Expression of beta-carotene 15,15' monooxygenase in retina and RPE-choroid.
OBJECTIVE
Beta-carotene 15,15' monooxygenase (beta-CM) catalyzes the central cleavage of beta-carotene to all-trans-retinal, the first step in vitamin A synthesis. This study was conducted to determine the expression of beta-CM in the mammalian retina and RPE, to assess its relevance in carotenoid-retinoid metabolism in the retina and RPE.


METHODS
RT-PCR was used to detect expression of beta-CM mRNA in the retina and RPE-choroid of the mouse, cow, human, and monkey and in RPE cells and other cell lines. Immunofluorescence microscopy was used to localize beta-CM in mouse and monkey retina with an anti-peptide antibody specific for beta-CM.


RESULTS
By RT-PCR, beta-CM mRNA was detected at a low level in mouse and monkey retina and in the RPE-choroid of the monkey but not of the mouse. Conversely, beta-CM mRNA was expressed at a low level in both human and bovine RPE-choroid, but not in the retina of either. RPE primary cultured cells of the monkey also showed beta-CM mRNA expression, although the three human lines did not. In addition, of nine other cell lines tested, only COS-7 was positive for beta-CM. Immunofluorescence microscopy showed weak immunoreactivity in the inner retina in both the mouse and monkey. beta-CM immunoreactivity was not detectable in RPE of the mouse. Use of a long-wavelength exciting and emitting secondary probe to mitigate lipofuscin autofluorescence, facilitated the detection of a low level of beta-CM immunoreactivity in monkey RPE.


CONCLUSIONS
Beta-CM mRNA and protein are expressed at low levels in the mammalian retina and RPE-choroid. Given the low and variable expression of beta-CM in the retina and RPE, it can be concluded that beta-CM is not necessary for a conserved retina or RPE-specific function, but may be necessary for a species-specific function.
Bhatti
Rizwan A
RA
Laboratory of Retinal Cell and Molecular Biology, National Eye Institute, National Institutes of Health, 6 Center Drive, Building 6 Room 339, Bethesda, MD 20892, USA.
Yu
Shirley
S
Boulanger
Ana
A
Fariss
Robert N
RN
Guo
Yan
Y
Bernstein
Steven L
SL
Gentleman
Susan
S
Redmond
T Michael
TM
D000818:Animals; D002417:Cattle; D002460:Cell Line; D002829:Choroid / Q000201:enzymology*; D019084:Fluorescent Antibody Technique, Indirect; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology*; D006801:Humans; D008251:Macaca; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D008856:Microscopy, Fluorescence; D010105:Oxygenases / Q000235:genetics* / Q000378:metabolism*; D010857:Pigment Epithelium of Eye / Q000201:enzymology*; D012333:RNA, Messenger / Q000378:metabolism; D012160:Retina / Q000201:enzymology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D050559:beta-Carotene 15,15'-Monooxygenase
D012333:RNA, Messenger; D010105:Oxygenases; C493359:Bco1 protein, mouse; C493358:BCO1 protein, human; D050559:beta-Carotene 15,15'-Monooxygenase