Pigment Epithelium-derived Factor (PEDF) Prevents Retinal Cell Death via PEDF Receptor (PEDF-R): IDENTIFICATION OF A FUNCTIONAL LIGAND BINDING SITE*

The extracellular pigment epithelium-derived factor (PEDF) displays retina survival activity by interacting with receptor proteins on cell surfaces. We have previously reported that PEDF binds and stimulates PEDF receptor (PEDF-R), a transmembrane phospholipase. However, the PEDF binding site of PEDF-R and its involvement in survival activity have not been identified. The purpose of this work is to identify a biologically relevant ligand-binding site on PEDF-R. PEDF bound the PEDF-R ectodomain L4 (Leu¹⁵⁹–Met³²⁵) with affinity similar to the full-length PEDF-R (Met¹–Leu⁵⁰⁴). Binding assays using synthetic peptides spanning L4 showed that PEDF selectively bound E5b (Ile¹⁹³–Leu²³²) and P1 (Thr²¹⁰–Leu²⁴⁹) peptides. Recombinant C-terminal truncated PEDF-R4 (Met¹–Leu²³²) and internally truncated PEDF-R and PEDF-R4 (ΔHis²⁰³–Leu²³²) retained phospholipase activity of the full-length PEDF-R. However, PEDF-R polypeptides without the His²⁰³–Leu²³² region lost the PEDF affinity that stimulated their enzymatic activity. Cell surface labeling showed that PEDF-R is present in the plasma membranes of retina cells. Using siRNA to selectively knock down PEDF-R in retina cells, we demonstrated that PEDF-R is essential for PEDF-mediated cell survival and antiapoptotic activities. Furthermore, preincubation of PEDF with P1 and E5b peptides blocked the PEDF·PEDF-R-mediated retina cell survival activity, implying that peptide binding to PEDF excluded ligand-receptor interactions on the cell surface. Our findings establish that PEDF-R is required for the survival and antiapoptotic effects of PEDF on retina cells and has determinants for PEDF binding within its L4 ectodomain that are critical for enzymatic stimulation.     

Pigment epithelium-derived factor (PEDF) shares binding sites in collagen with heparin/heparan sulfate proteoglycans

Pigment epithelium-derived factor (PEDF) is a collagen-binding protein that is abundantly distributed in various tissues, including the eye. It exhibits various biological functions, such as anti-angiogenic, neurotrophic, and neuroprotective activities. PEDF also interacts with extracellular matrix components such as collagen, heparan sulfate proteoglycans (HSPGs), and hyaluronan. The collagen-binding property has been elucidated to be important for the anti-angiogenic activity in vivo (Hosomichi, J., Yasui, N., Koide, T., Soma, K., and Morita, I. (2005) Biochem. Biophys. Res. Commun. 335, 756-761). Here, we investigated the collagen recognition mechanism by PEDF. We first narrowed down candidate PEDF-binding sequences by taking advantage of previously reported structural requirements in collagen. Subsequent searches for PEDF-binding sequences employing synthetic collagen-like peptides resulted in the identification of one of the critical binding sites for PEDF, human α1(I)(929-938) (IKGHRGFSGL). Further analysis revealed that the collagen recognition by PEDF is sequence- and conformation-specific, and the high affinity binding motif is KGXRGFXGL in the triple helix. The PEDF-binding motif significantly overlapped with the heparin/HSPG-binding motif, KGHRG(F/Y). The interaction of PEDF with collagen I was specifically competed with by heparin but not by chondroitin sulfate-C or hyaluronan. The binding sequences for PEDF and heparin/HSPG also overlapped with the covalent cross-linking sites between collagen molecules. These findings imply a functional relationship between PEDF and HSPGs during angiogenesis, and the interaction of these molecules is regulated by collagen modifications.     

Small Retinoprotective Peptides Reveal a Receptor-binding Region on Pigment Epithelium-derived Factor

The cytoprotective effects of pigment epithelium-derived factor (PEDF) require interactions between an as of a yet undefined region with a distinct ectodomain on the PEDF receptor (PEDF-R). Here we characterized the area in PEDF that interacts with PEDF-R to promote photoreceptor survival. Molecular docking studies suggested that the ligand binding site of PEDF-R interacts with the neurotrophic region of PEDF (44-mer, positions 78–121). Binding assays demonstrated that PEDF-R bound the 44-mer peptide. Moreover, peptide P1 from the PEDF-R ectodomain had affinity for the 44-mer and a shorter fragment within it, 17-mer (positions 98–114). Single residue substitutions to alanine along the 17-mer sequence were designed and tested for binding and biological activity. Altered 17-mer[R99A] did not bind to the P1 peptide, whereas 17-mer[H105A] had higher affinity than the unmodified 17-mer. Peptides 17-mer, 17-mer[H105A], and 44-mer exhibited cytoprotective effects in cultured retina R28 cells. Intravitreal injections of these peptides and PEDF in the rd1 mouse model of retinal degeneration decreased the numbers of dying photoreceptors, 17-mer[H105A] being most effective. The blocking peptide P1 hindered their protective effects both in retina cells and in vivo. Thus, in addition to demonstrating that the region composed of positions 98–114 of PEDF contains critical residues for PEDF-R interaction that mediates survival effects, the findings reveal distinct small PEDF fragments with neurotrophic effects on photoreceptors.     

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.     

Ablation of pigment epithelium-derived factor receptor (PEDF-R/Pnpla2) causes photoreceptor degeneration

Photoreceptor cells express the patatin-like phospholipase domain-containing 2 (PNPLA2) gene that codes for pigment epithelium-derived factor receptor (PEDF-R) (also known as ATGL). PEDF-R exhibits phospholipase activity that mediates the neurotrophic action of its ligand PEDF. Because phospholipids are the most abundant lipid class in the retina, we investigated the role of PEDF-R in photoreceptors by generating CRISPR Pnpla2 knock-out mouse lines in a retinal degeneration-free background. Pnpla2^−/− mice had undetectable retinal Pnpla2 gene expression and PEDF-R protein levels as assayed by RT-PCR and immunofluorescence, respectively. The photoreceptors of mice deficient in PEDF-R had deformities as examined by histology and transmission electron microscopy. Pnpla2 knockdown diminished the PLA₂ enzymatic activity of PEDF-R in the retina. Lipidomic analyses revealed the accumulation of lysophosphatidyl choline-DHA and lysophosphatidyl ethanolamine-DHA in PEDF-R-deficient retinas, suggesting a possible causal link to photoreceptor dysfunction. Loss of PEDF-R decreased levels of rhodopsin, opsin, PKCα, and synaptophysin relative to controls. Pnpla2^−/− photoreceptors had surface-exposed phosphatidylserine, and their nuclei were TUNEL positive and condensed, revealing an apoptotic onset. Paralleling its structural defects, PEDF-R deficiency compromised photoreceptor function in vivo as indicated by the attenuation of photoreceptor a- and b-waves in Pnpla2^−/− and Pnpla2^+/− mice relative to controls as determined by electroretinography. In conclusion, ablation of PEDF-R in mice caused alteration in phospholipid composition associated with malformation and malperformance of photoreceptors. These findings identify PEDF-R as an important component for photoreceptor structure and function, highlighting its role in phospholipid metabolism for retinal survival and its consequences.     

Pigment epithelium derived factor regulates human Sost/Sclerostin and other osteocyte gene expression via the receptor and induction of Erk/GSK-3beta/beta-catenin signaling

Mutations in Serpinf1 gene which encodes pigment epithelium derived factor (PEDF) lead to osteogenesis imperfecta type VI whose hallmark is defective mineralization. We reported that PEDF suppressed expression of Sost/Sclerostin and other osteocyte related genes in mineralizing osteoblast cultures and suggested that this could be part of the mechanisms by which PEDF regulates matrix mineralization (Li et al. J Cellular Phys. 2014). We have used a long-term differentiated mineralizing osteoblast culture (LTD) to define mechanisms by which PEDF regulates osteocyte gene expression. LTD cultures were established by culturing human osteoblasts in an osteogenic medium for 4?months followed by analysis of osteocytes related genes and encoded proteins. LTD cells synthesized Sclerostin, matrix extracellular phosphoglycoprotein (MEPE) and dentin matrix protein (DMP-1) and their synthesis was reduced by treatment with PEDF. Treatment of the cultures with PEDF induced phosphorylation of Erk and glycogen synthase kinase 3-beta (GSK-3β), and accumulation of nonphosphorylated β-catenin. Inhibition of Erk activation and neutralizing antibodies to the pigment epithelium derived receptor (PEDF-R) suppressed GSK-3β phosphorylation and accumulation of nonphosphorylated β-catenin in presence of PEDF. Topflash assays demonstrated that PEDF activated luciferase reporter activity and this activity was inhibited by treatment with Erk inhibitor or neutralizing antibodies to PEDF-R. Dickkopf-related protein 1 treatment of the cells in presence of PEDF had minimal effect suggesting that GSK-3β phosphorylation and accumulation of nonphosphorylayted β-catenin may not involve LRP5/6 in osteocytes. Taken together, the data demonstrate that PEDF regulates osteocyte gene expression through its receptor and possible involvement of Erk/GSK-3β/β-catenin signaling pathway.     

Identification of different receptor types for toxic phospholipases A2 in rabbit skeletal muscle.

Oxyuranus scutellatus scutellatus toxins 1 (OS1) and 2 (OS2) are two phospholipase A2S (PLA2) isolated from the venom of the Australian Taipan snake. Their iodinated derivatives have been used to characterize PLA2 binding sites on rabbit skeletal muscle. Competition and cross-linking experiments indicate that 125I-labelled OS2 binding sites in rabbit skeletal muscle in vivo are distributed into two classes of receptors. One class binds OS2 and OS1 and is insensitive to the bee venom PLA2. It is composed of a 180 kDa binding protein. This class of PLA2 receptor is expressed at a high level in rabbit myotube membranes. The other class of PLA2 receptor identified with 125I-OS2 also binds with high affinity the bee venom PLA2 but not OS1 and is composed of major polypeptides of 34, 48 and 82 kDa. This second class of receptor is similar to the one found in brain membranes. The density of the two classes of receptors varies during muscle development.     

Some properties of prostaglandin E2 receptors in rabbit alveolar bone cell membranes

[3H]prostaglandin E2 (PGE2) binding receptors exist in rabbit alveolar bone cell membranes. The presence of high (Kd = 3.9 X 10(-9) M) and low (Kd = 8.8 X 10(-8) M) affinity binding sites of [3H]PGE2 was demonstrated. The saturation values of [3H]PGE2 for high and low affinity binding sites were 0.13 pmol/mg protein and 1.22 pmol/mg protein, respectively. The digestion of the membranes with pronase, phospholipase C, D and neuraminidase led to a decrease of [3H]PGE2 binding but phospholipase A2 did not.     

Identification of a soluble form phospholipase A2 receptor as a circulating endogenous inhibitor for secretory phospholipase A2

Venomous snakes have various types of phospholipase A(2) inhibitory proteins (PLIs) in their circulatory system to protect them from attack by their own phospholipase A(2)s (PLA(2)s). Here we show the first evidence for the existence of circulating PLI against secretory PLA(2)s (sPLA(2)s) in mammals. In mouse serum, we detected specific binding activities of group IB and X sPLA(2)s, which was in contrast with the absence of binding activities in serum prepared from mice deficient in PLA(2) receptor (PLA(2)R), a type I transmembrane glycoprotein related to the C-type animal lectin family. Western blot analysis after partial purification with group IB sPLA(2) affinity column confirmed the identity of serum sPLA(2)-binding protein as a soluble form of PLA(2)R (sPLA(2)R) that retained all of the extracellular domains of the membrane-bound receptor. Both purified sPLA(2)R and the recombinant soluble receptor having all of the extracellular portions blocked the biological functions of group X sPLA(2), including its potent enzymatic activity and its binding to the membrane-bound receptor. Protease inhibitor tests with PLA(2)R-overexpressing Chinese hamster ovary cells suggested that sPLA(2)R is produced by cleavage of the membrane-bound receptor by metalloproteinases. Thus, sPLA(2)R is the first example of circulating PLI that acts as an endogenous inhibitor for enzymatic activities and receptor-mediated functions of sPLA(2)s in mice.     

Differential Sensitivity of "Central" and "Peripheral" Type Benzodiazepine Receptors to Phospholipase A2

The effects of preincubating cerebral cortical membranes with phospholipase A2 (PLA2) were examined on radioligand binding to benzodiazepine receptors of the "central" and "peripheral" types. PLA2 (0.005-0.1 U/ml) increased [3H]flunitrazepam and [3H]3-carboethoxy-beta-carboline binding by increasing the apparent affinities of these ligands with no concomitant change in the maximum number of binding sites. In contrast, neither gamma-aminobutyric acid (GABA)-enhanced [3H]flunitrazepam binding nor [3H]Ro 15-1788 binding was altered by preincubation with PLA2 at concentrations as high as 2 U/ml. Both pyrazolopyridine (SQ 65,396)- and barbiturate (pentobarbital)-enhanced [3H]flunitrazepam binding and [35S]t-butylbicyclophosphorothionate (TBPS) binding were markedly reduced by as little as 0.0025-0.005 U/ml of PLA2. These findings suggest that PLA2 inactivates the TBPS binding site on the benzodiazepine-GABA receptor chloride ionophore complex, which results in a selective loss of allosteric "regulation" between the components of this complex. PLA2 also reduced the apparent affinity of [3H]Ro 5-4864 to peripheral-type benzodiazepine receptors in cerebral cortical, heart, and kidney membranes, but increased the number of [3H]PK 11195 binding sites with no change in apparent affinity. These data demonstrate that PLA2 can differentially affect the lipid microenvironment of "central" and "peripheral" types of benzodiazepine receptors.     

Catalytically inactive phospholipase A2 homologue binds to vascular endothelial growth factor receptor-2 via a C-terminal loop region

VEGF (vascular endothelial growth factor) regulates neovascularization through binding to its receptor KDR (kinase insert domain-containing receptor; VEGF receptor-2). We recently identified a catalytically inactive PLA(2) (phospholipase A(2)) homologue (KDR-bp) in the venom of eastern cottonmouth (Agkistrodon piscivorus piscivorus) as a third KDR-binding protein, in addition to VEGF(165) and tissue inhibitor of metalloproteinase-3. KDR-bp binds to the extracellular domain of KDR with a K(d) of 10(-8) M, resulting in specific blockade of endothelial cell growth induced by VEGF(165). Inactive PLA(2) homologues are widely distributed in the venoms of Viperidae snakes and are known to act as myotoxins. In the present study, we demonstrated that KDR-binding ability is a common characteristic for inactive PLA(2) homologues in snake venom, but not for active PLA(2)s such as neurotoxic and platelet aggregation-modulating PLA(2)s. To understand better the KDR and KDR-bp interaction, we resolved the binding region of KDR-bp using eight synthetic peptides designed based on the structure of KDR-bp. A synthetic peptide based on the structure of the C-terminal loop region of KDR-bp showed high affinity for KDR, but other peptides did not, suggesting that the C-terminal loop region of KDR-bp is involved in the interaction with KDR. The results of the present study provide insight into the binding of inactive PLA(2) homologues to KDR, and may also assist in the design of novel anti-KDR molecules for anti-angiogenic therapy.     

Enhanced binding of phospholipase-A2-modified low density lipoprotein by human adipocytes

Recognition of low density lipoprotein (LDL) by human adipocytes is not dependent on the classical LDL (apoprotein B-E) receptor. To assess whether LDL phospholipids have a role in adipocyte-LDL interactions, binding studies were carried out with human LDL modified with cobra venom phospholipase A2 (PLA2) and freshly isolated adipocytes and purified adipocyte plasma membranes prepared from surgical biopsies. LDL incubated with PLA2 showed increased monoacylphospholipid content, decreased diacylphospholipid content, and increased anodic migration on agarose gel electrophoresis. LDL cholesterol, triglyceride, and protein content remained unchanged. Typically, modification of 16 and 47% of LDL phospholipids enhanced specific binding of 125I-labelled LDL to plasma membranes progressively from 3.1 micrograms LDL bound/mg membrane protein (control) to 5.8 and 28.2 micrograms LDL bound/mg membrane protein, respectively. Nonspecific binding was not altered significantly. Excess unlabelled native LDL and high density lipoprotein (HDL3) effectively inhibited binding of PLA2-modified LDL. Freshly isolated adipocytes also showed enhanced binding and uptake of PLA2-modified LDL (0.1 vs. 0.9 micrograms LDL/10(6) cells x 2 h), control vs. modified). The results demonstrate that alterations of LDL phospholipids significantly enhance LDL binding and suggest a regulatory role for phospholipids in lipoprotein-cell interaction. Furthermore, the results support the view that human adipose tissue may be involved in the metabolism of modified lipoproteins, in vivo.     

Roles of Trp31 in high membrane binding and proinflammatory activity of human group V phospholipase A2

Group V phospholipase A2 is a recently discovered secretory phospholipase A2 (PLA2) that has been shown to be involved in eicosanoid formation in inflammatory cells, such as macrophages and mast cells. We have demonstrated that human group V PLA2 (hsPLA2-V) can bind phosphatidylcholine (PC) membranes and hydrolyze PC substrates much more efficiently than human group IIa PLA2, which makes it better suited for acting on the outer plasma membrane (Han, S.-K., Yoon, E. T., and Cho, W. (1998) Biochem. J. 331, 353-357). In this study, we demonstrate that exogenous hsPLA2-V has much greater activity than does group IIa PLA2 to release fatty acids from various mammalian cells and to elicit leukotriene B4 formation from human neutrophils. To understand the molecular basis of these activities, we mutated two surface tryptophans of hsPLA2-V to alanine (W31A and W79A) and measured the effects of these mutations on the kinetic activity toward various substrates, on the binding affinity for vesicles and phospholipid-coated beads, on the penetration into phospholipid monolayers, and on the activity to release fatty acids and elicit eicosanoid formation from various mammalian cells. These studies show that the relatively high ability of hsPLA2-V to induce cellular eicosanoid formation derives from its high affinity for PC membranes and that Trp31 on its putative interfacial binding surface plays an important role in its binding to PC vesicles and to the outer plasma membrane.     

Pigment epithelium-derived factor (PEDF) interacts with transportin SR2, and active nuclear import is facilitated by a novel nuclear localization motif

PEDF (Pigment epithelium-derived factor) is a non-inhibitory member of the serpin gene family (serpinF1) that displays neurotrophic and anti-angiogenic properties. PEDF contains a secretion signal sequence, but although originally regarded as a secreted extracellular protein, endogenous PEDF is found in the cytoplasm and nucleus of several mammalian cell types. In this study we employed a yeast two-hybrid interaction trap screen to identify transportin-SR2, a member of the importin-β family of nuclear transport karyopherins, as a putative PEDF binding partner. The interaction was supported in vitro by GST-pulldown and co-immunoprecipitation. Following transfection of HEK293 cells with GFP-tagged PEDF the protein was predominantly localised to the nucleus, suggesting that active import of PEDF occurs. A motif (YxxYRVRS) shared by PEDF and the unrelated transportin-SR2 substrate, RNA binding motif protein 4b, was identified and we investigated its potential as a nuclear localization signal (NLS) sequence. Site-directed mutagenesis of this helix A motif in PEDF resulted in a GFP-tagged mutant protein being excluded from the nucleus, and mutation of two arginine residues (R67, R69) was sufficient to abolish nuclear import and PEDF interaction with transportin-SR2. These results suggest a novel NLS and mechanism for serpinF1 nuclear import, which may be critical for anti-angiogenic and neurotrophic function.     

Effects of trypsin on binding of [3H]epinephrine and [3H]-dihydroergocryptine to rat liver plasma membranes. Evidence for interconversion of binding sites

Treatment of liver plasma membranes with trypsin at low concentrations (1 to 2 microgram/mg of protein) caused at 3- to 4-fold increase in alpha-specific [3H]epinephrine binding. The change was due to an increase in the number of high affinity binding sites, with no change in the dissociation constant. With increasing trypsin concentrations, the dissociation constant was decreased and there was a progressive loss of binding. Elastase, papain, and thermolysin caused similar effects, whereas the thrombin, leucine aminopeptidase, phospholipase A2, phospholipase C, phospholipase D, and detergents did not cause an increase in [EH]epinephrine binding. The increase in epinephrine high affinity binding sites was correlated with a loss of high affinity [3H]-dihydroergocryptine binding sites which also bind [3H]epinephrine with low affinity (El-Refai, M. F., Blackmore, P. F., and Exton, J. H. (1979) J. Biol. Chem. 254, 4375-4386). Incubation of membranes with the alpha blockers dihydroergocryptine (50 nM) and phenoxybenzamine (20 nM) prior to protease treatment diminished the increase in [3H]epinephrine binding induced by trypsin (1.5 microgram/mg). The concentration dependence and time course of trypsin actions on 70 nM [3H]epinephrine binding and 10 nM [3H]dihydroergocryptine binding are consistent with a trypsin-mediated conversion of low affinity epinephrine binding sites to high affinity epinephrine binding sites.     

Pigment epithelium-derived factor binds to hyaluronan. Mapping of a hyaluronan binding site

Pigment epithelium-derived factor (PEDF) is a multifunctional serpin with antitumorigenic, antimetastatic, and differentiating activities. PEDF is found within tissues rich in the glycosaminoglycan hyaluronan (HA), and its amino acid sequence contains putative HA-binding motifs. We show that PEDF coprecipitation with glycosaminoglycans in media conditioned by human retinoblastoma Y-79 cells decreased after pretreatments with hyaluronidase, implying an association between HA and PEDF. Direct binding of human recombinant PEDF to highly purified HA was demonstrated by coprecipitation in the presence of cetylpyridinium chloride. Binding of PEDF to HA was concentration-dependent and saturable. The PEDF-HA interactions were sensitive to increasing NaCl concentrations, indicating an ionic nature of these interactions and having affinity higher than PEDF-heparin. Competition assays showed that PEDF can bind heparin and HA simultaneously. PEDF chemically modified with fluorescein retained the capacity for interacting with HA but lacked heparin affinity, suggesting one or more distinct HA-binding regions on PEDF. The HA-binding region was examined by site-directed mutagenesis. Single-point and cumulative alterations at basic residues within the putative HA-binding motif K189A/K191A/R194A/K197A drastically reduced the HA-binding activity without affecting heparin- or collagen I binding of PEDF. Cumulative alterations at sites critical for heparin binding (K146A/K147A/R149A) decreased HA affinity but not collagen I binding. Thus these clusters of basic residues (BXBXXBXXB and BX3AB2XB motifs) in PEDF are functional regions for binding HA. In the spatial PEDF structure they are located in distinct areas away from the collagen-binding site. The HA-binding activity of PEDF may contribute to deposition in the extracellular matrix and to its reported antitumor/antimetastatic effects.     

Purification and characterization of a high-affinity binding protein for pancreatic-type phospholipase A2.

A high-affinity and specific binding site for mammalian group I phospholipase A2 (PLA2-I) was found on the membranes of bovine corpus luteum. Affinity labeling experiments revealed that PLA2-I binds to a single polypeptide with a mass of 190-200 kDa. The PLA2-I binding protein in the membranes was solubilized in an active form with n-octyl beta-D-thioglucoside, and then purified approx. 16,000-fold. The purification procedures consisted of diethylaminoethyl-Sephacel chromatography, PLA2-I-affinity gel chromatography and gel-filtration high-performance liquid chromatography on a TSKgel G3,000SWXL column. The final preparation migrated as a single molecular species of 190 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and identification of the 190 kDa protein as the PLA2-I binding protein was demonstrated by ligand blotting analysis. The purified protein possessed a binding capacity with high affinity and specificity for a mammalian mature type of PLA2-I. Treatment of the purified material with N-glycosidase F resulted in increased mobility of the protein on SDS-PAGE as well as considerable abolition of the PLA2-I binding activity, thus suggesting the requirement of the carbohydrate moiety of the PLA2-I binding protein for receptor-ligand interactions.     

A novel prokaryotic phospholipase A2. Characterization, gene cloning, and solution structure

Until now, phospholipase A(2) (PLA(2); EC 3.1.14) has been found only from eukaryotic sources. In the present study, we found a secreted PLA(2), which is produced by a soil bacterium, Streptomyces violaceoruber A-2688, demonstrating that the enzyme is the first phospholipase A(2) identified in prokaryote. After characterization of the novel PLA(2), a gene encoding the enzyme was cloned, sequenced, and overexpressed using a Streptomyces host-vector system. The amino acid sequence showed that the prokaryotic PLA(2) has only four cysteines and less homology to the eukaryotic ones, which have 12-16 cysteines. The solution structures of the prokaryotic PLA(2), bound and unbound with calcium(II) ion, were determined by using the NMR technique and structure calculation. The overall structure of the S. violaceoruber PLA(2), which is composed of only five alpha-helices, is completely different from those of eukaryotic PLA(2)s, which consist of beta-sheets and alpha-helices. The structure of the calcium-binding domain is obviously distinct from that without the ion; the ligands for the calcium(II) ion are the two carboxylates of Asp(43) (monodentate) and Asp(65) (bidentate), the carbonyl oxygen of Leu(44), and three water molecules. A calcium-binding experiment showed that the calcium dissociation constant ( approximately 5 mm) for the prokaryotic PLA(2) is much larger than those of eukaryotic ones.     

Folate binding in intestinal brush border membranes: evidence for the presence of two binding activities

Binding of [(3)H]folic acid by isolated rat jejunal brush border membranes (BBMs) was analyzed by chromatography on small Biogel P-30 columns. Folic acid binding to BBMs exhibited a prominent pH effect with a sharp maximum at pH 5.5 to 6.0. After acid treatment to strip the BBMs of bound folate, the membranes demonstrated a wider pH optimum (5.5 to 7.5) of folate binding and a higher binding capacity. Scatchard analysis of binding experiments performed at pH 6.0 revealed the existence of two components: one with a high affinity (kd = 12 to 25 nM) and low capacity (V(max) for non-acidified BBMs = 0.259 to 0.264 pmol/mg protein, V(max) for acidified BBMs = 0.41 to 0.71 pmol/mg protein) and the other with a low affinity (kd = 1.1 to 5.1 microM and high capacity (V(max) for non-acidified BBMs = 0.93 to 1.93 pmol/mg protein, V(max) for acidified BBMs = 4.05 to 7.69 pmol/mg protein). Phosphatidylinositol-specific phospholipase C preferentially detached the high affinity component from jejunal BBMs. Phosphatidylinositol-specific phospholipase C-released folate binding protein was precipitated by antibodies to the high-affinity folate-binding protein from rat kidney. These data suggest the existence of two different folate-binding proteins in isolated rat jejunal BBMs. The high-affinity folate-binding protein shares epitopes with the folate-binding protein in the kidney.