Distribution of CESP-1 protein in the corneal endothelium and other tissues.
OBJECTIVE
The gene expression profile of human corneal endothelium (CE) was established with the gene signature system. A novel gene, GS3582, was abundantly transcribed in the CE compared with other tissues according to a human gene expression database. This protein was designated corneal endothelium-specific protein (CESP)-1. The tissue distribution and subcellular localization of CESP-1 was assessed in humans and mice, to investigate its physiological function.


METHODS
Rabbit and mouse CESP-1 cDNAs were cloned, and a polyclonal anti-human CESP-1 antibody (Ab) and anti-mouse N- or C-terminal ovary-specific acidic protein (OSAP)-1 Ab were produced. CESP-1 expression was investigated in human and mouse corneas by Western blot and/or immunohistochemical analysis. The distribution of CESP-1 in human tissues was also examined by Western blot analysis. To identify the subcellular localization of CESP-1, cultured human CE was colabeled with anti-human CESP-1 Ab and anti-cytochrome c monoclonal Ab or anti-GRP78 monoclonal Ab for confocal microscopy.


RESULTS
The rabbit and mouse CESP-1 cDNA sequences contained an open reading frame coding 242 and 283 amino acids, respectively. Mouse CESP-1 was entirely consistent with mouse OSAP. Western blot analysis showed that CESP-1 was expressed in the human corneal epithelium, CE, cultured CE, brain, testis, and ovary. Mouse CESP-1 was also expressed in mouse corneal epithelium and CE with anti-mouse C- but not N-terminal OSAP Ab according to immunohistochemical analysis. Subcellular localization of CESP-1 to the mitochondria was demonstrated in cultured human CE. The N-terminal of CESP-1, possessing a mitochondrial targeting sequence, may be processed after the protein is imported into the mitochondria.


CONCLUSIONS
CESP-1 was distributed in the corneal epithelium, the CE and cultured human CE, as well as the brain, testis, and ovary. CESP-1 was localized in the mitochondria of cultured human CE. These findings may provide some clues about the physiological function of CESP-1.
Department of Ophthalmology, Jichi Medical School, Tochigi, Japan.
Rieko
R
Kinouchi
Tadatoshi
T
Kinouchi
Toshirou
T
Hamamoto
Takakazu
T
Saito
Adriano
A
Tavares
Tadahiko
T
Tsuru
Satoru
S
Yamagami
D000328:Adult; D000368:Aged; D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D001921:Brain / Q000378:metabolism*; D018929:Cell Culture Techniques; D003001:Cloning, Molecular; D000091342:Endoplasmic Reticulum Chaperone BiP; D004728:Endothelium, Corneal / Q000378:metabolism*; D019573:Epithelium, Corneal; D005136:Eye Proteins / Q000235:genetics / Q000378:metabolism*; D005260:Female; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D008297:Male; D008565:Membrane Proteins; D051379:Mice; D008810:Mice, Inbred C57BL; D018613:Microscopy, Confocal; D008875:Middle Aged; D024101:Mitochondrial Proteins; D008969:Molecular Sequence Data; D010053:Ovary / Q000378:metabolism*; D011506:Proteins; D011817:Rabbits; D013737:Testis / Q000378:metabolism*
D000091342:Endoplasmic Reticulum Chaperone BiP; D005136:Eye Proteins; C000717247:HSPA5 protein, human; C000717248:Hspa5 protein, mouse; C485853:MGARP protein, human; C509731:MGARP protein, mouse; D008565:Membrane Proteins; D024101:Mitochondrial Proteins; D011506:Proteins