS Lett. 423: 133-137.
Link to PubMed
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Marlhens,F., Bareil,C., Griffoin,J.M., Zrenner,E., Amalric,P., Eliaou,C., Liu,S.Y., Harris,E., Redmond,T.M., Arnaud,B., Claustres,M., and Hamel,C.P. Mutations in RPE65 cause Leber's congenital amaurosis. 1997; Nat.Genet. 17: 139-141.
Link to PubMed
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Morimura,H., Fishman,G.A., Grover,S.A., Fulton,A.B., Berson,E.L., and Dryja,T.P. Mutations in the RPE65 gene in patients with autosomal recessive retinitis pigmentosa or Leber congenital amaurosis. 1998; Proc.Natl.Acad.Sci.U.S.A. 95: 3088-3093.
Link to PubMed
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Nicoletti,A., Kawase,K., and Thompson,D.A. Promoter analysis of RPE65, the gene encoding a 61-kDa retinal pigment epithelium-specific protein. 1998; Invest Ophthalmol Vis.Sci. 39: 637-644.
Link to PubMed
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Nicoletti,A., Swain,P.K., Swaroop,A., and Thompson,D.A. Regulation Of Gene Expression In The Retinal Pigment Epithelium: NRL Enhances Transcription From The RPE65 Promoter. 1998; Invest.Ophthalmol.Vis.Sci. 39: S197
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Nicoletti,A., Wong,D.J., Kawase,K., Gibson,L.H., Yang-Feng,T.L., Richards,J.E., and Thompson,D.A. Molecular characterization of the human gene encoding an abundant 61 kDa protein specific to the retinal pigment epithelium. 1995; Hum.Mol.Genet. 4: 641-649.
Link to PubMed
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Porto,F.B., Perrault,I., Hicks,D., Rozet,J.M., Hanoteau,N., Hanein,S., Kaplan,J., and Sahel,J.A. Prenatal human ocular degeneration occurs in Leber's congenital amaurosis (LCA2). 2002; J Gene Med. 4: 390-396.
Link to PubMed
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Redmond,T.M. and Hamel,C.P. Genetic analysis of RPE65: from human disease to mouse model. 2000; Methods Enzymol. 316:705-24.: 705-724.
Link to PubMed
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Redmond,T.M., Harris,E.W., Yu,S., Liu,S.Y., Kapsis,A., and Hamel,C.P. Analysis of the human gene for the retinal pigment epithelium specific protein RPE65. 1995; Invest.Ophthalmol.Vis.Sci. 37: S598
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Redmond,T.M., Yu,S., Lee,E., Bok,D., Hamasaki,D., Chen,N., Goletz,P., Ma,J.X., Crouch,R.K., and Pfeifer,K. Rpe65 is necessary for production of 11-cis-vitamin A in the retinal visual cycle. 1998; Nat.Genet. 20: 344-351.
Link to PubMed
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Tsilou,E., Hamel,C.P., Yu,S., and Redmond,T.M. RPE65, the major retinal pigment epithelium microsomal membrane protein, associates with phospholipid liposomes. 1997; Arch.Biochem.Biophys. 346: 21-27.
Link to PubMed
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Veske,A., Nilsson,S.E., Narfstr�m,K., and Gal,A. Retinal dystrophy of Swedish Briard/Briard-beagle dogs is due to a 4-bp deletion in RPE65. 1999; Genomics. 57: 57-61.
Link to PubMed
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Znoiko,S.L., Crouch,R.K., Moiseyev,G., and Ma,J.X. Identification of the RPE65 protein in mammalian cone photoreceptors. 2002; Invest Ophthalmol.Vis.Sci. 43: 1604-1609.
Link to PubMed
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Contact the editor	This site is maintained and edited by Dr. rer. medic. Markus Preising, Dipl.Biol. Molecular Genetics Laboratory Department of Paediatric Ophthalmology, Strabismology and Ophthalmogenetics University of Regensburg Head: Prof. Dr. med. Birgit Lorenz