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Reelin Is a Serine Protease of the Extracellular Matrix

Reelin Is a Serine Protease of the Extracellular Matrix THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 277, No. 1, Issue of January 4, pp. 303–309, 2002 © 2002 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Received for publication, July 24, 2001, and in revised form, October 18, 2001 Published, JBC Papers in Press, October 31, 2001, DOI 10.1074/jbc.M106996200 a,b,c d,e a d Carlo C. Quattrocchi, Francesca Wannenes, Antonio M. Persico, Silvia Anna Ciafre ´, f,g,h,i f a, j Gabriella D’Arcangelo, Maria G. Farace, and Flavio Keller From the Laboratory of Neuroscience, Department of Physiology and Neuroscience, Universita ` “Campus Bio-Medico,” Via Longoni 83, 00155 Roma, Italy, the Program in Neuroscience, Faculty of Medicine, University of Brescia, Via Valsabbina 19, 25123 Brescia, Italy, the Department of Experimental Medicine and Biochemical Sciences, Universita ` di Tor Vergata, Via di Tor Vergata 135, 00133 Roma, Italy, the Department of Internal Medicine, Universita ` di Tor Vergata, Via di Tor f g h Vergata 135, 00133 Roma, Italy, and The Cain Foundation Laboratories, Department of Pediatrics, Program in Developmental Biology and Division of Neuroscience, Baylor College of Medicine, Houston, Texas 77030 Reelin is an extracellular matrix protein that plays a contacts with the radial glial fibers (6). Several hypotheses have been suggested regarding the function of Reelin: (i) Reelin pivotal role in development of the central nervous sys- tem. Reelin is also expressed in the adult brain, notably may act as an attractant molecule for migrating neurons; (ii) it in the cerebral cortex, where it might play a role in may act as a repulsive molecule; or (iii) Reelin may interrupt synaptic plasticity. The mechanism of action of reelin at the association between migrating neurons and radial glia (7, the molecular level has been the subject of several hy- 8), thus allowing migrating neurons to switch from a “glio- potheses. Here we show that reelin is a serine protease philic” to a “neurophilic” state (9). Furthermore, Reelin has and that proteolytic activity is relevant to its function, been recently shown to be expressed in several adult neuronal since (i) Reelin expression in HEK 293T cells impairs cells, including glutamatergic cerebellar granule neurons and their ability to adhere to fibronectin-coated surfaces, specific GABAergic interneurons of the cerebral cortex and and adhesion to fibronectin is restored by micromolar hippocampus (10), and in the adult mammalian blood, liver, concentrations of diisopropyl phosphorofluoridate, a pituitary pars intermedia, and adrenal chromaffin cells (11, serine hydrolase inhibitor; (ii) purified Reelin binds FP- 12). The cellular function of Reelin in the adult organism is Peg-biotin, a trap probe which irreversibly binds to ser- unknown. Evidence is accumulating for involvement of Reelin ine residues located in active catalytic sites of serine in human diseases such as autosomal recessive lissencephaly hydrolases; (iii) purified Reelin rapidly degrades fi- (13), schizophrenia (14), and autistic disorder (15). bronectin and laminin, while collagen IV is degraded at The mouse Reelin sequence (1) encompasses 3461 amino a much slower rate; fibronectin degradation is inhibited acids and possesses a signal peptide followed by a domain with by inhibitors of serine proteases, and by monoclonal 28% sequence identity with F-spondin (as assessed by -Blast antibody CR-50, an antibody known to block the func- software), a protein secreted by floor plate cells and promoting tion of Reelin both in vitro and in vivo. The proteolytic cell adhesion and neurite growth (16). This region is followed activity of Reelin on adhesion molecules of the extracel- lular matrix and/or receptors on neurons may explain by a unique region with no sequence homology, and then by how Reelin regulates neuronal migration and synaptic eight internal repeats of 350 –390 amino acids, each repeat plasticity. containing two related subdomains flanking a cystein-rich se- quence similar to the epidermal growth factor-like motif. The carboxyl terminus region contains many positively charged Reelin (1, 2) is an extracellular matrix protein that plays a amino acids required for secretion (5). Human Reelin (2) is pivotal role in neuronal migration during development of lam- 94.8% identical to the mouse protein at the amino acid level, inar structures of the mammalian brain including the cerebral indicating strong functional conservation. Recent findings sug- cortex, hippocampus, cerebellum, and several brainstem nu- gest that the Reelin signal transduction involves binding to the clei, as shown by spontaneous Reelin null mutations (i.e. the very low density lipoprotein receptor and to apoE receptor 2 reeler mouse) (3, 4). In the developing cerebral cortex, Reelin is followed by intracellular activation of the adapter protein dis- secreted by Cajal-Retzius cells, located in the marginal zone. abled-1 (17–19). Other possible Reelin signal transduction Reelin must be secreted into the extracellular matrix to exert pathways may involve interaction with the   integrin recep- 3 1 its biological effect (5). tor (8, 9) and with cadherin-related neuronal receptors (20). In the reeler mouse, migrating neurons fail to pass through We have analyzed the primary amino acid sequence of hu- earlier-generated neurons, possibly because they are unable to man Reelin, and found several hints that Reelin might be a penetrate the subplate, or because they maintain extensive serine protease, since: (i) Reelin contains the sequence GKSDG (amino acids 1280 –1284 of human Reelin) (2), corresponding to the serine hydrolase consensus sequence GXSXG; this se- * This work was supported by Consiglio Nazionale delle Ricerche Programma “Biomolecole per la salute umana” Grant 99.00555.PF33. quence is 100% conserved among mouse, chicken, and human The costs of publication of this article were defrayed in part by the Reelin; (ii) Reelin shows significant structural similarities with payment of page charges. This article must therefore be hereby marked serine hydrolases, such as the extracellular serine protease “advertisement” in accordance with 18 U.S.C. Section 1734 solely to precursor (EC 3.4.21) of Serratia marcescens, and the probable indicate this fact. Present address: Dept. of Pediatrics-Neurology, Baylor College of ubiquitin carboxyl-terminal hydrolases FAM and FAF-Y (EC Medicine, 1102 Bates St., MC 3– 6365, Houston, TX 77030. 3.1.2.15); (iii) Reelin contains eight epidermal growth factor- To whom correspondence should be addressed: Laboratory of Neu- like repeats; epidermal growth factor-like repeats are observed roscience, Universita ` “Campus Bio-Medico,” Via Longoni 83, 00155 in serine proteases, for example, coagulation factors VII, IX, Roma, Italy. Tel.: 39-06-2254-1335; Fax: 39-06-22-54-14-56; E-mail: f.keller@unicampus.it. and X, and protein C, Z (21), calcium-dependent serine protein- This paper is available on line at http://www.jbc.org 303 This is an Open Access article under the CC BY license. 304 Reelin Is a Serine Protease of the Extracellular Matrix balanced salt solution. Attached cells were fixed with 4% formaldehyde ase (22) which degrades extracellular matrix proteins, and in PBS, pH 7.4, and counted manually using an inverted microscope by complement C1s and C1r components (23); (iv) several serine two different observers. The counting area was defined by a grid (12 hydrolases, such as lipoprotein lipase and the urokinase-type mm area) placed under the wells. Attached cells were defined as cells plasminogen activator, bind very low density lipoprotein recep- that had spread and grown at least 1 process. All data are expressed as tor and apoE receptor 2 (24, 25). In this study we present mean  S.E. Differences between groups were tested by one-way converging evidence that purified Reelin acts as a serine pro- ANOVA followed by the LSD post-hoc test, using the SPSS statistics package (SPSS, Chicago, IL, version 9.0). tease, and that this enzymatic activity may be relevant for its Gel Electrophoresis and Immunoblotting—Samples were mixed with cellular function. 2  sample buffer and heated at 100 °C for 2 min. Six percent resolving gel, pH 8.8, and 5% stacking gel, pH 6.8, were prepared and run in a EXPERIMENTAL PROCEDURES MiniProtean II assembly (Bio-Rad, Hercules, CA) according to standard Chemicals—Cell culture media, antibiotics, and media supplements protocols (29). Proteins were transferred to nitrocellulose filter were purchased from Invitrogen (Gaithersburg, MD). All other chemi- (Schleicher & Schuell, GmbH, Dassel, Germany) by a semi-dry blotting cals were from Sigma, unless otherwise specified. apparatus (Hoefer Scientific, Amersham Bioscience, Inc., San Fran- Amino acid Sequence Analysis of Reelin—All sequence analyses are cisco, CA) for 90 min at constant current of 70 mA. After blotting, the based on human Reelin (accession number: NP005036) (2). Homologies nitrocellulose filter was blocked for 30 min in tris-buffered saline with were evaluated by using -Blast 2 sequences software (www.ncbi.nih. 0.1% Tween 20 (TBST), containing 1% bovine serum albumin. Reelin gov/blast). Also, PropSearch software (EMBL, Heidelberg) was used to was revealed with 1:2000 mAb 142, overnight, followed by 1:5000 alka- find homologies with other serine hydrolases. PattinProt software line phosphatase-labeled anti-mouse IgG (Promega Italia, Milan, Italy). (PBIL, NPSA, Lyon) was used to identify putative consensus sequences Filters were developed with nitro blue tetrazolium-5-bromo-4-chloro-3- for serine proteases. M MgCl indolyl phosphate in Tris-HCl, pH 9.5, containing 5 m . The Cell Culture, Transfection, and Expression of Recombinant Reelin— M EDTA. Alternatively, reaction was stopped with PBS containing 2 m Human embryonic kidney (HEK) 293T cells (ATCC, Q401) were grown Reelin bands were revealed using a chemiluminescence method: filters in Dulbecco’s modified Eagle’s medium supplemented with 10% heat- were incubated with biotinylated anti-mouse secondary antibody (1: inactivated fetal bovine serum, penicillin/streptomycin (100 interna- 25.000 in TBST) for 1 h. Then, after three washes, 1:500 peroxidase- tional units/ml and 100 g/ml, respectively), and 10 mML-glutamine. conjugated streptavidin (CHEMICON Int., Temecula, CA) was added All cells were cultured at 37 °C, 5% CO and 97% relative humidity. for 15 min. Finally filters was washed, incubated in a freshly made Passaging was routinely performed with trypsin-EDTA. Cells were solution containing 200 mM Tris-HCl, pH 8.5, 250 mM 3-aminophthal- stably transfected with 2 g of plasmid DNA pCrl (5), which contains hydrazide, 40 mM p-coumaric acid, and 0.0005% H O , dried, and ex- 2 2 the entire mouse Reelin open reading frame (accession number posed to Kodak X-OMAT film. Filters or films were scanned with a NP_035391), or with 2 g of pCDNA3 empty vector in 10 l of Lipo- SCANJET ADF digital scanner using Twain-32 software (Hewlett- fectAMINE in 4 ml of Opti-MEM serum-free medium, according to the Packard). The specificity of staining was checked by preincubating mAb manufacturer’s instructions. Cells were transfected in 60-mm plates at 142 with protein SP, the Reelin fragment recognized by mAb 142 (27). a density of 5  10 /dish and transferred to selection medium (0.6 Labeling of Reelin with FP-Peg-biotin—Fluorophosphate biotin (FP- mg/ml G418) 48 h after transfection. For Reelin purification, a stable M Peg-biotin) (a gift of Dr. Benjamin Cravatt) (30), stored as a 100 m cell line (CER) was generated by stable transfection of 293-EBNA cells stock solution in Me SO at 20 °C, was added directly to protein (Invitrogen) with pCER followed by selection in medium containing M. The reaction mixture was samples to a final concentration of 2– 4 0.25 mg/ml G418 and 0.4 mg/ml hygromycin B. The pCER episomal incubated at room temperature for 30 min, and stopped by adding an plasmid contains a reelin insert identical to that of pCrl, cloned into the equal volume of 2  reducing sample buffer. As a control for the pCEP4 vector (Invitrogen). specificity of FP-Peg-biotin labeling, replica samples were incubated for Cultures were harvested for RNA preparation (26). Total RNA (3 g) 1 h with 11.4 M diisopropylphosphofluoridate (DFP), a potent and was incubated in reaction buffer containing 5 M random hexamer specific serine-hydrolase inhibitor, before incubation with FP-Peg-bio- (Amersham Bioscience, Inc.), Moloney murine leukemia virus reverse tin. Samples were separated by SDS-PAGE and transferred by electro- transcriptase, reaction buffer, and RNase inhibitor, according to the blotting to nitrocellulose membranes; the membranes were blocked in manufacturer’s specifications (Invitrogen). Reverse transcription was TBST with 3% bovine serum albumin for1hat25 °C or overnight at performed for1hat37 °C, and stopped by incubating the samples for 4 °C, and then incubated for 15 min with an avidin-horseradish perox- 10 min at 95 °C. Expression of Reelin was checked by PCR amplification idase conjugate (Pierce) diluted 1:300 in blocking solution. The labeled with specific primers: forward 5-GGAAAGTCAGATGGAGAC-3, re- bands were revealed by chemiluminescence (see above). verse 5-CATCTAAGCCAAACG-3 corresponding to nucleotides 4123– Purification of Reelin—For gel filtration chromatography purifica- 4498 of mouse Reelin mRNA (Ref. (1), accession number U24703). PCR tion, 50 l of supernatant from Reelin secreting CER cells were con- amplification was carried out in a total volume of 50 l, with 10 lof centrated first by osmotic dyalisis with AQUACIDE I (Calbiochem, La reverse transcription reaction and 1.25 units of Taq polymerase (M- Jolla, CA). A 5-ml concentrated sample was loaded on a FPLC system Medical Genenco-Life Science). Amplification was performed for 35 AKTAprime and passed through a HiLoad Superdex 200 26/60 column cycles (1 min denaturing at 94 °C, 1 min annealing at 58 °C, and 1 min (Amersham Bioscience, Inc., Uppsala, Sweden). The run was performed extension at 72 °C) in a thermal cycler (Oracle BioSystems, Delphi at a constant flow rate of 3 ml/min and maximal pressure limit of 0.5 Pa. 1500). The 376-bp amplified product was analyzed by 2% agarose gel The eluate was collected in 1.5-ml fractions. After dot immunoblot electrophoresis and visualized under UV illumination after staining screening and Western blot, positive fractions for Reelin were pooled with ethidium bromide. Reelin secretion into supernatants was ana- and concentrated again as described above. lyzed by plating 1  10 cells onto 90-mm dishes. After 24 h conditioned For purification by SDS-PAGE, Reelin-containing supernatant from media were removed, cells were washed twice with phosphate-buffered transfected 293T cells was concentrated as above and separated on a 5% saline (PBS), and serum-free Dulbecco’s modified Eagle’s medium was gel. The gel was run for3h30minat125Vto achieve a good separation in the 250 kDa range. Thereafter a thin vertical slice of gel was cut added. Forty-eight hours later, the supernatants were collected, cleared and stained with silver to reveal the 400-kDa Reelin band. A 5-mm wide by a brief centrifugation (10 min, 1000 rpm) at 4 °C, concentrated by horizontal slice was cut from the remaining gel, using the stained slice Microcon YM-100 (Millipore Corp. Bedford, MA) and stored at 80 °C as a reference, and 400-kDa Reelin was electroeluted at 60 V into 25 mM for analysis by SDS-PAGE and immunoblotting. Reelin secretion into Tris, 250 mM glycine, 0,1% SDS buffer. Electroelution was carried out the cell culture medium was assessed by Western blots using mAb 142, overnight at 4 °C. Purified Reelin was, then, transferred to PBS, pH 7.2, an antibody that recognizes the NH -terminal sequence of Reelin (27). by overnight dialysis in a Slide-A-Lyzer cassette (Pierce). Finally the Cell Adhesion Assay—Cell adhesion assay was performed according protein was concentrated. The final protein concentration was esti- to published protocols (28). Ninety-six-well plates were coated over- mated to be 0.2 g/ml (Bradford Reagent, Sigma). Purified Reelin was night with fibronectin (2.5 g/ml) in carbonate/bicarbonate buffer, pH re-electrophoresed on a SDS gel to check the purity of the protein. 9.7. Cells were harvested, washed three times with serum-free Dulbec- co’s modified Eagle’s medium, and then resuspended in sterile attach- ment solution (calcium- and magnesium-free Hanks’ balanced salt so- lution, 20 mM HEPES, 1 mg/ml heat-inactivated bovine serum albumin, The abbreviations used are: DFP, diisopropylphosphorofluoridate; 1mM CaCl ,1mM MgCl . Mock- and pCrl-transfected 293T cells (1 HEK, human embryonic kidney; mAb, monoclonal antibody; PBS, phos- 2 2 10 in 200 l/well) were allowed to attach for2hat37 °C in a humid- phate-buffered saline; PMSF, phenylmethylsulfonyl fluoride; tPA, tis- ified 5% CO incubator. Unattached cells were removed with Hanks’ sue plasminogen activator. 2 Reelin Is a Serine Protease of the Extracellular Matrix 305 FIG.2. HEK 293T cells transfected with the pCrl vector ex- press Reelin mRNA and secrete Reelin in the supernatant. A, total RNA was extracted from cell homogenates and pCrl mRNA expression was probed with RT-PCR, using primers corresponding to exon 27 of Reelin. Lanes 1, PCR of pCrl vector; 2, RT-PCR of mock-(pCDNA3)-transfected cells; 3, RT-PCR of pCrl-transfected cells. The position of the 350-bp marker is indicated on the left. B, Western blot of cell culture supernatants, probed with anti-Reelin mAb 142. Lanes 1, supernatant from mock-transfected cells; 2, supernatant from pCrl-transfected cells; 1 and 2 replica samples of lanes 1 and 2, after silver stain. The position of 250- and 150-kDa markers is indicated. stained bands (lane 1). HEK 293T cells endogenously express   integrin which 5 1 specifically mediates adhesion to fibronectin (28). Titration experiments showed dose-dependent adhesion to fibronectin, with maximal adhesion occurring at 2.5–5 g/ml fibronectin (data not shown). Reelin-transfected HEK 293T showed signif- icantly less adhesion as compared with mock-transfected HEK 293T cells. After a 2-h incubation, 44.7  6.3 (mean  S.E.) pCrl-transfected cells were attached to the substrate, as com- pared with 96.3  10.8 mock-transfected cells (Fig. 3A, p 0.001). Furthermore, cell morphology was markedly different: Reelin-secreting cells appeared unable to spread on fibronectin and their processes were diminished in number and length, as compared with mock-transfected cells (Fig. 3B). To assess the FIG.1. Consensus sequence analysis of Reelin around hypo- biological significance of the enzymatic activity of Reelin, the thetically catalytic amino acids (serine, histidine, and aspartic effect of DFP, a potent and specific inhibitor of serine hydro- acid) of serine proteases. Identical or homologous residues are lases, on cell adhesion was studied. Micromolar concentrations shaded. of DFP partially restored adhesion of Reelin-expressing cells on fibronectin, without affecting mock-transfected cells (Fig. 3, A Degradation of Extracellular Matrix Proteins—Reelin aliquots (10 and B). The effect of DFP was dose-dependent, starting at ng) were incubated with 1 g of fibronectin from human plasma (Sig- ma), or with laminin or collagen type IV from basement membrane of concentrations 0.54 M; maximal increase in adhesion of Ree- Engelbreth-Holm-Swarm mouse sarcoma (Sigma) for 0, 10, 30, or 120 lin-expressing cells was seen at 5.4 M DFP, while 5.4 mM was min at 37 °C, in PBS, pH 7.9. The reaction was stopped by adding equally toxic for pCrl- and mock-transfected cells, inhibiting sample buffer and heating the samples at 100 °C for 2 min. Samples adhesion of 99% of the cells (Fig. 3C). were separated in a 8% SDS gel. After electrophoresis, the gel was fixed, Reelin Binds a Serine Hydrolase Probe—FP-Peg-biotin is and silver-stained. described to behave as a specific and irreversible probe for RESULTS serine hydrolases, showing properties similar to those of com- Reelin Contains Regions of Homology with Serine Pro- mon FP inhibitors, such as DFP (30). To explore Reelin labeling teases—Human Reelin contains the sequence GKSDG (amino with FP-Peg-biotin, aliquots of transfected 293T cell culture acids 1280 –1284), homologous to the consensus sequence GX- supernatants incubated with 5 M FP-Peg-biotin were sepa- SXG of serine proteases (Fig. 1). Furthermore, searches of the rated on standard SDS-PAGE gels, blotted, and probed with Reelin sequence for consensus patterns (PROSITE www.ich/ avidin peroxidase; replica samples were stained with the mono- ucl.ac.uk/cmgs/serpro.htm) around hypothetical amino acids of clonal antibody 142. The supernatant of Reelin-expressing cells the catalytic triad (Ser, His, and Asp) using PattinProt (PBIL, showed distinct Reelin bands at approximately 400, 300, and NPSA, Lyon), yielded several sequences sharing 60% homol- 140 kDa (Fig. 4A, lane 2). The 400- and 300-kDa band showed ogy with serine proteases (Fig. 1). faint labeling with FP-Peg-biotin, while the 140-kDa band Reelin Inhibits Cell Attachment in Vitro—To obtain recom- showed strong labeling with FP-Peg-biotin (Fig. 4B, lane 1; binant Reelin protein, HEK 293T cells were transfected with arrows indicate corresponding bands in the two blots). Labeling pCrl plasmid, and Reelin mRNA expression was assessed by of these three bands was inhibited by DFP (Fig. 4B, lane 2). The RT-PCR, using primers complementary to exon 27 sequences. supernatant of mock-transfected cells showed a completely dif- RT-PCR of pCrl-transfected cells revealed the expected 376-bp ferent labeling pattern with FP-Peg-biotin: the most evidently band (Fig. 2A, lane 3), while the band was absent in mock- labeled band was a 150-kDa band, while no labeled bands were transfected cells (lane 2). Reelin secretion into the supernatant visible at 400 and 140 kDa (Fig. 4B, lane 3). Given the complex was confirmed by Western blotting. The supernatant of pCrl- pattern of labeling with FP-Peg-biotin in supernatants, we transfected cells showed a major Reelin band at approximately decided to perform FP-Peg-biotin labeling on partially purified 400 kDa, and minor bands at 350 and 140 kDa (Fig. 2B, lane 2). Reelin. The supernatant of the stable cell line CER, expressing The supernatant of mock-transfected cells did not show any high levels of Reelin, was concentrated and partially purified 306 Reelin Is a Serine Protease of the Extracellular Matrix FIG.4. Reelin can be labeled with FP-Peg-biotin, a serine trap probe, and labeling is inhibited by DFP. A, aliquots of superna- tants were separated by SDS-PAGE, blotted, and stained with mAb 142. Lanes 1, Mock-transfected HEK 293T cells; 2, pCrl-transfected cells; arrows indicate Reelin bands. B, FP-Peg-biotin labels several bands in the supernatants. Lanes 1, supernatant of pCrl-transfected cells; arrows indicate bands corresponding to the 400-, 300-, and 140- kDa Reelin bands; 2, replica sample as in lane 1, but preincubated with 11 M DFP; 3, supernatant of mock-transfected cells; 4, replica sample as in lane 3, but preincubated in the presence of 11 M DFP. Two- hundred and fifty ng of total protein were applied to each lane. FIG.3. Expression of Reelin inhibits adhesion of HEK 293T cells to fibronectin, and DFP treatment reverses the effect of Reelin expression. A, quantification of the effect of various treat- ments. Bars show the numbers of cells attached to fibronectin-coated FIG.5. Purification of Reelin by fast protein liquid chromatog- wells under different conditions. Each bar represent the mean and S.E. raphy and SDS-PAGE electroelution, and labeling of purified of five wells. Double-headed arrows indicate the statistical difference Reelin with FP-Peg-biotin. A, the concentrated supernatant from between groups (one-way ANOVA followed by LSD post-hoc test). B, CER cells was purified on a Sephadex-200 gel filtration column; the phase-contrast images of cells grown in different conditions. DFP was Reelin-positive eluate from the column was separated on a 4 –12% applied at a concentration of 5.4 M. C, dose-dependent effect of DFP on gradient SDS-PAGE. Lanes 1 and 2, immunoblot (mAb E4) of the cell adhesion of mock- or pCrl-transfected HEK 293T cells. Each point sample incubated with FP-Peg-biotin after pretreatment without and represents the percent ratio between the number of attached cells after with PMSF, respectively. Lanes 3 and 4, the blot was stripped and DFP treatment and the number of attached cells in the absence of DFP. developed with horseradish peroxidase-conjugated streptavidin to re- veal bound FP-Peg-biotin. The 250-kDa calibration marker shown next by gel filtration chromatography. The Reelin-positive eluate to lane 1 is valid for all 4 lanes. B, supernatants of mock-transfected (lane 1) and pCrl-transfected cells (lane 2) were separated with SDS- from the Superdex 200 gel filtration column was concentrated PAGE (5% gel) for3h30minandthegelwas stained with silver and then incubated in the absence or presence of the serine nitrate. The arrow next to lane 2 indicates the 400-kDa Reelin band. protease inhibitor phenylmethylsulfonyl fluoride (PMSF). The Lanes 3 and 4, blot corresponding to lanes 1 and 2, respectively, stained samples were separated by SDS-PAGE on a 4 –12% gradient with anti-Reelin mAb 142. The arrow next to lane 4 points to 400-kDa Reelin. The 250-kDa calibration marker next to lane 1 is valid for lanes gel, blotted, and incubated with the monoclonal antibody E4 to 2– 4 as well. Lane 5, the 400-kDa Reelin band shown in lane 2 was reveal Reelin (Fig. 5A, lanes 1 and 2). The blot was then electroeluted, electrophoresed in a second gel, blotted, and stained with stripped and incubated with streptavidin to reveal the binding mAb 142. The asterisks mark degradation products of Reelin at approx- to FP-Peg-biotin (Fig. 5A, lanes 3 and 4). As with the crude imately 180 and 140 kDa. The 400-kDa band has practically disappeared. supernatant (Figs. 2 and 4), the immunoblot showed two major Reelin-positive bands, one higher than the 250-kDa marker, corresponding to the 400- and 300-kDa isoforms, and a smaller higher molecular weight isoform (Fig. 5A, lane 3). band at about 140 kDa (Fig. 5A, lanes 1 and 2). FP-Peg-biotin Reelin Shows Protease Activity on Extracellular Matrix Pro- binds to both major Reelin bands in the absence, but not in the teins in Vitro—To investigate the catalytic activity of Reelin, presence of PMSF (Fig. 5A, lanes 3 and 4). The smaller band we first further purified the high molecular weight isoforms by appeared to bind FP-Peg-biotin with a higher affinity than the SDS-PAGE and electroelution. As shown in Fig. 5B, separation Reelin Is a Serine Protease of the Extracellular Matrix 307 FIG.6. Purified Reelin rapidly degrades ECM proteins fibronectin and laminin, while collagen IV is degraded at a much slower rate. Protein degradation was assessed with SDS-PAGE and silver staining of gels. A, time course of fibronectin degradation by Reelin; human fibronectin (1 g) was incubated with purified Reelin (5 ng) for 0, 10, 30, and 120 min at 37 °C. C: intact fibronectin. B, degradation of fibronectin by Reelin is inhibited by serine protease inhibitors, but not by inhibitors of other classes of proteases; fibronectin was incubated with Reelin for 120 min at 37 °C in the presence of different protease inhibitors; control, intact fibronectin; concentrations of inhibitors are: DFP, 21.6 mM; PMSF, 2 mg/ml; aprotinin, 1 mg/ml; leupeptin, 1 mg/ml; pepstatin, 0.1 mg/ml; EDTA, 10 mM. C, degradation of fibronectin by Reelin is partially inhibited by mAb CR-50. Lane 1, purified CR-50, arrows point to the heavy and light chains of the antibody. Lanes 2– 4, fibronectin incubated with Reelin for 120 min at 37 °C in the absence (lane 2) or presence (lanes 3 and 4) of mAb CR-50 (lane 3, 0.098 g/ml; lane 4, 9.8 g/ml). Arrow next to lane 4 points to intact fibronectin. D, degradation of laminin by Reelin; C, laminin alone. E, degradation of collagen IV by Reelin; C, collagen IV alone. The positions of molecular weight markers are indicated on the left of the gels. Molecular weight markers indicated in A are also for B and C. of the concentrated cell culture supernatant using a 5% SDS bronectin degradation was seen at a CR-50 concentration of 9.8 gel resulted in a good separation of the Reelin isoforms. Silver g/ml, the highest concentration tested. Interestingly, this con- nitrate staining indicated that only the supernatant of Reelin centration is comparable with the concentrations that have expressing cells contains a band at approximately 400 kDa been reported in the literature to inhibit Reelin function (20 – corresponding to the Reelin isoforms of 400 (and 300) kDa (Fig. 200 g/ml, Ref. 31). 5B, lanes 2 and 4). Therefore, the high molecular weight Reelin DISCUSSION band was electroeluted from the gel to achieve a high degree of purification. The electroeluted sample was reanalyzed by SDS- In this paper we present converging biochemical and cellular PAGE and Western blotting (Fig. 5B, lane 5). However, we evidence that Reelin is a serine protease of the extracellular found that the purified high molecular weight Reelin protein matrix, and that its enzymatic activity is important for the quickly disappeared and smaller bands appeared around 180 modulation of cell adhesion. The fact that CR-50, a monoclonal and 140 kDa, probably corresponding to self-degradation prod- antibody known to inhibit Reelin function both in vitro and in ucts. The major proteolytic product of 140 kDa that we ob- vivo, blocks the proteolytic activity of Reelin further supports served in this study may correspond to the 180-kDa degrada- the hypothesis that proteolytic activity is of fundamental im- tion product that has been described by other investigators portance for the function of Reelin. These findings appear in- (8, 35). teresting in view of the fact that serine proteases, such as To test for proteolytic activity on extracellular matrix pro- tissue plasminogen activator (tPA), are already known to be teins, purified Reelin was incubated with pure fibronectin, important modulators of cell migration and axon growth (32). laminin, or collagen IV, and breakdown products were ana- HEK 293T cells adhere quickly to fibronectin, due to endog- lyzed by SDS-PAGE and silver staining of gels. Fibronectin and enous expression of   integrin (28), which is a selective 5 1 laminin breakdown fragments were seen already after 10 min fibronectin receptor (33). In this paper we show that expression incubation (Fig. 6, A and D), while collagen IV was degraded at of Reelin leads to a marked decrease of adhesion of HEK 293T a much slower rate (Fig. 6E). Fibronectin degradation was cells to fibronectin. These data, together with the demonstra- blocked by inhibitors of serine proteases (DFP, PMSF, and tion that purified Reelin degrades fibronectin in vitro, are aprotinin), but not by inhibitors of other families of proteases consistent with the hypothesis that Reelin, secreted by HEK (Fig. 6B). Fibronectin degradation was also partially inhibited 293T cells, inhibits cell adhesion by degrading the fibronectin by monoclonal antibody CR-50 (Fig. 6C), an antibody directed substrate. Alternatively, Reelin might activate other targets, against the NH -terminal portion of Reelin that has been dem- for example, cell membrane receptors or other proteases, which are in turn directly responsible for cell detachment. A third onstrated to inhibit Reelin function both in vitro and in vivo (see Ref. 31 and references quoted therein). Inhibition of fi- hypothesis is that Reelin induces the expression of another 308 Reelin Is a Serine Protease of the Extracellular Matrix serine protease, which is in turn responsible for cell detach- expression and distribution of Reelin and adhesion molecules of ment. This hypothesis appears remote, since we have demon- the extracellular matrix in structures other than the develop- strated that Reelin binds FP-Peg-biotin, and degrades fibronec- ing cerebral cortex. Reelin is co-expressed with integrins in tin in vitro. dendritic spines of GABAergic neurons in the adult cerebral We found that, after purification, Reelin appears to undergo cortex, and of glutamatergic neurons in the cerebellum and rapid self-degradation. Our data suggest that the major 140- olfactory bulb (10, 11, 36). Dendritic spine density is decreased kDa fragment is enzymatically active, since its binding to FP- in fronto-parietal cortex and CA1 pyramidal neurons of het- Peg-biotin is even stronger than that of full-length Reelin. erozygous reeler mice (37), and heterozygous reeler mice show Interestingly, we also observed strong labeling with FP-Peg- abnormalities in complex behavior, like neophobia and in- biotin of the smaller fragment after immunoaffinity purifica- creased anxiety (38), a finding that led investigators to propose tion of Reelin from mouse brain. These data support the idea these mice as an animal model of schizophrenia. We suggest that the proteolytic processing of Reelin is functionally impor- that the synaptic role of Reelin as a serine protease might tant, and that full activity of Reelin might require degradation consist in a rapid and local modulation of adhesive forces be- of the 400-kDa full-length precursor to generate smaller, more tween pre- and post-synaptic elements, thus modulating the active isoforms. efficiency of synaptic transmission at the local level. In fact, Reelin appears to behave as a specific serine protease, as other serine proteases, like tPA and plasmin, have already collagen IV is degraded at a much slower rate than fibronectin been suspected to be involved in synaptic plasticity. tPA con- or laminin. However, this hypothesis needs further confirma- tributes to the late phase of long-term potentiation in hip- tion, using model peptide substrates. pocampal slices and stimulates synapse formation in hip- Reelin has been suggested to allow migrating neurons to pocampal cell culture (39). Plasmin cleaves laminin and grow past previously migrated cells and to promote detachment appears to regulate long-term potentiation (40). In conclusion, of neurons from radial glial fibers (6 – 8). The  our findings may help to better understand the roles of Reelin integrin sub- unit, expressed in HEK 293T cells, shows high homology with both in physiology and in disease, by bringing this protein into the  the complex and exciting scenario of protease-regulated signal- subunit, which is expressed on migrating neurons, and appears to be involved in the inhibitory effect of Reelin on ing networks. neuronal migration along radial glial processes. Reelin has Acknowledgments—We are deeply grateful to Dr. A. M. Goffinet for been demonstrated to bind to   integrin (8). In situ hybrid- 3 1 the gift of monoclonal antibodies 142 and G10 and Dr. Benjamin ization experiments and double immunolabeling with antibod- Cravatt for the gift of FP-Peg-biotin. Furthermore, we thank Graziano ies against fibronectin and antibodies against radial glia dem- Bonelli, Marco De Luca, and Ramona Marino for technical assistance, and Flavia Mancuso for editorial assistance. onstrate transient fibronectin expression on radial glia processes during early stages of cortical development, until REFERENCES completion of corticogenesis (34). On the basis of the available 1. D‘Arcangelo, G., Miao, G. G., Chen, S., Soares, H. D., Morgan, J. I. & Curran, evidence, we propose that   integrin might immobilize ex- 3 1 T. (1995) Nature 374, 719 –723 tracellular Reelin on the surface of migrating neurons and thus 2. De Silva, U., D’Arcangelo, G., Braden, V. 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Reelin Is a Serine Protease of the Extracellular Matrix

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THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 277, No. 1, Issue of January 4, pp. 303–309, 2002 © 2002 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Received for publication, July 24, 2001, and in revised form, October 18, 2001 Published, JBC Papers in Press, October 31, 2001, DOI 10.1074/jbc.M106996200 a,b,c d,e a d Carlo C. Quattrocchi, Francesca Wannenes, Antonio M. Persico, Silvia Anna Ciafre ´, f,g,h,i f a, j Gabriella D’Arcangelo, Maria G. Farace, and Flavio Keller From the Laboratory of Neuroscience, Department of Physiology and Neuroscience, Universita ` “Campus Bio-Medico,” Via Longoni 83, 00155 Roma, Italy, the Program in Neuroscience, Faculty of Medicine, University of Brescia, Via Valsabbina 19, 25123 Brescia, Italy, the Department of Experimental Medicine and Biochemical Sciences, Universita ` di Tor Vergata, Via di Tor Vergata 135, 00133 Roma, Italy, the Department of Internal Medicine, Universita ` di Tor Vergata, Via di Tor f g h Vergata 135, 00133 Roma, Italy, and The Cain Foundation Laboratories, Department of Pediatrics, Program in Developmental Biology and Division of Neuroscience, Baylor College of Medicine, Houston, Texas 77030 Reelin is an extracellular matrix protein that plays a contacts with the radial glial fibers (6). Several hypotheses have been suggested regarding the function of Reelin: (i) Reelin pivotal role in development of the central nervous sys- tem. Reelin is also expressed in the adult brain, notably may act as an attractant molecule for migrating neurons; (ii) it in the cerebral cortex, where it might play a role in may act as a repulsive molecule; or (iii) Reelin may interrupt synaptic plasticity. The mechanism of action of reelin at the association between migrating neurons and radial glia (7, the molecular level has been the subject of several hy- 8), thus allowing migrating neurons to switch from a “glio- potheses. Here we show that reelin is a serine protease philic” to a “neurophilic” state (9). Furthermore, Reelin has and that proteolytic activity is relevant to its function, been recently shown to be expressed in several adult neuronal since (i) Reelin expression in HEK 293T cells impairs cells, including glutamatergic cerebellar granule neurons and their ability to adhere to fibronectin-coated surfaces, specific GABAergic interneurons of the cerebral cortex and and adhesion to fibronectin is restored by micromolar hippocampus (10), and in the adult mammalian blood, liver, concentrations of diisopropyl phosphorofluoridate, a pituitary pars intermedia, and adrenal chromaffin cells (11, serine hydrolase inhibitor; (ii) purified Reelin binds FP- 12). The cellular function of Reelin in the adult organism is Peg-biotin, a trap probe which irreversibly binds to ser- unknown. Evidence is accumulating for involvement of Reelin ine residues located in active catalytic sites of serine in human diseases such as autosomal recessive lissencephaly hydrolases; (iii) purified Reelin rapidly degrades fi- (13), schizophrenia (14), and autistic disorder (15). bronectin and laminin, while collagen IV is degraded at The mouse Reelin sequence (1) encompasses 3461 amino a much slower rate; fibronectin degradation is inhibited acids and possesses a signal peptide followed by a domain with by inhibitors of serine proteases, and by monoclonal 28% sequence identity with F-spondin (as assessed by -Blast antibody CR-50, an antibody known to block the func- software), a protein secreted by floor plate cells and promoting tion of Reelin both in vitro and in vivo. The proteolytic cell adhesion and neurite growth (16). This region is followed activity of Reelin on adhesion molecules of the extracel- lular matrix and/or receptors on neurons may explain by a unique region with no sequence homology, and then by how Reelin regulates neuronal migration and synaptic eight internal repeats of 350 –390 amino acids, each repeat plasticity. containing two related subdomains flanking a cystein-rich se- quence similar to the epidermal growth factor-like motif. The carboxyl terminus region contains many positively charged Reelin (1, 2) is an extracellular matrix protein that plays a amino acids required for secretion (5). Human Reelin (2) is pivotal role in neuronal migration during development of lam- 94.8% identical to the mouse protein at the amino acid level, inar structures of the mammalian brain including the cerebral indicating strong functional conservation. Recent findings sug- cortex, hippocampus, cerebellum, and several brainstem nu- gest that the Reelin signal transduction involves binding to the clei, as shown by spontaneous Reelin null mutations (i.e. the very low density lipoprotein receptor and to apoE receptor 2 reeler mouse) (3, 4). In the developing cerebral cortex, Reelin is followed by intracellular activation of the adapter protein dis- secreted by Cajal-Retzius cells, located in the marginal zone. abled-1 (17–19). Other possible Reelin signal transduction Reelin must be secreted into the extracellular matrix to exert pathways may involve interaction with the   integrin recep- 3 1 its biological effect (5). tor (8, 9) and with cadherin-related neuronal receptors (20). In the reeler mouse, migrating neurons fail to pass through We have analyzed the primary amino acid sequence of hu- earlier-generated neurons, possibly because they are unable to man Reelin, and found several hints that Reelin might be a penetrate the subplate, or because they maintain extensive serine protease, since: (i) Reelin contains the sequence GKSDG (amino acids 1280 –1284 of human Reelin) (2), corresponding to the serine hydrolase consensus sequence GXSXG; this se- * This work was supported by Consiglio Nazionale delle Ricerche Programma “Biomolecole per la salute umana” Grant 99.00555.PF33. quence is 100% conserved among mouse, chicken, and human The costs of publication of this article were defrayed in part by the Reelin; (ii) Reelin shows significant structural similarities with payment of page charges. This article must therefore be hereby marked serine hydrolases, such as the extracellular serine protease “advertisement” in accordance with 18 U.S.C. Section 1734 solely to precursor (EC 3.4.21) of Serratia marcescens, and the probable indicate this fact. Present address: Dept. of Pediatrics-Neurology, Baylor College of ubiquitin carboxyl-terminal hydrolases FAM and FAF-Y (EC Medicine, 1102 Bates St., MC 3– 6365, Houston, TX 77030. 3.1.2.15); (iii) Reelin contains eight epidermal growth factor- To whom correspondence should be addressed: Laboratory of Neu- like repeats; epidermal growth factor-like repeats are observed roscience, Universita ` “Campus Bio-Medico,” Via Longoni 83, 00155 in serine proteases, for example, coagulation factors VII, IX, Roma, Italy. Tel.: 39-06-2254-1335; Fax: 39-06-22-54-14-56; E-mail: f.keller@unicampus.it. and X, and protein C, Z (21), calcium-dependent serine protein- This paper is available on line at http://www.jbc.org 303 This is an Open Access article under the CC BY license. 304 Reelin Is a Serine Protease of the Extracellular Matrix balanced salt solution. Attached cells were fixed with 4% formaldehyde ase (22) which degrades extracellular matrix proteins, and in PBS, pH 7.4, and counted manually using an inverted microscope by complement C1s and C1r components (23); (iv) several serine two different observers. The counting area was defined by a grid (12 hydrolases, such as lipoprotein lipase and the urokinase-type mm area) placed under the wells. Attached cells were defined as cells plasminogen activator, bind very low density lipoprotein recep- that had spread and grown at least 1 process. All data are expressed as tor and apoE receptor 2 (24, 25). In this study we present mean  S.E. Differences between groups were tested by one-way converging evidence that purified Reelin acts as a serine pro- ANOVA followed by the LSD post-hoc test, using the SPSS statistics package (SPSS, Chicago, IL, version 9.0). tease, and that this enzymatic activity may be relevant for its Gel Electrophoresis and Immunoblotting—Samples were mixed with cellular function. 2  sample buffer and heated at 100 °C for 2 min. Six percent resolving gel, pH 8.8, and 5% stacking gel, pH 6.8, were prepared and run in a EXPERIMENTAL PROCEDURES MiniProtean II assembly (Bio-Rad, Hercules, CA) according to standard Chemicals—Cell culture media, antibiotics, and media supplements protocols (29). Proteins were transferred to nitrocellulose filter were purchased from Invitrogen (Gaithersburg, MD). All other chemi- (Schleicher & Schuell, GmbH, Dassel, Germany) by a semi-dry blotting cals were from Sigma, unless otherwise specified. apparatus (Hoefer Scientific, Amersham Bioscience, Inc., San Fran- Amino acid Sequence Analysis of Reelin—All sequence analyses are cisco, CA) for 90 min at constant current of 70 mA. After blotting, the based on human Reelin (accession number: NP005036) (2). Homologies nitrocellulose filter was blocked for 30 min in tris-buffered saline with were evaluated by using -Blast 2 sequences software (www.ncbi.nih. 0.1% Tween 20 (TBST), containing 1% bovine serum albumin. Reelin gov/blast). Also, PropSearch software (EMBL, Heidelberg) was used to was revealed with 1:2000 mAb 142, overnight, followed by 1:5000 alka- find homologies with other serine hydrolases. PattinProt software line phosphatase-labeled anti-mouse IgG (Promega Italia, Milan, Italy). (PBIL, NPSA, Lyon) was used to identify putative consensus sequences Filters were developed with nitro blue tetrazolium-5-bromo-4-chloro-3- for serine proteases. M MgCl indolyl phosphate in Tris-HCl, pH 9.5, containing 5 m . The Cell Culture, Transfection, and Expression of Recombinant Reelin— M EDTA. Alternatively, reaction was stopped with PBS containing 2 m Human embryonic kidney (HEK) 293T cells (ATCC, Q401) were grown Reelin bands were revealed using a chemiluminescence method: filters in Dulbecco’s modified Eagle’s medium supplemented with 10% heat- were incubated with biotinylated anti-mouse secondary antibody (1: inactivated fetal bovine serum, penicillin/streptomycin (100 interna- 25.000 in TBST) for 1 h. Then, after three washes, 1:500 peroxidase- tional units/ml and 100 g/ml, respectively), and 10 mML-glutamine. conjugated streptavidin (CHEMICON Int., Temecula, CA) was added All cells were cultured at 37 °C, 5% CO and 97% relative humidity. for 15 min. Finally filters was washed, incubated in a freshly made Passaging was routinely performed with trypsin-EDTA. Cells were solution containing 200 mM Tris-HCl, pH 8.5, 250 mM 3-aminophthal- stably transfected with 2 g of plasmid DNA pCrl (5), which contains hydrazide, 40 mM p-coumaric acid, and 0.0005% H O , dried, and ex- 2 2 the entire mouse Reelin open reading frame (accession number posed to Kodak X-OMAT film. Filters or films were scanned with a NP_035391), or with 2 g of pCDNA3 empty vector in 10 l of Lipo- SCANJET ADF digital scanner using Twain-32 software (Hewlett- fectAMINE in 4 ml of Opti-MEM serum-free medium, according to the Packard). The specificity of staining was checked by preincubating mAb manufacturer’s instructions. Cells were transfected in 60-mm plates at 142 with protein SP, the Reelin fragment recognized by mAb 142 (27). a density of 5  10 /dish and transferred to selection medium (0.6 Labeling of Reelin with FP-Peg-biotin—Fluorophosphate biotin (FP- mg/ml G418) 48 h after transfection. For Reelin purification, a stable M Peg-biotin) (a gift of Dr. Benjamin Cravatt) (30), stored as a 100 m cell line (CER) was generated by stable transfection of 293-EBNA cells stock solution in Me SO at 20 °C, was added directly to protein (Invitrogen) with pCER followed by selection in medium containing M. The reaction mixture was samples to a final concentration of 2– 4 0.25 mg/ml G418 and 0.4 mg/ml hygromycin B. The pCER episomal incubated at room temperature for 30 min, and stopped by adding an plasmid contains a reelin insert identical to that of pCrl, cloned into the equal volume of 2  reducing sample buffer. As a control for the pCEP4 vector (Invitrogen). specificity of FP-Peg-biotin labeling, replica samples were incubated for Cultures were harvested for RNA preparation (26). Total RNA (3 g) 1 h with 11.4 M diisopropylphosphofluoridate (DFP), a potent and was incubated in reaction buffer containing 5 M random hexamer specific serine-hydrolase inhibitor, before incubation with FP-Peg-bio- (Amersham Bioscience, Inc.), Moloney murine leukemia virus reverse tin. Samples were separated by SDS-PAGE and transferred by electro- transcriptase, reaction buffer, and RNase inhibitor, according to the blotting to nitrocellulose membranes; the membranes were blocked in manufacturer’s specifications (Invitrogen). Reverse transcription was TBST with 3% bovine serum albumin for1hat25 °C or overnight at performed for1hat37 °C, and stopped by incubating the samples for 4 °C, and then incubated for 15 min with an avidin-horseradish perox- 10 min at 95 °C. Expression of Reelin was checked by PCR amplification idase conjugate (Pierce) diluted 1:300 in blocking solution. The labeled with specific primers: forward 5-GGAAAGTCAGATGGAGAC-3, re- bands were revealed by chemiluminescence (see above). verse 5-CATCTAAGCCAAACG-3 corresponding to nucleotides 4123– Purification of Reelin—For gel filtration chromatography purifica- 4498 of mouse Reelin mRNA (Ref. (1), accession number U24703). PCR tion, 50 l of supernatant from Reelin secreting CER cells were con- amplification was carried out in a total volume of 50 l, with 10 lof centrated first by osmotic dyalisis with AQUACIDE I (Calbiochem, La reverse transcription reaction and 1.25 units of Taq polymerase (M- Jolla, CA). A 5-ml concentrated sample was loaded on a FPLC system Medical Genenco-Life Science). Amplification was performed for 35 AKTAprime and passed through a HiLoad Superdex 200 26/60 column cycles (1 min denaturing at 94 °C, 1 min annealing at 58 °C, and 1 min (Amersham Bioscience, Inc., Uppsala, Sweden). The run was performed extension at 72 °C) in a thermal cycler (Oracle BioSystems, Delphi at a constant flow rate of 3 ml/min and maximal pressure limit of 0.5 Pa. 1500). The 376-bp amplified product was analyzed by 2% agarose gel The eluate was collected in 1.5-ml fractions. After dot immunoblot electrophoresis and visualized under UV illumination after staining screening and Western blot, positive fractions for Reelin were pooled with ethidium bromide. Reelin secretion into supernatants was ana- and concentrated again as described above. lyzed by plating 1  10 cells onto 90-mm dishes. After 24 h conditioned For purification by SDS-PAGE, Reelin-containing supernatant from media were removed, cells were washed twice with phosphate-buffered transfected 293T cells was concentrated as above and separated on a 5% saline (PBS), and serum-free Dulbecco’s modified Eagle’s medium was gel. The gel was run for3h30minat125Vto achieve a good separation in the 250 kDa range. Thereafter a thin vertical slice of gel was cut added. Forty-eight hours later, the supernatants were collected, cleared and stained with silver to reveal the 400-kDa Reelin band. A 5-mm wide by a brief centrifugation (10 min, 1000 rpm) at 4 °C, concentrated by horizontal slice was cut from the remaining gel, using the stained slice Microcon YM-100 (Millipore Corp. Bedford, MA) and stored at 80 °C as a reference, and 400-kDa Reelin was electroeluted at 60 V into 25 mM for analysis by SDS-PAGE and immunoblotting. Reelin secretion into Tris, 250 mM glycine, 0,1% SDS buffer. Electroelution was carried out the cell culture medium was assessed by Western blots using mAb 142, overnight at 4 °C. Purified Reelin was, then, transferred to PBS, pH 7.2, an antibody that recognizes the NH -terminal sequence of Reelin (27). by overnight dialysis in a Slide-A-Lyzer cassette (Pierce). Finally the Cell Adhesion Assay—Cell adhesion assay was performed according protein was concentrated. The final protein concentration was esti- to published protocols (28). Ninety-six-well plates were coated over- mated to be 0.2 g/ml (Bradford Reagent, Sigma). Purified Reelin was night with fibronectin (2.5 g/ml) in carbonate/bicarbonate buffer, pH re-electrophoresed on a SDS gel to check the purity of the protein. 9.7. Cells were harvested, washed three times with serum-free Dulbec- co’s modified Eagle’s medium, and then resuspended in sterile attach- ment solution (calcium- and magnesium-free Hanks’ balanced salt so- lution, 20 mM HEPES, 1 mg/ml heat-inactivated bovine serum albumin, The abbreviations used are: DFP, diisopropylphosphorofluoridate; 1mM CaCl ,1mM MgCl . Mock- and pCrl-transfected 293T cells (1 HEK, human embryonic kidney; mAb, monoclonal antibody; PBS, phos- 2 2 10 in 200 l/well) were allowed to attach for2hat37 °C in a humid- phate-buffered saline; PMSF, phenylmethylsulfonyl fluoride; tPA, tis- ified 5% CO incubator. Unattached cells were removed with Hanks’ sue plasminogen activator. 2 Reelin Is a Serine Protease of the Extracellular Matrix 305 FIG.2. HEK 293T cells transfected with the pCrl vector ex- press Reelin mRNA and secrete Reelin in the supernatant. A, total RNA was extracted from cell homogenates and pCrl mRNA expression was probed with RT-PCR, using primers corresponding to exon 27 of Reelin. Lanes 1, PCR of pCrl vector; 2, RT-PCR of mock-(pCDNA3)-transfected cells; 3, RT-PCR of pCrl-transfected cells. The position of the 350-bp marker is indicated on the left. B, Western blot of cell culture supernatants, probed with anti-Reelin mAb 142. Lanes 1, supernatant from mock-transfected cells; 2, supernatant from pCrl-transfected cells; 1 and 2 replica samples of lanes 1 and 2, after silver stain. The position of 250- and 150-kDa markers is indicated. stained bands (lane 1). HEK 293T cells endogenously express   integrin which 5 1 specifically mediates adhesion to fibronectin (28). Titration experiments showed dose-dependent adhesion to fibronectin, with maximal adhesion occurring at 2.5–5 g/ml fibronectin (data not shown). Reelin-transfected HEK 293T showed signif- icantly less adhesion as compared with mock-transfected HEK 293T cells. After a 2-h incubation, 44.7  6.3 (mean  S.E.) pCrl-transfected cells were attached to the substrate, as com- pared with 96.3  10.8 mock-transfected cells (Fig. 3A, p 0.001). Furthermore, cell morphology was markedly different: Reelin-secreting cells appeared unable to spread on fibronectin and their processes were diminished in number and length, as compared with mock-transfected cells (Fig. 3B). To assess the FIG.1. Consensus sequence analysis of Reelin around hypo- biological significance of the enzymatic activity of Reelin, the thetically catalytic amino acids (serine, histidine, and aspartic effect of DFP, a potent and specific inhibitor of serine hydro- acid) of serine proteases. Identical or homologous residues are lases, on cell adhesion was studied. Micromolar concentrations shaded. of DFP partially restored adhesion of Reelin-expressing cells on fibronectin, without affecting mock-transfected cells (Fig. 3, A Degradation of Extracellular Matrix Proteins—Reelin aliquots (10 and B). The effect of DFP was dose-dependent, starting at ng) were incubated with 1 g of fibronectin from human plasma (Sig- ma), or with laminin or collagen type IV from basement membrane of concentrations 0.54 M; maximal increase in adhesion of Ree- Engelbreth-Holm-Swarm mouse sarcoma (Sigma) for 0, 10, 30, or 120 lin-expressing cells was seen at 5.4 M DFP, while 5.4 mM was min at 37 °C, in PBS, pH 7.9. The reaction was stopped by adding equally toxic for pCrl- and mock-transfected cells, inhibiting sample buffer and heating the samples at 100 °C for 2 min. Samples adhesion of 99% of the cells (Fig. 3C). were separated in a 8% SDS gel. After electrophoresis, the gel was fixed, Reelin Binds a Serine Hydrolase Probe—FP-Peg-biotin is and silver-stained. described to behave as a specific and irreversible probe for RESULTS serine hydrolases, showing properties similar to those of com- Reelin Contains Regions of Homology with Serine Pro- mon FP inhibitors, such as DFP (30). To explore Reelin labeling teases—Human Reelin contains the sequence GKSDG (amino with FP-Peg-biotin, aliquots of transfected 293T cell culture acids 1280 –1284), homologous to the consensus sequence GX- supernatants incubated with 5 M FP-Peg-biotin were sepa- SXG of serine proteases (Fig. 1). Furthermore, searches of the rated on standard SDS-PAGE gels, blotted, and probed with Reelin sequence for consensus patterns (PROSITE www.ich/ avidin peroxidase; replica samples were stained with the mono- ucl.ac.uk/cmgs/serpro.htm) around hypothetical amino acids of clonal antibody 142. The supernatant of Reelin-expressing cells the catalytic triad (Ser, His, and Asp) using PattinProt (PBIL, showed distinct Reelin bands at approximately 400, 300, and NPSA, Lyon), yielded several sequences sharing 60% homol- 140 kDa (Fig. 4A, lane 2). The 400- and 300-kDa band showed ogy with serine proteases (Fig. 1). faint labeling with FP-Peg-biotin, while the 140-kDa band Reelin Inhibits Cell Attachment in Vitro—To obtain recom- showed strong labeling with FP-Peg-biotin (Fig. 4B, lane 1; binant Reelin protein, HEK 293T cells were transfected with arrows indicate corresponding bands in the two blots). Labeling pCrl plasmid, and Reelin mRNA expression was assessed by of these three bands was inhibited by DFP (Fig. 4B, lane 2). The RT-PCR, using primers complementary to exon 27 sequences. supernatant of mock-transfected cells showed a completely dif- RT-PCR of pCrl-transfected cells revealed the expected 376-bp ferent labeling pattern with FP-Peg-biotin: the most evidently band (Fig. 2A, lane 3), while the band was absent in mock- labeled band was a 150-kDa band, while no labeled bands were transfected cells (lane 2). Reelin secretion into the supernatant visible at 400 and 140 kDa (Fig. 4B, lane 3). Given the complex was confirmed by Western blotting. The supernatant of pCrl- pattern of labeling with FP-Peg-biotin in supernatants, we transfected cells showed a major Reelin band at approximately decided to perform FP-Peg-biotin labeling on partially purified 400 kDa, and minor bands at 350 and 140 kDa (Fig. 2B, lane 2). Reelin. The supernatant of the stable cell line CER, expressing The supernatant of mock-transfected cells did not show any high levels of Reelin, was concentrated and partially purified 306 Reelin Is a Serine Protease of the Extracellular Matrix FIG.4. Reelin can be labeled with FP-Peg-biotin, a serine trap probe, and labeling is inhibited by DFP. A, aliquots of superna- tants were separated by SDS-PAGE, blotted, and stained with mAb 142. Lanes 1, Mock-transfected HEK 293T cells; 2, pCrl-transfected cells; arrows indicate Reelin bands. B, FP-Peg-biotin labels several bands in the supernatants. Lanes 1, supernatant of pCrl-transfected cells; arrows indicate bands corresponding to the 400-, 300-, and 140- kDa Reelin bands; 2, replica sample as in lane 1, but preincubated with 11 M DFP; 3, supernatant of mock-transfected cells; 4, replica sample as in lane 3, but preincubated in the presence of 11 M DFP. Two- hundred and fifty ng of total protein were applied to each lane. FIG.3. Expression of Reelin inhibits adhesion of HEK 293T cells to fibronectin, and DFP treatment reverses the effect of Reelin expression. A, quantification of the effect of various treat- ments. Bars show the numbers of cells attached to fibronectin-coated FIG.5. Purification of Reelin by fast protein liquid chromatog- wells under different conditions. Each bar represent the mean and S.E. raphy and SDS-PAGE electroelution, and labeling of purified of five wells. Double-headed arrows indicate the statistical difference Reelin with FP-Peg-biotin. A, the concentrated supernatant from between groups (one-way ANOVA followed by LSD post-hoc test). B, CER cells was purified on a Sephadex-200 gel filtration column; the phase-contrast images of cells grown in different conditions. DFP was Reelin-positive eluate from the column was separated on a 4 –12% applied at a concentration of 5.4 M. C, dose-dependent effect of DFP on gradient SDS-PAGE. Lanes 1 and 2, immunoblot (mAb E4) of the cell adhesion of mock- or pCrl-transfected HEK 293T cells. Each point sample incubated with FP-Peg-biotin after pretreatment without and represents the percent ratio between the number of attached cells after with PMSF, respectively. Lanes 3 and 4, the blot was stripped and DFP treatment and the number of attached cells in the absence of DFP. developed with horseradish peroxidase-conjugated streptavidin to re- veal bound FP-Peg-biotin. The 250-kDa calibration marker shown next by gel filtration chromatography. The Reelin-positive eluate to lane 1 is valid for all 4 lanes. B, supernatants of mock-transfected (lane 1) and pCrl-transfected cells (lane 2) were separated with SDS- from the Superdex 200 gel filtration column was concentrated PAGE (5% gel) for3h30minandthegelwas stained with silver and then incubated in the absence or presence of the serine nitrate. The arrow next to lane 2 indicates the 400-kDa Reelin band. protease inhibitor phenylmethylsulfonyl fluoride (PMSF). The Lanes 3 and 4, blot corresponding to lanes 1 and 2, respectively, stained samples were separated by SDS-PAGE on a 4 –12% gradient with anti-Reelin mAb 142. The arrow next to lane 4 points to 400-kDa Reelin. The 250-kDa calibration marker next to lane 1 is valid for lanes gel, blotted, and incubated with the monoclonal antibody E4 to 2– 4 as well. Lane 5, the 400-kDa Reelin band shown in lane 2 was reveal Reelin (Fig. 5A, lanes 1 and 2). The blot was then electroeluted, electrophoresed in a second gel, blotted, and stained with stripped and incubated with streptavidin to reveal the binding mAb 142. The asterisks mark degradation products of Reelin at approx- to FP-Peg-biotin (Fig. 5A, lanes 3 and 4). As with the crude imately 180 and 140 kDa. The 400-kDa band has practically disappeared. supernatant (Figs. 2 and 4), the immunoblot showed two major Reelin-positive bands, one higher than the 250-kDa marker, corresponding to the 400- and 300-kDa isoforms, and a smaller higher molecular weight isoform (Fig. 5A, lane 3). band at about 140 kDa (Fig. 5A, lanes 1 and 2). FP-Peg-biotin Reelin Shows Protease Activity on Extracellular Matrix Pro- binds to both major Reelin bands in the absence, but not in the teins in Vitro—To investigate the catalytic activity of Reelin, presence of PMSF (Fig. 5A, lanes 3 and 4). The smaller band we first further purified the high molecular weight isoforms by appeared to bind FP-Peg-biotin with a higher affinity than the SDS-PAGE and electroelution. As shown in Fig. 5B, separation Reelin Is a Serine Protease of the Extracellular Matrix 307 FIG.6. Purified Reelin rapidly degrades ECM proteins fibronectin and laminin, while collagen IV is degraded at a much slower rate. Protein degradation was assessed with SDS-PAGE and silver staining of gels. A, time course of fibronectin degradation by Reelin; human fibronectin (1 g) was incubated with purified Reelin (5 ng) for 0, 10, 30, and 120 min at 37 °C. C: intact fibronectin. B, degradation of fibronectin by Reelin is inhibited by serine protease inhibitors, but not by inhibitors of other classes of proteases; fibronectin was incubated with Reelin for 120 min at 37 °C in the presence of different protease inhibitors; control, intact fibronectin; concentrations of inhibitors are: DFP, 21.6 mM; PMSF, 2 mg/ml; aprotinin, 1 mg/ml; leupeptin, 1 mg/ml; pepstatin, 0.1 mg/ml; EDTA, 10 mM. C, degradation of fibronectin by Reelin is partially inhibited by mAb CR-50. Lane 1, purified CR-50, arrows point to the heavy and light chains of the antibody. Lanes 2– 4, fibronectin incubated with Reelin for 120 min at 37 °C in the absence (lane 2) or presence (lanes 3 and 4) of mAb CR-50 (lane 3, 0.098 g/ml; lane 4, 9.8 g/ml). Arrow next to lane 4 points to intact fibronectin. D, degradation of laminin by Reelin; C, laminin alone. E, degradation of collagen IV by Reelin; C, collagen IV alone. The positions of molecular weight markers are indicated on the left of the gels. Molecular weight markers indicated in A are also for B and C. of the concentrated cell culture supernatant using a 5% SDS bronectin degradation was seen at a CR-50 concentration of 9.8 gel resulted in a good separation of the Reelin isoforms. Silver g/ml, the highest concentration tested. Interestingly, this con- nitrate staining indicated that only the supernatant of Reelin centration is comparable with the concentrations that have expressing cells contains a band at approximately 400 kDa been reported in the literature to inhibit Reelin function (20 – corresponding to the Reelin isoforms of 400 (and 300) kDa (Fig. 200 g/ml, Ref. 31). 5B, lanes 2 and 4). Therefore, the high molecular weight Reelin DISCUSSION band was electroeluted from the gel to achieve a high degree of purification. The electroeluted sample was reanalyzed by SDS- In this paper we present converging biochemical and cellular PAGE and Western blotting (Fig. 5B, lane 5). However, we evidence that Reelin is a serine protease of the extracellular found that the purified high molecular weight Reelin protein matrix, and that its enzymatic activity is important for the quickly disappeared and smaller bands appeared around 180 modulation of cell adhesion. The fact that CR-50, a monoclonal and 140 kDa, probably corresponding to self-degradation prod- antibody known to inhibit Reelin function both in vitro and in ucts. The major proteolytic product of 140 kDa that we ob- vivo, blocks the proteolytic activity of Reelin further supports served in this study may correspond to the 180-kDa degrada- the hypothesis that proteolytic activity is of fundamental im- tion product that has been described by other investigators portance for the function of Reelin. These findings appear in- (8, 35). teresting in view of the fact that serine proteases, such as To test for proteolytic activity on extracellular matrix pro- tissue plasminogen activator (tPA), are already known to be teins, purified Reelin was incubated with pure fibronectin, important modulators of cell migration and axon growth (32). laminin, or collagen IV, and breakdown products were ana- HEK 293T cells adhere quickly to fibronectin, due to endog- lyzed by SDS-PAGE and silver staining of gels. Fibronectin and enous expression of   integrin (28), which is a selective 5 1 laminin breakdown fragments were seen already after 10 min fibronectin receptor (33). In this paper we show that expression incubation (Fig. 6, A and D), while collagen IV was degraded at of Reelin leads to a marked decrease of adhesion of HEK 293T a much slower rate (Fig. 6E). Fibronectin degradation was cells to fibronectin. These data, together with the demonstra- blocked by inhibitors of serine proteases (DFP, PMSF, and tion that purified Reelin degrades fibronectin in vitro, are aprotinin), but not by inhibitors of other families of proteases consistent with the hypothesis that Reelin, secreted by HEK (Fig. 6B). Fibronectin degradation was also partially inhibited 293T cells, inhibits cell adhesion by degrading the fibronectin by monoclonal antibody CR-50 (Fig. 6C), an antibody directed substrate. Alternatively, Reelin might activate other targets, against the NH -terminal portion of Reelin that has been dem- for example, cell membrane receptors or other proteases, which are in turn directly responsible for cell detachment. A third onstrated to inhibit Reelin function both in vitro and in vivo (see Ref. 31 and references quoted therein). Inhibition of fi- hypothesis is that Reelin induces the expression of another 308 Reelin Is a Serine Protease of the Extracellular Matrix serine protease, which is in turn responsible for cell detach- expression and distribution of Reelin and adhesion molecules of ment. This hypothesis appears remote, since we have demon- the extracellular matrix in structures other than the develop- strated that Reelin binds FP-Peg-biotin, and degrades fibronec- ing cerebral cortex. Reelin is co-expressed with integrins in tin in vitro. dendritic spines of GABAergic neurons in the adult cerebral We found that, after purification, Reelin appears to undergo cortex, and of glutamatergic neurons in the cerebellum and rapid self-degradation. Our data suggest that the major 140- olfactory bulb (10, 11, 36). Dendritic spine density is decreased kDa fragment is enzymatically active, since its binding to FP- in fronto-parietal cortex and CA1 pyramidal neurons of het- Peg-biotin is even stronger than that of full-length Reelin. erozygous reeler mice (37), and heterozygous reeler mice show Interestingly, we also observed strong labeling with FP-Peg- abnormalities in complex behavior, like neophobia and in- biotin of the smaller fragment after immunoaffinity purifica- creased anxiety (38), a finding that led investigators to propose tion of Reelin from mouse brain. These data support the idea these mice as an animal model of schizophrenia. We suggest that the proteolytic processing of Reelin is functionally impor- that the synaptic role of Reelin as a serine protease might tant, and that full activity of Reelin might require degradation consist in a rapid and local modulation of adhesive forces be- of the 400-kDa full-length precursor to generate smaller, more tween pre- and post-synaptic elements, thus modulating the active isoforms. efficiency of synaptic transmission at the local level. In fact, Reelin appears to behave as a specific serine protease, as other serine proteases, like tPA and plasmin, have already collagen IV is degraded at a much slower rate than fibronectin been suspected to be involved in synaptic plasticity. tPA con- or laminin. However, this hypothesis needs further confirma- tributes to the late phase of long-term potentiation in hip- tion, using model peptide substrates. pocampal slices and stimulates synapse formation in hip- Reelin has been suggested to allow migrating neurons to pocampal cell culture (39). Plasmin cleaves laminin and grow past previously migrated cells and to promote detachment appears to regulate long-term potentiation (40). In conclusion, of neurons from radial glial fibers (6 – 8). The  our findings may help to better understand the roles of Reelin integrin sub- unit, expressed in HEK 293T cells, shows high homology with both in physiology and in disease, by bringing this protein into the  the complex and exciting scenario of protease-regulated signal- subunit, which is expressed on migrating neurons, and appears to be involved in the inhibitory effect of Reelin on ing networks. neuronal migration along radial glial processes. Reelin has Acknowledgments—We are deeply grateful to Dr. A. M. Goffinet for been demonstrated to bind to   integrin (8). In situ hybrid- 3 1 the gift of monoclonal antibodies 142 and G10 and Dr. Benjamin ization experiments and double immunolabeling with antibod- Cravatt for the gift of FP-Peg-biotin. Furthermore, we thank Graziano ies against fibronectin and antibodies against radial glia dem- Bonelli, Marco De Luca, and Ramona Marino for technical assistance, and Flavia Mancuso for editorial assistance. onstrate transient fibronectin expression on radial glia processes during early stages of cortical development, until REFERENCES completion of corticogenesis (34). On the basis of the available 1. D‘Arcangelo, G., Miao, G. G., Chen, S., Soares, H. D., Morgan, J. I. & Curran, evidence, we propose that   integrin might immobilize ex- 3 1 T. (1995) Nature 374, 719 –723 tracellular Reelin on the surface of migrating neurons and thus 2. De Silva, U., D’Arcangelo, G., Braden, V. 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Published: Jan 1, 2002

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