Sequence and expression analysis of bovine pigment epithelium-derived factor.
PEDF, a member of the serpin superfamily of proteins related through their highly conserved folded conformation, has neurotrophic properties, including promotion of neurite-outgrowth and neuronal survival. Previously, we have purified and characterized PEDF protein from extracellular matrixes of bovine eyes. Here, we show the cDNA sequence and expression analysis of bovine PEDF. Northern analysis of RNA from bovine retinal pigment epithelium (RPE) and neural retina using a human PEDF cDNA fragment reveals expression of the PEDF gene only for RPE. Sequence analysis of a cDNA clone isolated from bovine RPE predicts a polypeptide of 416 amino acid residues that shares 88.6% and 85% amino acid identity with human and mouse PEDF, respectively. It has an N-terminal signal peptide, a consensus glycosylation site and homology with serpins including the conserved residues required for maintaining the serpin tertiary structure. Cell-free expression of the bovine PEDF cDNA by in vitro transcription and translation yields a precursor polypeptide of 45,000-Mr that immunoprecipitates with an antibody to human PEDF. Expression analysis in stably transfected baby hamster kidney cells shows that the recombinant bovine protein is secreted to the culture media as a mature 50,000-Mr protein, which induces neurite-outgrowth on retinoblastoma cells, like the naturally-occurring PEDF protein. Thus, the bovine PEDF cDNA isolated here codes for a functional soluble secreted PEDF glycoprotein.
Laboratory of Retinal Cell and Molecular Biology, National Eye Institute, National Institutes of Health, Bethesda, MD 20892, USA.
L A
LA
Perez-Mediavilla
C
C
Chew
P A
PA
Campochiaro
R W
RW
Nickells
V
V
Notario
D J
DJ
Zack
S P
SP
Becerra
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D002460:Cell Line; D002474:Cell-Free System / Q000737:chemistry / Q000378:metabolism; D004247:DNA / Q000737:chemistry / Q000235:genetics*; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D005136:Eye Proteins; D015870:Gene Expression / Q000235:genetics; D008969:Molecular Sequence Data; D009414:Nerve Growth Factors; D010857:Pigment Epithelium of Eye / Q000737:chemistry / Q000378:metabolism; D011506:Proteins / Q000235:genetics*; D011994:Recombinant Proteins / Q000235:genetics; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D015843:Serpins / Q000235:genetics*; D000099174:Pigment Epithelium-Derived Factor
D004247:DNA; D018076:DNA, Complementary; D005136:Eye Proteins; D009414:Nerve Growth Factors; D011506:Proteins; D011994:Recombinant Proteins; D015843:Serpins; D000099174:Pigment Epithelium-Derived Factor