TY - JOUR
T1 - Sequence and expression analysis of bovine pigment epithelium-derived factor
AU - Perez-Mediavilla, L. Alberto
AU - Chew, Christina
AU - Campochiaro, Peter A.
AU - Nickells, Robert W.
AU - Notario, Vicente
AU - Zack, Donald J.
AU - Becerra, S. Patricia
N1 - Funding Information:
We thank Dr. Fintan Steele for kindly providing the human PEDF cDNA πFS17, Dr. Jorge Laborda for assistance in the use of a photoimager, Miss Emily Cartwright for technical assistance in isolation of recombinant proteins, Drs. Barbara Wiggert and Elena Alberdi for proofreading the manuscript. Dr. L. Alberto Perez-Mediavilla is a recipient of a fellowship from the North Atlantic Treaty Organization. This work was supported, in part, by funds from The Foundation Fighting Blindness and the Rebecca P. Moon, Charles M. Moon, Jr., and Dr. P. Thomas Manchester Research Fund. Dr. D.J. Zack is a recipient of a Career Development Award from Research to Prevent Blindness.
PY - 1998/6/16
Y1 - 1998/6/16
N2 - 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.
AB - 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.
KW - Expression
KW - PEDF
KW - Retinal pigment epithelium
KW - Sequence
KW - Serpin
KW - Translation
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U2 - 10.1016/S0167-4781(98)00055-4
DO - 10.1016/S0167-4781(98)00055-4
M3 - Article
C2 - 9689919
AN - SCOPUS:0032537459
SN - 0167-4781
VL - 1398
SP - 203
EP - 214
JO - Biochimica et Biophysica Acta - Gene Structure and Expression
JF - Biochimica et Biophysica Acta - Gene Structure and Expression
IS - 2
ER -