Biomedical subjects

S P Becerra

Publications and source records attributed to S P Becerra.

At least 19 recordsLinked to original sources

PubMed2001

Survival effects of pigment epithelium-derived factor expressed by a lentiviral vector in rat cerebellar granule cells.

We have previously shown that pigment epithelium-derived factor (PEDF) acts as a survival factor for cerebellar granule cells (CGCs), by blocking apoptotic death, and can also protect these cells against glutamate-induced neurotoxicity. In preparation for gene therapy studies, pseudotyped HIV-1-based lentiviral vectors containing the PEDF gene, as well as either green fluorescent protein or beta-galactosidase, were prepared. These bicistronic vectors are unique in that they express two genes efficiently under one promoter. Primary cell cultures of CGCs from postnatal day 8 rats were infected with the vectors encoding PEDF. RT-PCR demonstrated expression of mRNA and Western blot analysis confirmed that infected CGCs secrete PEDF protein to the medium. Assays for cell survival demonstrated that PEDF-infected cells were significantly more protected compared with mock-infected controls for 6-8 days in culture, as well as against induced apoptosis. The PEDF vectors expressing tat (trans-acting transcription factor) provided more protection than the tat(-) vectors. These results demonstrate that while the lentiviral vectors expressing PEDF are as neuroprotective as the protein itself for CGCs, the vectors have the advantage of providing long-lasting expression of PEDF protein, which will be more effective in in vivo studies. The present results suggest that this system may be useful for gene therapy for neurodegenerative disorders.

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PubMed2001

Evidence for pigment epithelium-derived factor receptors in the neural retina.

PURPOSE: The neurotrophic activity of pigment epithelium-derived factor (PEDF), an extracellular factor present in the retina, is mediated by binding to cell-surface receptors in responsive cell cultures. In the present study, the expression of PEDF receptors in native neural retinas from adult steers was examined. METHODS: Binding reactions were performed with (125)I-PEDF and fluoresceinated PEDF using plasma membranes, detergent-soluble membrane proteins, or cryosections of retina from adult bovine eyes. Radioligand-binding and competition analyses were performed with a computer-assisted program. Ligand blot analysis of detergent-soluble membrane proteins was performed with (125)I-PEDF followed by autoradiography. Ligand-affinity column chromatography of detergent-soluble membrane proteins was performed with PEDF-coupled resin followed by SDS-PAGE. Binding of fluoresceinated PEDF to retina cryosections was detected by confocal microscopy. RESULTS: Radioligand-binding assays showed that (125)I-PEDF bound in a specific and saturable fashion to one class of sites on retina membranes (K(d) = 2.5-6.5 nM; maximum binding [B(max)] = 1-48 x 10(10) sites/retina). A peptide of 44 amino acids (44-mer), identified as the receptor-binding region of PEDF, competed efficiently for (125)I-PEDF binding to retina membranes with kinetics similar to the full-length PEDF. Ligand blot analysis and ligand-affinity chromatography revealed a specific and high-affinity PEDF-binding protein of approximately 85 kDa in retina plasma membranes. Confocal microscopy showed that fluorescein-conjugated PEDF stained exclusively the inner segments of photoreceptors and cells of the ganglion cell layer in retinal cryosections. CONCLUSIONS: Altogether, these data conclusively demonstrate the existence of PEDF receptors discretely distributed on the surface of cells from the adult neural retina of bovine eyes. Furthermore, they provide evidence for the direct action of PEDF on photoreceptor and ganglion cell neurons and an anatomic basis for studies to assess PEDF neurotrophic effects on the adult retina.

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PubMed2000

Clusterin protein diversity in the primate eye.

PURPOSE: The clusterin gene encodes a multi-functional protein that has been identified in different tissues, including a number of different eye tissues, primarily in the mouse and to a much lesser extent in humans. Clusterin has been implicated in a number of cellular processes such as lipid transport, membrane integrity, apoptosis, and neurodegeneration, all of which could be important to the biology of the eye. In the current communication, we provide data that confirms the expression of clusterin in a number of different human eye tissues and establishes the expression profile of this gene in monkey derived eye tissues. The issue that we sought to examine is whether a broad profile of clusterin expression in the eye is consistent in primates (monkey and human). METHODS: The majority of our study was done using monkey eye tissues. Where possible, we have used human tissues in order to confirm published findings. Northern and western analysis was performed using tissues derived from monkey eyes. In situ hybridization and immunochemistry were carried out on human eye sections. RESULTS: Clusterin mRNA is expressed in primate lens, cornea, limbus, sclera, orbital muscle, ciliary body, retina, RPE/choroid, and RPE cells in culture. Western analysis revealed that two major groups of clusterin exist in the eye, a high molecular weight group (>100 kDa) and a second group consisting of at least five clusterin species that are all approximately 80 kDa. Analysis of conditioned media from RPE cells cultured on permeable supports suggests that different forms of clusterin display alternative patterns of secretion. CONCLUSIONS: Clusterin is expressed in a broad range of eye tissues in both human and monkey, suggesting that this is a characteristic feature in primates. We demonstrate for the first time that a diverse number of clusterin isoforms were observed in monkey eye tissues by western analysis. Meanwhile, the molecular size of clusterin mRNA detected in the array of tissues are identical in size, suggesting that the nature of the diversity in clusterin forms is due to post-translational modifications. In addition, new insights were made in defining clusterin expression in ciliary body, cornea, and the retinal pigment epithelium.

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PubMed1999

Binding of pigment epithelium-derived factor (PEDF) to retinoblastoma cells and cerebellar granule neurons. Evidence for a PEDF receptor.

Pigment epithelium-derived factor (PEDF) has neuronal differentiation and survival activity on retinoblastoma and cerebellar granule (CG) cells. Here, we investigated the presence of PEDF receptors on retinoblastoma Y-79 and CG cells. PEDF radiolabeled with (l25)I remained biologically active and was used for radioligand binding analysis. The binding was saturable and specific to a single class of receptors on both cells and with similar affinities (K(d) = 1.7-3.6 nM, B(max) = 0.5-2.7 x 10(5) sites/Y-79 cell; and K(d) = 3.2 nM, B(max) = 1.1 x 10(3) sites/CG cell). A polyclonal antiserum to PEDF, previously shown to block the PEDF neurotrophic activity, prevented the (125)I-PEDF binding. We designed two peptides from a region previously shown to confer the neurotrophic property to human PEDF, synthetic peptides 34-mer (positions 44-77) and 44-mer (positions 78-121). Only peptide 44-mer competed for the binding to Y-79 cell receptors (EC(50) = 5 nM) and exhibited neuronal differentiating activity. PEDF affinity column chromatography of membrane proteins from both cell types revealed a PEDF-binding protein of approximately 80 kDa. These results are the first demonstration of a PEDF-binding protein with characteristics of a PEDF receptor and suggest that the region comprising amino acid positions 78-121 of PEDF might be involved in ligand-receptor interactions.

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PubMed1999

Pigment epithelium-derived factor promotes the survival and differentiation of developing spinal motor neurons.

Pigment epithelium-derived factor (PEDF) is a member of the serine protease inhibitor (serpin) superfamily that has been shown previously to promote the survival and/or differentiation of rat cerebellar granule neurons and human retinoblastoma cells in vitro. However, in contrast to most serpins, PEDF has no inhibitory activity against any known proteases, and its described biological activities do not appear to require the serpin-reactive loop located toward the carboxy end of the polypeptide. Because another serpin, protease nexin-1, has been shown to promote the in vivo survival and growth of motor neurons, the authors investigated the potential neurotrophic effects of PEDF on spinal cord motor neurons in highly enriched cultures and in vivo after injury. Here, it is shown that native bovine and recombinant human PEDF promoted the survival and differentiation (neurite outgrowth) of embryonic chick spinal cord motor neurons in vitro in a dose-dependent manner. A truncated form of PEDF that lacks approximately 62% of the carboxy end of the polypeptide comprising the homologous serpin-reactive loop also exhibited neurotrophic activities similar to those of the full-length protein. Furthermore, the data here showed that PEDF was transported retrogradely and prevented the death and atrophy of spinal motor neurons in the developing neonatal mouse after axotomy. These results indicate that PEDF exerts trophic effects on motor neurons, and, together with previous reports, these findings suggest that this protein may be useful as a pharmacologic agent to promote the development and maintenance of motor neurons. J. Comp. Neurol. 412:506-514, 1999. Published 1999 Wiley-Liss, Inc.

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PubMed1999

Pigment epithelium-derived factor delays the death of photoreceptors in mouse models of inherited retinal degenerations.

Pigment epithelium-derived factor (PEDF) is a member of the serine protease inhibitor superfamily produced by retinal pigment epithelial cells in the developing and adult retina. In vitro, it induces neuronal differentiation of retinoblastoma cells and promotes survival of cerebellar granule neurons. The pedf gene is closely linked to an autosomal-dominant locus for retinitis pigmentosa, suggesting that PEDF could be a survival factor for photoreceptors. We have investigated this possibility by injecting PEDF into the eyes of homozygous retinal degeneration (rd) and retinal degeneration slow (rds) mice, two mutants displaying apoptotic photoreceptor loss. This procedure resulted in a transient delay of photoreceptor loss in the rd mouse and a reduction in apoptotic photoreceptor profiles in the rds mouse. We conclude that PEDF can act as a survival-promoting factor for photoreceptors in vivo and could potentially be useful for the treatment of photoreceptor diseases.

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PubMed1998

Pigment epithelium-derived factor (PEDF) binds to glycosaminoglycans: analysis of the binding site.

Pigment epithelium-derived factor (PEDF), a neurotrophic protein, is a secreted serpin identified in extracellular matrixes. We show that PEDF extractions from the interphotoreceptor matrix are more efficient with increasing NaCl concentrations, indicating that ionic interactions mediate its association with this polyanionic matrix. We have used affinity chromatography and ultrafiltration to probe for direct binding of PEDF to glycosaminoglycans/polyanions. Correctly folded PEDF bound to immobilized heparin, chondroitin sulfate-A, -B, -C, and dextran sulfate columns and eluted from each with an increase in NaCl concentration. However, in the presence of urea, the protein lost its affinity for heparin. Binding of PEDF to heparan sulfate proteoglycan in solution was in a concentration-dependent fashion (half-maximal specific binding EC50 = 40 micrograms/mL) and was sensitive to increasing NaCl concentrations. The glycosaminoglycan-binding region was analyzed using chemical modification and limited proteolysis. PEDF chemically modified on lysine residues by biotinylation lost its capacity for interacting with heparin, implicating the involvement of PEDF lysine residues in heparin binding. Cleavage of the serpin-exposed loop with chymotrypsin did not affect the heparin-binding property. A limited proteolysis product containing residues 21-approximately 260 bound to heparin with similar affinity as the intact PEDF. Homology modeling of PEDF based on the X-ray crystal structures of antithrombin III and ovalbumin shows a region at the center of beta-sheet A-strands 2 and 3- and helix F that has a basic electrostatic surface potential and is densely populated with lysines exposed to the surface (K134, K137, K189, K191, H212, and K214) that are available to interact with various glycosaminoglycans/polyanions. This region represents a novel site for glycosaminoglycan binding in a serpin, which in PEDF, is distinct and nonoverlapping from the PEDF neurotrophic active region.

Amino Acid Sequence↗

PubMed1998

Pigment epithelium-derived factor (PEDF) differentially protects immature but not mature cerebellar granule cells against apoptotic cell death.

We have shown previously that pigment epithelium-derived factor (PEDF) acts as a survival factor for cerebellar granule cell neurons in culture, as well as protecting them against glutamate toxicity. In this study we have examined effects of PEDF on apoptotic cell death. We find that the granule cells die of apoptosis throughout the culture period, what we have termed "natural" apoptosis. PEDF prevents this natural apoptosis if added to immature cells, within the first 2 days in vitro (DIV), and the effect is maintained for up to DIV12. However, PEDF has no effect if added to mature cells at DIV5. Similar results are obtained when apoptosis is induced by shifting the cells from a serum- and 25 mM KCl-containing medium to serum-free medium with 5 mM KCl. PEDF most effectively blocks induced apoptosis in immature cells (DIV2) when added 24 hr prior to the change of medium, but still provides some protection when added simultaneously. However, 24 hr pretreatment with PEDF has a minimal effect when apoptosis is induced in mature DIV6 cells; addition at the same time is completely ineffective. Two polypeptide fragments of PEDF, only one of which contains the serine-protease inhibitory site, are equally active, supporting previous results which suggest that the neurotrophic effects of PEDF are not mediated by protease inhibition. We conclude that PEDF protects immature but not mature granule cells against both natural and induced apoptosis.

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PubMed1998

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.

Amino Acid Sequence↗

PubMed1997

Pigment epithelium-derived factor (PEDF) has direct effects on the metabolism and proliferation of microglia and indirect effects on astrocytes.

Pigment epithelium-derived factor (PEDF), a neurotrophic agent first identified in conditioned medium from cultured human retinal pigment epithelial cells, induces neuronal differentiation with neurite outgrowth in Y-79 retinoblastoma cells and has a neurotrophic survival effect on cerebellar granule cells in culture. In the present study, we investigated the effects of human recombinant PEDF (rPEDF) on proliferation and activation of microglia and astrocytes isolated from newborn rat brain. rPEDF treatment caused microglia to round up morphologically, increased their metabolic activity (measured by both MTS conversion and acid phosphatase activity), but blocked proliferation (mitosis). This blocking effect could be demonstrated in cultures stimulated to proliferate by addition of granulocyte-macrophage colony stimulating factor. The effect of rPEDF on microglial metabolic activity showed a dose-response relationship both in serum-containing medium and in chemically defined medium and was blocked with anti-PEDF antibody. rPEDF had no direct effect on the metabolic activity or proliferation of cultured astrocytes but blocked their proliferation in astrocyte-microglia co-cultures. Proliferation of isolated astrocytes was also blocked by conditioned medium from microglia treated with PEDF (PMCM). The effect of PMCM on astrocytes was not blocked by an antibody to transforming growth factor-beta. These results demonstrate that PEDF activates microglial metabolism while blocking proliferation and suggest that a soluble factor(s) released by rPEDF-stimulated microglia blocks the proliferation of astrocytes. Thus, PEDF could play an important role in regulation of glial function and proliferation in the central nervous system.

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PubMed1997

Pigment epithelium-derived factor protects cultured cerebellar granule cells against glutamate-induced neurotoxicity.

Pigment epithelium-derived factor (PEDF) is a survival factor for cerebellar granule cells in culture. In the present study, we have investigated the ability of a recombinant form of PEDF (rPEDF) to protect against glutamate neurotoxicity. When rPEDF was added to cerebellar granule cell cultures 30 min before addition of 100 microM glutamate, glutamate-induced neuronal death was significantly reduced. The protective effect of rPEDF was dose-dependent in the range from 0.023 to 7.0 nM (1-500 ng/ml), with a half-maximal dose of 0.47 nM. An antibody to rPEDF blocked this protective effect. Measurement of intraneuronal free calcium levels demonstrated that rPEDF raised the basal calcium content. However, after the elevation of intracellular calcium in response to administration of glutamate, rPEDF reduced the plateau level seen in the presence of glutamate. These data show that PEDF can protect neurons against glutamate-induced neurotoxicity, possibly via a calcium-related pathway. The finding that only 30 min of preincubation is required for the neuroprotective effect, significantly faster than other known neurotrophic factors, suggests that PEDF may be useful clinically as a neuroprotective agent in the CNS.

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PubMed1997

Structure-function studies on PEDF. A noninhibitory serpin with neurotrophic activity.

The neuronal differentiating and survival activities and presence of PEDF next to the neural retina support the idea that this serpin plays a neurotrophic role in vivo. The knowledge of the PEDF structure has provided information on the determinants for the noninhibitory and neurotrophic activities. PEDF has characteristics of a substrate rather than an inhibitor of serine proteases. Further studies are needed to identify the missing structural elements on PEDF that would confer serpin inhibitory activity. An N-terminus peptide region provides the neurotrophic function to the PEDF protein while other structural characteristics are dispensable (e.g. signal peptide, oligosaccharides on the polypeptide backbone, serpin exposed loop). During evolution PEDF might have lost its inhibitory activity and gained its neurotrophic function. Particular activities on other serpins have been reported (e.g. angiotensinogen, maspin, etc.). PEDF is an example of the separation of inhibitory and particular activities in a serpin.

Amino Acid Sequence↗

PubMed1996

Recombinant human pigment epithelium-derived factor (PEDF): characterization of PEDF overexpressed and secreted by eukaryotic cells.

Pigment epithelium-derived factor (PEDF) is a serpin found in the interphotoreceptor matrix of the eye, which, although not a proteinase inhibitor, possesses a number of important biological properties, including promotion of neurite outgrowth and differential expression in quiescent versus senescent states of certain cell types. The low amounts present in the eye, together with the impracticality of using the eye as a source for isolation of the human protein, make it important to establish a system for overexpression of the recombinant protein for biochemical and biological studies. We describe here the expression and secretion of full-length glycosylated human recombinant PEDF at high levels (> 20 micrograms/ mL) into the growth medium of baby hamster kidney cells and characterization of the purified rPEDF by circular dichroism and fluorescence spectroscopies and neurite outgrowth assay. By these assays, the recombinant protein behaves as expected for a correctly folded full-length human PEDF. The availability of milligram amounts of PEDF has permitted quantitation of its heparin binding properties and of the effect of reactive center cleavage on the stability of PEDF towards thermal and guanidine hydrochloride denaturation.

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PubMed1996

Proteolytic activity directed toward pigment epithelium-derived factor in vitreous of bovine eyes. Implications of proteolytic processing.

PURPOSE: Experiments were designed to identify proteolytic activities that cleave pigment epithelium-derived factor (PEDF), a member of the serpin (serine protease inhibitor) family. METHODS: Proteins in vitreous humor from bovine eyes were analyzed by Western blot with antiserum to human recombinant PEDF protein. Protein fractionation was by ammonium sulfate saturation and by S-Sepharose column chromatography. Proteolytic activities were determined by gelatin zymography and by solution assays against PEDF or chromogenic peptide substrates. RESULTS: PEDF protein was identified and purified to near homogeneity from vitreous humor of bovine eyes. Limited proteolysis showed that the vitreal protein has a protease-sensitive region at its serpin-exposed peptide loop. Proteolytic activities that cleave the PEDF 49.5 kDa-polypeptide were identified only when proteins from these extracts were separated by 45% to 70% ammonium sulfate fractionation (P70). The degradation product had an apparent molecular weight of 46 kDa. This result is consistent with cleavage at the serpin-exposed loop. The PEDF-cleavage activity in P70 was inhibited specifically by the serine protease inhibitor 4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF), but not by aprotinin, EDTA, or pepstatin. The vitreal P70 extracts contained 49- and 53-kDa gelatinolytic activities that also were inhibited by AEBSF and not by EDTA, aprotinin, or pepstatin. The PEDF-cleavage activity did not hydrolyze substrates for thrombin, factor Xa, alpha-chymotrypsin, trypsin, or plasmin, nor did it immunoreact with antibody to urokinase plasminogen activator. CONCLUSIONS: These data indicated that vitreous has a serine-proteolytic activity associated with a novel 49/53-kDa enzyme that cleaves the PEDF protein in a serpinase fashion. In addition to cleavage in vitro, these proteases might play a role in modulating PEDF in vivo.

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PubMed1996

Gene expression of the neurotrophic pigment epithelium-derived factor in the human ciliary epithelium. Synthesis and secretion into the aqueous humor.

PURPOSE: To study the expression of the neurotrophic pigment epithelium-derived factor (PEDF), a protein with neurotrophic and neuronal-survival activities, by the human ocular ciliary epithelium. METHODS: Total RNA extracted from human and bovine ocular tissues were screened by Northern blot analysis with cDNA probes for PEDF. Antibodies to PEDF were used to monitor its synthesis and secretion by metabolically labeling ciliary processes in vitro with 35S-methionine, followed by immunoprecipitation. Pigment epithelium-derived factor antibodies also were used to visualize the cellular distribution of PEDF along the human and bovine ciliary epithelium. Polymerase chain reaction (PCR) and reverse transcription (RT)-PCR was used to screen cDNA libraries of tissue and cell lines derived from the ciliary epithelium to demonstrate PEDF expression. RESULTS: From a subtractive library of the human ocular ciliary body, the authors identified a cDNA clone exhibiting nucleotide homology with the PEDF. Northern blot analysis indicated that PEDF transcripts are present in all the ocular tissues in the human eye; in the bovine eye, it is expressed preferentially in the retinal pigment epithelium. RT-PCR and PCR demonstrated that the PEDF gene is still transcriptionally active in cultured cell lines derived from the bilayer of the ciliary epithelium. Immunoprecipitation and Western blot (immunoblot) analyses with antisera to the PEDF protein demonstrated that a predominant PEDF form of 46 kDa is synthesized in the ciliary body and is secreted as a glycoprotein of 50 kDa. By indirect immunofluorescence and immunocytochemistry, PEDF antibodies decorated both cell types that comprise the ciliary epithelium (nonpigmented and pigmented) and, more distinctively, the plasma-membrane domain of nonpigmented cells in the pars plicata region. CONCLUSIONS: These results reveal a new site of synthesis (ciliary epithelium) and accumulation (aqueous humor) of PEDF, and they emphasize its potential importance as a trophic factor in the neuro-differentiated functions of the human ciliary epithelium.

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PubMed1995

Pigment epithelium-derived factor behaves like a noninhibitory serpin. Neurotrophic activity does not require the serpin reactive loop.

Pigment epithelium-derived factor (PEDF), a neurite-promoting factor, has an amino acid primary structure that is related to members of the serine protease inhibitor (serpin) family. Controlled proteolysis of native PEDF (50 kDa) with either trypsin, chymotrypsin, elastase, or subtilisin yields in each case one major limited product of 46 kDa as analyzed by SDS-polyacrylamide gel electrophoresis. N-terminal sequence analysis of the isolated 46-kDa products indicates a favored cleavage region located toward the C-terminal end of PEDF. A proteolyzed PEDF protein reaction mixture reveals two overlapping sequences: that of the N terminus of intact PEDF and that of an internal region, consistent with cleavage of PEDF about position 382. These data indicate that PEDF protein has a globular conformation with one protease-sensitive exposed loop that contains the homologous serpin-reactive site. Cleavage within the reactive-site loop of PEDF does not cause a conformational change in the molecules (the stressed (S)-->relaxed (R) transition) and results in heat denaturation identical to its native counterpart. This lack of conformational change is also seen upon cleavage within the reactive-site loop of the noninhibitory serpin ovalbumin. Furthermore, the PEDF neurite-promoting function is not lost with cleavage of the exposed loop. Recombinant PEDF polypeptide fragments with larger truncations from the C-terminal end show neurotrophic activity. Our results clearly indicate that integrity of the PEDF homologous serpin reactive center is dispensable for neurotrophic activity. Thus, the PEDF induction of neurites must be mediated by a mechanism other than serine protease inhibition. Altogether our data indicate that PEDF belongs to the subgroup of noninhibitory serpins and that its N-terminal region confers a neurite-promoting activity to the protein. The neurotrophic active site of PEDF is separated from the serpin reactive-site loop, not only in the primary structure, but also in the folded protein structure.

Amino Acid Sequence↗

PubMed1995

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.

Amino Acid Sequence↗

PubMed1995

Pigment epithelium-derived factor is a survival factor for cerebellar granule cells in culture.

Pigment epithelium-derived factor (PEDF), purified from human fetal retinal pigment epithelium cell culture medium, was shown to potentiate the differentiation of human Y-79 retinoblastoma cells. To investigate potential neurotrophic effects of PEDF on neurons other than those of retinal derivation, we used cultures of cerebellar granule cells. The number of cerebellar granule cells was significantly larger in the presence of PEDF, as demonstrated by an assay for viable cells that uses 3-(4,5-dimethylthiazol-2-yl)-5-(3- carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt, conversion, by cell count, and by immunocytochemistry. The effect of PEDF showed a dose-response relationship, with a larger effect in chemically defined medium than in serum-containing medium [ED50 = 30 ng/ml (0.70 nM) in chemically defined medium and 100 ng/ml (2.3 nM) in serum-containing medium]. PEDF had no effect on incorporation of bromodeoxyuridine (cell proliferation) or on neurofilament content (neurite outgrowth) measured by an enzyme-linked immunoadsorbent assay. These results demonstrate that PEDF has a neurotrophic survival effect on cerebellar granule cells in culture and suggest the possibility that it may affect other CNS neurons as well.

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