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Department of Molecular Neurobiology, Institute of Basic Medical Sciences, Graduate School of Comprehensive Human Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8577, Japan
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Introduction |
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ENPP1-3 possess a common structural architecture: an N-terminal hydrophobic domain, two somatomedin-B-like domains, a catalytic domain, and a nuclease-like domain (Goding et al. 1998; Bollen et al. 2000). A well-characterized member of this group of proteins, ENPP1 (also known as PC-1), was demonstrated to be a type II transmembrane protein that is expressed on the cell surface and is released into the extracellular space by proteolytic cleavage (Belli et al. 1993; Goding et al. 1998; Bollen et al. 2000). Because autotaxin shows overall structural similarity to PC-1, it has been assumed to be a type II transmembrane protein.
Here, contrary to this assumption, we demonstrate that autotaxin is synthesized as a secreted protein, not as an integral membrane protein. We show that:
These data unequivocally demonstrate that the N-terminal hydrophobic region of autotaxin functions as a signal peptide, not as a transmembrane segment.
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Results |
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Autotaxin has been reported to be expressed at high levels in the adult brain (Narita et al. 1994; Fuss et al. 1997), but its roles in the brain remain unknown. In order to investigate the physiological functions of autotaxin in the adult nervous system, we first examined the expression pattern of autotaxin mRNA using in situ hybridization. This analysis showed that autotaxin mRNA was abundantly expressed in the epithelial cells of the choroid plexus in the adult mouse and rat brains (Fig. 1A–C, data not shown), as reported previously (Narita et al. 1994; Fuss et al. 1997; Bächner et al. 1999). In addition, punctate signals with much weaker intensity were detected throughout the brain (data not shown). Because the choroid plexus is a secretory organ that produces cerebrospinal fluid (CSF) in the brain, we wondered whether autotaxin is secreted into CSF. We therefore performed immunoblotting of rat CSF with anti-autotaxin antibody, which we generated in this study by immunizing a rabbit with a peptide common to human, rat, and mouse autotaxin. We found that autotaxin was detected in the CSF as a band of about 120 kDa (Fig. 1D), as reported previously in the CSF from rats, dogs, and humans (Tanaka et al. 2004; Sato et al. 2005). It is thus likely that autotaxin is expressed in the choroid plexus and secreted into CSF.
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Because autotaxin has been assumed to be a type II transmembrane protein, it was important to clarify how and when autotaxin is proteolytically released. In order to investigate the molecular mechanism regulating the secretion of autotaxin, we first transfected COS-7 cells with expression constructs for autotaxin as well as for other members of the ENPP family, PC-1 and B10 (also known as ENPP3) as controls, and examined the localization of these proteins using immunostaining. All the cDNAs were tagged with a FLAG sequence at the C-termini of their open reading frames in order to detect their expression with the same antibody. To identify the transfected cells, enhanced green fluorescent protein (EGFP) cDNA was co-transfected. Because these ENPP family proteins are thought to be exposed extracellularly, cell surface expression would be detected by incubating the live cells with anti-FLAG antibody. Unexpectedly, we failed to detect autotaxin immunoreactivity on the cell surface (Fig. 2A), whereas we clearly observed the cell surface expression of PC-1 and B10 (Fig. 2B,C). The same results were obtained using 293EBNA cells (data not shown), indicating that autotaxin protein, unlike PC-1 and B10, is not present on the cell surface.
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Autotaxin is efficiently secreted
To investigate whether autotaxin was secreted, we examined the protein in the culture supernatants and cell lysates of the autotaxin-transfected cells using immunoblotting with anti-autotaxin antibody. This analysis revealed that a robust band of 120 kDa was detected in the supernatants after 5-min exposure of the immunoblot to film, while a band of similar intensity was not obtained in the cell lysates until the immunoblot was exposed for 6 h (Fig. 3A), suggesting that autotaxin was much more abundant in the supernatants. The size of the band in the cell lysates (115 kDa) was slightly smaller than that in the supernatants, as previously reported (Jansen et al. 2005), suggesting that some additional post-translational modification(s) occur in the secreted form.
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Because matrix metalloproteinases (MMPs) are known to be involved in ectodomain shedding of many important cell surface proteins (Sternlicht & Werb 2001; Stamenkovic 2003), we tested the effects of three kinds of MMP inhibitors (MMPI-I, II, and III) on autotaxin secretion. Incubation with these inhibitors did not change the amounts of autotaxin in the cell lysates or supernatants (Fig. 3B), suggesting that MMPs are not necessary for autotaxin secretion. In contrast, treatment with monensin, a secretion inhibitor, almost completely abolished the secretion of autotaxin into the supernatants, and increased the retention of autotaxin in the cell lysates (Fig. 3B).
Autotaxin is proteolytically processed
Although both autotaxin and PC-1 have one hydrophobic domain near their N-termini, a hydrophilic domain of 60 amino acid residues precedes the hydrophobic region in PC-1, while such a long hydrophilic sequence is not present in autotaxin (Fig. 4A), suggesting that the roles of the hydrophobic regions in autotaxin and PC-1 are different. Indeed, a signal peptide prediction program, SignalP 3.0 (Bendtsen et al. 2004), predicted that the signal peptide cleavage occurs in rat autotaxin between Gly27 and Phe28 with 93.3% probability, whereas the hydrophobic sequence of rat PC-1 functions as a signal anchor with 94.6% probability. Essentially the same predictions were made using human and mouse sequences, suggesting that the N-terminal hydrophobic domain in autotaxin might function as a signal peptide, whereas that in PC-1 is a transmembrane segment, as reported previously (Clair et al. 1997; Goding et al. 1998; Bollen et al. 2000).
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The N-terminal hydrophobic sequence functions as a signal peptide
In order to directly examine whether the N-terminal sequence of autotaxin functions as a signal peptide, we performed in-vitro translation analysis. To this end, we made an artificial RNA encoding amino acid residues 1–243 of autotaxin tagged with a FLAG sequence at its C-terminus (Fig. 5A). The translation product contains a putative signal sequence (amino acid residues 1–27) and one potential N-glycosylation site (Fig. 5A). This mini-autotaxin construct, ATX(1–243)-FLAG enabled us to sensitively detect small changes in molecular masses of the translation products.
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Discussion |
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These results indicate that the N-terminal hydrophobic region of autotaxin functions as a signal peptide, not as a transmembrane segment, and thus autotaxin is synthesized as a secreted protein. Recently, using domain swapping and mutagenesis experiments, Jansen et al. (2005) reached the similar conclusion that autotaxin is synthesized as a prepro-enzyme and the proteolytically processed protein is secreted. Our direct results using in vitro translation analysis further strengthen the notion that autotaxin possesses a cleavable signal sequence.
Since autotaxin mRNA is robustly expressed in the choroid plexus of the adult mouse and rat brain (this study; Narita et al. 1994; Fuss et al. 1997; Bächner et al. 1999) and autotaxin protein is detected in the CSF from rats, dogs, and humans (this study; Tanaka et al. 2004; Sato et al. 2005), it is likely that autotaxin produced in the choroid plexus is efficiently secreted into CSF in many species. Recently, Sato et al. (2005) showed that autotaxin is responsible for the long-lasting response of CSF that induces neurite retraction of differentiated PC12 cells over several hours. In addition, they found that leptomeningeal cells expressed the highest levels of lysophospholipase D activity and autotaxin protein. Therefore, autotaxin expressed in the choroid plexus and leptomeninges may exert pleiotropic effects and also function as a barrier for extending neurites. Because autotaxin was also detected in human serum and seminal fluid (Tanaka et al. 2004) and LPA was found to be produced in biological fluids such as human follicular fluid, saliva, and ascites (Tokumura 2002), autotaxin may in general be secreted and function in the fluid environment.
By determining the N-terminal sequence of the secreted autotaxin, we showed that rat autotaxin is cleaved between Gly27 and Phe28, and between Arg35 and Ala36. Jansen et al. (2005) reported that proteolytic cleavage occurred at exactly the same sites in rat autotaxin that was purified from the culture medium of autotaxin-transfected HEK293 cells. However, the N-terminus of autotaxin that was originally purified from human melanoma cells started from Asp49 (Murata et al. 1994), whereas the protein purified from human plasma started from both Ala36 (Tokumura et al. 2002). Therefore, 36Ala-autotaxin produced by furin cleavage, which we detected as the major form of autotaxin in the supernatant of the 293EBNA cells, is indeed present in the native protein, but the molecular mechanism that generates 49Asp-autotaxin remains to be elucidated. In addition, Jansen et al. (2005) reported that specific lysophospholipase-D activity of the mature autotaxin protein was about 30% higher than that of the pro-autotaxin, but the activity of 49Asp-autotaxin remains to be determined. Therefore, it will be necessary to determine the functional difference of three forms of autotaxin in future. Because MMPs, a growing family of zinc-dependent proteolytic enzymes that regulate many biological processes, are known to play a pivotal role in the functional regulation of growth factors and their receptors, cytokines, cell surface proteoglycans, and a variety of enzymes (Sternlicht & Werb 2001; Stamenkovic 2003), we tested the effect of MMP inhibitors on autotaxin secretion, but we did not observe any effects. In contrast, monensin, a secretion inhibitor, almost completely abolished the secretion of autotaxin. Because monensin is known to disrupt the trans-Golgi apparatus cisternae near the exit point of secretory vesicles (Mollenhauer et al. 1990), autotaxin is thought to be secreted via a regulatory secretion pathway. Further studies will be necessary to elucidate the molecular mechanism that regulates the proteolytic processing, secretion, and activation of autotaxin.
Taken together, our findings indicate that the N-terminal hydrophobic region of autotaxin functions as a signal peptide, and thus autotaxin is synthesized as a secreted protein. These findings should have implications regarding the functions and regulation of autotaxin, and should provide insights useful for the development of inhibitors of autotaxin for possible therapeutic use.
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Experimental procedures |
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In our previous study (Ohto et al. 2002), we isolated autotaxin cDNA on the basis of its specific expression in the floor plate of the E13 rat embryonic spinal cord. A full-length autotaxin cDNA was isolated from an E18 rat forebrain cDNA library (a kind gift of K. Moriyoshi and S. Nakanishi, Kyoto University). Three positive clones were obtained from about 100 000 plaques. Sequence analysis, including Kyte-Doolittle hydrophobicity plot construction, was performed using GENETYX-MAC (Genetyx Co., Japan). The signal peptidase cleavage site was predicted using the SignalP 3.0 server (Bendtsen et al. 2004; http://www.cbs.dtu.dk/services/SignalP/).
In situ hybridization
In situ hybridization was performed as previously described (Ohto et al. 2002). Briefly, adult mice were perfusion-fixed with 4% paraformaldehyde (PFA) in phosphate buffered saline (PBS) under deep anesthesia induced by intraperitoneal injection of an excess of sodium pentobarbital. After their brains were incubated in 30% sucrose/PBS at 4 °C overnight and embedded in OCT compound (Sakura Finetek Japan), 10-µm-thick sections were cut using a cryostat CM1850 (Leica). The sections were treated with 1 µg/mL proteinase K in PBT (PBS with 0.1% Tween-20) at 37 °C for 5 min, washed and fixed with 4% PFA, and hybridized with 1 µg/mL digoxigenin (DIG)-labeled anti-sense RNA probe (nt 678–1323 of mouse autotaxin cDNA; GENBANK accession number NM015744) in a hybridization solution (50% formamide, 5x SSC pH 4.5, 1% SDS, 50 µg/mL heparin, 50 µg/mL yeast RNA) at 65 °C for 16 h. Slides were washed with 50% formamide, 5x SSC, 1% SDS at 65 °C for 30 min, and with 50% formamide, 2x SSC at 65 °C for 30 min three times, and then incubated with an alkaline phosphatase-conjugated anti-DIG antibody (Roche Diagnostics) at 4 °C overnight. After washing with Tris-buffered saline with 0.1% Tween-20, signals were detected using BM purple (Roche Diagnostics) in the presence of 2 mM levamisole (Sigma) at room temperature.
Production of anti-autotaxin polyclonal antibodies
Rabbits were immunized with synthetic peptides, KVMPNIEKLRSC (ATX-N1) and LKTYLHTYESEIC (ATX-C1), which were conjugated to keyhole lympet hemocyanin (Transgenic Inc.). These two sequences are commonly found in human, rat, and mouse autotaxin. The antisera obtained were purified using peptide affinity columns. Anti-ATX-N1 and anti-ATX-C1 antibodies proved to be useful for immunoblotting and immunohistochemistry, respectively.
Preparation of cerebrospinal fluid
After an adult rat was deeply anesthetized with pentobarbital, cerebrospinal fluid was collected from the cerebromedullary cisterna with a fine needle. All the experiments using animals were approved by the animal care and use committee of the University of Tsukuba, and performed under its guidelines.
Cell culture and transfection
COS-7 (RIKEN cell bank) and 293EBNA (Invitrogen) cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and antibiotics. Cells grown in 35-mm dishes were transfected using Lipofectamine Transfection Reagent (Invitrogen) according to the manufacturer's instructions. To examine the mechanism that regulates the secretion of autotaxin, the cells were incubated with 1–1000 µM matrix metalloproteinase inhibitor I, II, or III (Calbiochem), or with 0.1–10 µM monensin (Nacalai Tesque Inc.) overnight.
Cell surface staining
For cell surface staining experiments, autotaxin, PC-1, and B10 cDNAs tagged with a FLAG sequence (DYKDDDDK) at the C-termini of their open reading frames were subcloned into pCEP4 vector (Invitrogen). Mouse PC-1 and human B10 cDNAs were provided by J.W. Goding and Y. Hayashi, respectively. Twenty-four hours after COS-7 cells grown on cover slips were transfected, live cells were incubated with anti-FLAG (M2) monoclonal antibody (1 : 2000, Sigma) in PBS with 1% heat-inactivated normal goat serum (HINGS) at room temperature for 1 h. After washing, the cells were fixed with 4% PFA and then incubated with Cy3-conjugated anti-mouse IgG antibody (1 : 1000, Jackson ImmunoResearch Laboratories). Fluorescent images were recorded using a laser scanning microscope LSM510 (Zeiss).
Immunohistochemistry
Immunohistochemical analysis of autotaxin in the adult mouse choroid plexus was performed using anti-ATX-C1 antibody. For cell surface staining, the choroid plexus was dissected out and incubated with anti-ATX-C1 antibody (1 : 500) in PBS with 1% HINGS at 4 °C for 6 h. After washing, it was fixed with 4% PFA, permeabilized in PBS supplemented with 1% HINGS and 0.1% Tween-20 (PHT), and incubated with Cy3-conjugated anti-rabbit IgG antibody (1 : 1000, Jackson ImmunoResearch Laboratories). For cell staining, the choroid plexus was fixed with 4% PFA, permeabilized in PHT, and incubated with anti-ATX-C1 antibody at 4 °C for 6 h, and then with Cy3-conjugated anti-rabbit IgG antibody. Fluorescent images were observed after making flat-mounted preparations or cryostat sections. To examine the intracellular localization, cryostat sections of the fixed choroid plexus were incubated with anti-ATX-C1 and anti-PDI antibodies (1 : 500, Stressgen Bioreagents) in PHT solution. After washing, the slides were incubated with Cy3-conjugated anti-rabbit IgG antibody, Alexa-488-conjugated anti-mouse IgG antibody (1 : 1000, Molecular probes), and 4',6-Diamidino-2-phenylindole dihyrochloride (1 µg/mL, Wako).
Immunoblot analysis
For immunoblotting of autotaxin, cells grown in 35-mm dishes were transfected with autotaxin cDNA subcloned into pCEP4, and pEGFP-N1 (BD Biosciences) was used as a negative control. Twenty-four hours after transfection, the culture medium was replaced with serum-free Opti-MEM I medium (Invitrogen) supplemented with GlutaMax I (Invitrogen) and the cells were further cultured. Cells were harvested 48 h after transfection and lyzed in ice-cold buffer containing 10 mM Tris-HCl, pH 8.0, 1 mM CaCl2, 0.1% Triton X-100 and Complete Protease Inhibitor (Roche Diagnostic). The lysates were centrifuged at 20 000 g for 15 min at 4 °C, and the supernatants were used for immunoblotting. Culture supernatants were centrifuged at 20 000 g for 15 min at 4 °C to remove cell debris. Protein concentrations were determined using a Micro BCA protein assay kit (Pierce). Five micrograms of cell lysates and 1 µg of culture supernatants were separated by SDS-PAGE and transferred to Immobilon-P PVDF membranes (Millipore). After blocking in a buffer containing 5% skim milk (Nacalai Tesque Inc.) in PBS supplemented with 0.1% Tween-20, the membranes were incubated with anti-ATX-N1 polyclonal antibody (1 : 1000) and then with horseradish peroxidase (HRP)-conjugated anti-rabbit IgG antibody (1 : 10 000, Bio-Rad). The signal was detected using ECL Western Blotting Detection Reagents (Amersham) according to the manufacturer's instructions.
Enzyme assay
For measuring phosphodiesterase activity, transfection and preparation of the cell lysates and supernatants were done as described in ‘Immunoblot analysis.’ Three micrograms of the cell lysate (approximately 1% of the total cell lysate) and 100 ng of the supernatant were incubated with p-nitrophenyl thymidine-5'-monophosphate (Sigma) at 37 °C for 30 min and the OD at 410 nm was measured (Gijsbers et al. 2003).
Protein sequencing of autotaxin
For protein sequencing of autotaxin, the culture supernatant of the 293EBNA cells transfected with pCEP4-autotaxin was prepared as described in ‘Cell culture and transfection’ above. The collected medium (3 mL) was precipitated with trichloroacetate, subjected to SDS-PAGE, and transferred to an Immobilon-P membrane, which was then stained with Coomassie Brilliant Blue R-250 (Bio-Rad). A protein band of about 120 kDa, which was detected in the supernatant of the autotaxin-transfected cells but not of untransfected cells, was excised and subjected to Edman degradation analysis to determine its N-terminal amino acid sequence.
In vitro translation
In vitro translation was performed using the Rabbit Reticulocyte Lysate System (nuclease treated, Promega) and Canine Pancreatic Microsome Membranes (Promega) according to the manufacturer's instructions. First, the 5'ß-globin leader sequence and a FLAG sequence with a stop codon were added to the 5'- and 3'-ends, respectively, of the cDNA encoding the autotaxin N-terminal fragment (amino acid residues 1–243) and the resulting construct was subcloned into pBluescript II KS+ (Stratagene). The resultant cDNA was linearized with NotI and transcribed in vitro using an mMessage mMachine Capped RNA Transcription Kit (Ambion) and T3 RNA polymerase (Roche). The in-vitro transcribed mRNA (500 ng) was translated using reticulocyte lysate in the presence or absence of microsome membranes at 30 °C for 2 h. In protease digestion experiments, aliquots of the translation products were treated with 25 ng/µL proteinase K at 37 °C for 30 min, in the presence or absence of 1% Triton X-100. To remove the N-linked oligosaccharides, aliquots of the translation products were incubated with 0.5 mU of endoglycosidase H (Roche) in the presence of 1% Triton X-100 at 37 °C for 2 h. The products were subjected to immunoblotting using anti-FLAG (M2) antibody (1 : 1000), HRP-conjugated anti-mouse IgG antibody (1 : 10 000, Chemicon), and ECL Western Blotting Detection Reagents.
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Acknowledgements |
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The nucleotide sequence reported in this paper has been submitted to the GENBANK database with accession number DQ131564.
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Footnotes |
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* Correspondence: E-mail: mmasu{at}md.tsukuba.ac.jp
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References |
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Received: 1 September 2005
Accepted: 6 November 2005
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PDE), which was calculated by subtracting the endogenous PDE activity in the GFP-transfected cells, are indicated below. PDE activity was measured using p-nitrophenyl thymidine-5'-monophosphate and is shown as nmol degradation per h per µg protein. (B) Effects of matrix metalloproteinase inhibitors and monensin on autotaxin secretion. The supernatants and cell lysates of the autotaxin-transfected 293EBNA cells that were incubated with one of three different inhibitors for matrix metalloproteinases (MMPI-I, II, and III) or monensin at the indicated concentrations were analyzed by immunoblotting with anti-autotaxin antibody. MMP inhibitors had no effect on autotaxin secretion, while monensin effectively inhibited the secretion of autotaxin. C, control with no drug treatment.
