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Molecular cloning and expression of RPE65, a novel retinal pigment epithelium-specific microsomal protein that is
C P Hamel1, E Tsilou, B A Pfeffer
1Laboratory of Retinal Cell and Molecular Biology, National Eye Institute, National Institutes of Health, Bethesda, Maryland 20892.
Abstract:
Studies reported previously from this laboratory have shown that microsomal membranes of the vertebrate retinal pigment epithelium (RPE) contain an RPE-specific 65-kDa protein, RPE65, which bears the determinant recognized by the strictly tissue-specific monoclonal antibody RPE9, and which is developmentally regulated (Hamel, C. P., Tsilou, E., Harris, E., Pfeffer, B. A., Hooks, J. J., Detrick, B., and Redmond, T. M. (1993) J. Neurosci. Res. 34, 414-425). Microsequencing of 17 tryptic and chymotryptic peptides obtained from the in situ digestion of the RPE65 blotted on nitrocellulose yielded primary sequences that were used to generate oligonucleotide probes. An 84-nucleotide guessmer was used to isolate two clones from a bovine RPE lambda Zap II cDNA library. Rapid amplification of cDNA ends was used to complete the 5' and 3' ends, resulting in a 3,115-base pair composite cDNA. The open reading frame encodes a novel protein of 533 amino acid residues with a computed molecular weight of 60,940. This protein does not match any other sequence in the data bases. The 231 amino acids obtained from peptide sequencing match 43% of the amino acid sequence deduced from the cDNA. The protein has a calculated pI of 6.41 and is not predicted to have any transmembrane segments. The open reading frame expressed in Escherichia coli has an apparent molecular weight identical to that of the native protein and is recognized by the monoclonal antibody RPE9, further corroborating its validity. Northern blot analysis detected a major mRNA species of 3.15 kilobases for RPE65, as well as shorter species, only in RPE and not in other tissues (including other ocular tissues). Cultured RPE cells (7 weeks in primary culture) contained RPE65 mRNA in amounts equivalent to fresh RPE. Such cells, however, contained no immunodetectable RPE65. The possible structure of this RPE-specific protein and hypotheses for the absence of translation in vitro are discussed.
Insights
Researchers identified and sequenced RPE65, a novel 60.9 kDa protein specific to the retinal pigment epithelium (RPE). This discovery provides a molecular basis for understanding RPE-specific functions and potential disease mechanisms.
Area of Science:
- Molecular Biology
- Ophthalmology
- Protein Biochemistry
Background:
- Microsomal membranes of vertebrate retinal pigment epithelium (RPE) contain RPE65, a 65-kDa protein recognized by monoclonal antibody RPE9.
- RPE65 is developmentally regulated and tissue-specific within the RPE.
Purpose of the Study:
- To clone and characterize the RPE65 protein.
- To determine the full-length cDNA sequence and encoded amino acid sequence of RPE65.
- To investigate the expression of RPE65 at the mRNA and protein levels.
Main Methods:
- Microsequencing of tryptic and chymotryptic peptides from digested RPE65.
- Oligonucleotide probe generation and screening of a bovine RPE cDNA library.
- Rapid amplification of cDNA ends (RACE) to obtain full-length cDNA.
- Open reading frame expression in Escherichia coli and Western blot analysis.
- Northern blot analysis to detect RPE65 mRNA in various tissues and cultured cells.
Main Results:
- A composite cDNA of 3,115 base pairs was obtained, encoding a novel 533-amino acid protein (60,940 Da).
- The deduced amino acid sequence showed a 43% match with the sequenced peptides.
- The expressed protein in E. coli was recognized by the RPE9 antibody.
- RPE65 mRNA (3.15 kb) was detected exclusively in RPE, with shorter species also present.
- Cultured RPE cells expressed RPE65 mRNA but lacked immunodetectable RPE65 protein.
Conclusions:
- The study successfully cloned and characterized the RPE65 protein, revealing it as a novel, RPE-specific protein.
- The findings provide a molecular basis for RPE-specific functions.
- The absence of detectable RPE65 protein in cultured cells despite mRNA presence suggests post-transcriptional regulation.