MOLECULAR-CLONING AND EXPRESSION OF RPE65, A NOVEL RETINAL-PIGMENT EPITHELIUM-SPECIFIC MICROSOMAL PROTEIN THAT IS POSTTRANSCRIPTIONALLY REGULATED IN-VITRO

Studies reported previously from this laboratory have shown that micro somal membranes of the vertebrate retinal pigment epithelium (RPE) con tain an RPE-specific 65-kDa protein, RPE65, which bears the determinan t recognized by the strictly tissue-specific monoclonal antibody RPE9, and which is developmentally regulated (Hamel, C. P., Tsilou, E., Har ris, 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 R PE65 blotted on nitrocellulose yielded primary sequences that were use d to generate oligonucleotide probes. An 84-nucleotide guessmer was us ed to isolate two clones from a bovine RPE lambdaZap II cDNA library. Rapid amplification of cDNA ends was used to complete the 5' and 3' en ds, resulting in a 3,115-base pair composite cDNA. The open reading fr ame encodes a novel protein of 533 amino acid residues with a computed molecular weight of 60,940. This protein does not match any other seq uence in the data bases. The 231 amino acids obtained from peptide seq uencing match 43% of the amino acid sequence deduced from the cDNA. Th e protein has a calculated pI of 6.41 and is not predicted to have any transmembrane segments. The open reading frame expressed in Escherich ia coli has an apparent molecular weight identical to that of the nati ve protein and is recognized by the monoclonal antibody RPE9, further corroborating its validity. Northern blot analysis detected a major mR NA species of 3.15 kilobases for RPE65, as well as shorter species, on ly in RPE and not in other tissues (including other ocular tissues). C ultured RPE cells (7 weeks in primary culture) contained RPE65 mRNA in amounts equivalent to fresh RPE. Such cells, however, contained no im munodetectable RPE65. The possible structure of this RPE-specific prot ein and hypotheses for the absence of translation in vitro are discuss ed.