Identification of pigment epithelium-derived factor in the interphotoreceptor matrix of bovine eyes.
Pigment epithelium-derived factor (PEDF) is a neurotrophic protein and a member of the serine protease inhibitor superfamily. Here we describe the identification of PEDF in bovine eyes and optimization of its purification from this natural source. We have developed a polyclonal antibody to recombinant human PEDF, Ab-rPEDF, that immunoreacts in a specific, sensitive, and linear fashion with PEDF protein, and furthermore, blocks its neurotrophic activity. We show that Ab-rPEDF specifically recognizes a 49,500-M(r) polypeptide on Western transfers of a wash of the extracellular matrix between the retinal pigment epithelium and the neural retina-termed interphotoreceptor matrix (IPM). PEDF is present as approximately 1% of total soluble IPM protein. Starting with an IPM wash, PEDF protein is purified 164-fold to near homogeneity by ammonium sulfate fractionation and cation-exchange chromatography, with a recovery of 47%. The highly purified protein has an apparent M(r) of 49,500 +/- 1,500 as assessed by SDS-polyacrylamide gel electrophoresis, and a native pI of 7.0-7.7. It elutes as a single peak on gel-filtration chromatography with a retention time immediately behind that of ovalbumin (43,000 M(r)). N-glycosidase treatment indicates that each PEDF molecule has a 5% carbohydrate content attached to internal asparagine residue(s). Amino terminal sequence of the purified PEDF reveals removal of an amino-terminal peptide region for the mature protein. Purified PEDF has neurotrophic activity on human retinoblastoma cells, as previously observed for IPM. The neurotrophic activities of both PEDF and IPM are blocked by antiserum Ab-rPEDF. Altogether, PEDF is present in the bovine IPM as a soluble, extracellular, monomeric glycoprotein that by itself confers neurotrophic activity to the IPM. Thus, native PEDF isolated and purified as described here should prove useful for biochemical studies as well as other approaches.
Wu
Y Q
YQ
Laboratory of Retinal Cell and Molecular Biology, National Eye Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.
Notario
V
V
Chader
G J
GJ
Becerra
S P
SP
D000595:Amino Acid Sequence; D000818:Animals; D019138:Antibodies, Blocking; D002417:Cattle; D005109:Extracellular Matrix / Q000737:chemistry; D005136:Eye Proteins / Q000276:immunology / Q000302:isolation & purification* / Q000494:pharmacology; D006031:Glycosylation; D006801:Humans; D007120:Immunochemistry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009414:Nerve Growth Factors / Q000276:immunology / Q000302:isolation & purification* / Q000494:pharmacology; D016501:Neurites / Q000187:drug effects / Q000648:ultrastructure; D010786:Photoreceptor Cells / Q000737:chemistry*; D011506:Proteins / Q000276:immunology / Q000302:isolation & purification* / Q000494:pharmacology; D011994:Recombinant Proteins / Q000276:immunology / Q000302:isolation & purification / Q000494:pharmacology; D017386:Sequence Homology, Amino Acid; D015843:Serpins / Q000276:immunology / Q000302:isolation & purification* / Q000494:pharmacology; D014407:Tumor Cells, Cultured; D000099174:Pigment Epithelium-Derived Factor
D019138:Antibodies, Blocking; D005136:Eye Proteins; D009414:Nerve Growth Factors; D011506:Proteins; D011994:Recombinant Proteins; D015843:Serpins; D000099174:Pigment Epithelium-Derived Factor