1 Division of Biochemistry, Flanders Interuniversity Institute for Biotechnology, Faculty of Medicine, K.U.Leuven, B-3000 Leuven, Belgium
2 Department for Human Genetics, Flanders Interuniversity Institute for Biotechnology, Faculty of Medicine, K.U.Leuven, B-3000 Leuven, Belgium
Author for correspondence (e-mail: Mathieu.Bollen{at}med.kuleuven.ac.be)
Accepted 13 April 2005
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Summary |
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Key words: Autotaxin, NPP2, Lysophospholipase D, Furin, Signal peptide
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Introduction |
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NPP2 is one of seven known members of the nucleotide pyrophosphatase/phosphodiesterase (NPP) family (reviewed by Bollen et al., 2000; Goding et al., 2003
; Duan et al., 2003
). The NPPs have a structurally related catalytic domain and an identical catalytic mechanism, but they differ in their substrate specificity (Gijsbers et al., 2001
; Gijsbers et al., 2003a
; Koh et al., 2003). The best-characterized NPP is NPP1, which releases nucleoside 5'-monophosphates from a variety of nucleotides and nucleotide derivatives. For example, NPP1 releases pyrophosphate from ATP, an inhibitor of bone mineralization and tissue calcification (Bollen et al., 2000
; Goding et al., 2003
). NPP1 is a type-II transmembrane protein that accumulates in the plasma membrane. It contains a short N-terminal cytoplasmic domain that is involved in the targeting of NPP1 to the basolateral membrane (Bello et al., 2001
), a single transmembrane domain and a large extracellular domain. The extracellular domain consists consecutively of two somatomedin-B like domains that mediate homodimerization via disulfide bonds, the catalytic domain and a C-terminal nuclease-like domain with an unknown function (Goding et al., 2003
; Gijsbers et al., 2003b
).
Since NPP1 and NPP2 have a similar domain structure, it has always been assumed that NPP2 is also an integral membrane protein and that the extracellular form originates from proteolysis of the plasma membrane-associated precursor, resulting in the release of an N-terminally nicked polypeptide (Clair et al., 1997; Moolenaar, 2002
; Goding et al., 2003
). We have analyzed the origin of extracellular NPP2 and, unexpectedly, we have found that NPP1 and NPP2 follow a different trafficking and maturation pathway. NPP2 appears to be synthesized as a pre-pro-enzyme and the removal of the pro-peptide by furin-like proteases is required for its full activation.
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Materials and Methods |
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Site-directed mutagenesis of the hydrophobic domain was performed using the QuickChangeTM kit (Stratagene). Four amino acids of NPP2 were changed for the corresponding amino acids of NPP1, such that each mutant had an overlap of one amino acid with the previous one. To monitor the cleavage of NPP2 at a specific site, a Flag-tag was inserted after Gly27, Phe28 or Arg35. This was done by PCR with a sense primer starting at Phe28, Thr29 or Ala36 and containing the Flag-tag at its 5' end, and an antisense primer starting at Gly27, Phe28 or Arg35. After the PCR reaction, the mixture was treated with DpnI, phosphorylated and ligated to obtain circular DNA for transformation of DH5 bacteria. All chimaeric and mutated constructs were verified by sequence analysis.
Cell culture and RNA-interference
HEK293 cells were maintained at 37°C under a humidified atmosphere containing 5% CO2 in Dulbecco's modified Eagle's medium, supplemented with 10% (v/v) heat-inactivated foetal bovine serum, penicillin (100 units/ml) and streptomycin (100 µg/ml). Mouse insulinoma ß-TC3 cells were maintained in DMEM F12 medium under the same conditions. Cells were transiently transfected at 30-40% confluency using the FugeneTM 6 transfection system (Roche Diagnostics). The cells were harvested 24-72 hours after transfection, washed once in PBS and lysed in 50 mM Tris/HCl at pH 7.5, 0.5 mM phenylmethanesulphonyl fluoride, 0.5 mM benzamidine, 150 mM NaCl and 1% (v/v) Triton X-100. After ultracentrifugation (45 minutes at 100,000 g), the supernatant (cell lysate) was used for western blot analysis.
For the RNAi-mediated knockdown of furin or PACE4, short hairpin RNAs (shRNAs), consisting of 19 sense and antisense nucleotides separated by a hairpin loop and complementary to the target mRNA, were cloned into the mU6pro-vector (Yu et al., 2002). The targeted sequences were 5' GACCATTCGACCAAACAGT 3' and 5' GCTCTTCATCCAGTTTTGC 3' for the knockdown of furin and PACE4, respectively. Mouse insulinoma ß-TC3 cells were transiently co-transfected with HA-NPP2-Myc with a Flag-tag inserted after Arg35 and an RNAi plasmid, as indicated. The levels of endogenous furin and PACE4 are too low to be detected by immunoblotting. Therefore, as a control for the RNAi, we overexpressed furin and PACE4 in ß-TC3 cells and showed that the expression of these proteins was silenced with the respective shRNAs. All cells were harvested 48 hours after transfection and analysed by immunoblotting.
Immunofluorescence
HEK293 cells were seeded in four-well chambers at a density of 30,000 cells/well. Cells were transfected using the FugeneTM 6 transfection agent. After 24 hours, the medium was removed and the cells were washed twice in phosphate-buffered saline (PBS). Cells transfected with NPP2-EGFP were fixed and looked at immediately. For the visualisation of NPP1, the endoplasmic reticulum, the Golgi apparatus and the trans-Golgi network, cells were fixed in 2% formaldehyde during 10 minutes. Subsequently, the cells were permeabilised with 40 µg/ml digitonin for 10 minutes. After washes in PBS, non-specific binding was reduced by washing the cells with 3% bovine serum albumin in PBS for 20 minutes. Following this blocking step, the cells were incubated with anti-Myc (clone 9E10) or anti-Golgin-97 (clone CDF4, Molecular Probes, Invitrogen) anti-BiP (donated by L. Hendershot), anti-TGN38 (Transduction Laboratories, Lexington, KY), anti--adaptin-1 (Sigma) antibodies for 90 minutes. Cells were washed again and incubated for 60 minutes with a secondary antibody, i.e. Alexa Fluor 594 (Molecular Probes, Invitrogen) for fluorescence microscopy and anti-mouse FITC (Sigma) or Alexa Fluor 543 for confocal microscopy. After washing in PBS, the fluorescence was visualized with an LSM 510 axiovert 100M laser-scanning microscope or an Olympus fluorescence microscope (pictures not shown), as indicated.
Lysophospholipase-D assay and immunoblotting
Lysophospholipase-D assays were done on aliquots of cell lysates, culture medium or purified autotaxin, as described by Gijsbers et al. (Gijsbers et al., 2003a), with slight modifications. Briefly, the substrate lysophosphatidylcholine (14:0) or sphingosylphosphorylcholine was prepared in 12 mM chloroformmethanol (3:1) and stored at 20°C. Before the assay, an aliquot of the substrate was dried with N2 and reconstituted to 4 mM in 200 mM Tris at pH 9, 10 mM MgCl2, and 10 mM CaCl2. 10 µl of the substrate was mixed with the same volume of sample and incubated at 37°C for 5-60 minutes. Subsequently, the released choline was quantified spectrophotometrically at 540 nm after incubation for 5 minutes with 50 µl of each the peroxidase reagent (50 mM Tris at pH 9.0, 2 mM TOOS, 5 U/ml peroxidase and 0.01% Triton X-100) and the choline oxidase reagent (50 mM Tris at pH 9.0, 2 mM aminoantipyrine, 5 U/ml choline oxidase and 0.01% Triton X-100). The production of choline was linear within the time frame that we adopted and was, because of the high substrate concentration (2 mM) and the limited conversion to product, not hampered by product inhibition (van Meeteren et al., 2005
).
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Purification and N-terminal sequencing of NPP2 fusions
Hybridoma cells (clone 9E10) were used for the production of anti-c-Myc monoclonal antibodies. Cells were grown in DMEM containing 10% foetal bovine serum, 2 mM glutamine, penicillin 100 U/ml, streptomycin 100 µg/ml and 25 mM glucose. When the cells were 80% confluent the medium was replaced by serum-free medium and the cells were maintained in culture for one additional week. Antibodies were purified from the medium by affinity chromatography on Protein-A Sepharose (Amersham). The purified anti-Myc antibodies were randomly coupled to CNBr-activated Sepharose 4B and the matrix was used for affinity purification of NPP2 fusions with a C-terminal Myc tag. The retained NPP2 fusions were eluted with 0.1 M triethanolamine at pH 12. Following SDS-PAGE and blotting onto a polyvinylidene fluoride membrane (Amersham) the NPP2 fusions were visualized by Amido-black 10B (Biorad) staining and N-terminally sequenced the by Edman degradation.
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Results |
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Immunoblotting showed an accumulation of NPP1 in HEK293 cells 48-72 hours after transfection but did not visualize NPP1 in the non-concentrated culture medium (Fig. 1B). By contrast, similar levels of NPP2 were detected in the cell lysates and the non-concentrated culture medium 48-72 hours after transfection. Since the cell volume is small compared to the culture-medium volume, this implies that the large majority of NPP2 was extracellular. Indeed, lysophospholipase-D activity assays revealed that 98.8% of the NPP2 that had been synthesized by 72 hours was present in the culture medium (not illustrated).
To map the protein fragments that are responsible for the different maturation pathways of NPP1 and NPP2, we used a domain-swapping approach (Fig. 2A). Chimaeras of the N-terminal, catalytic and nuclease-like domains of NPP1 and NPP2 were expressed with an N-terminal HA-tag and a C-terminal Myc-tag. All NPP chimaeras with the N-terminal domain of NPP2 were efficiently secreted, whereas the chimaeras with the N-terminus of NPP1 remained cell-associated (Fig. 2B). Swapping of the catalytic and nuclease-like domains did not affect the localization of the resulting chimaeras. Thus, the N-terminal domain emerges as the sole determinant for the targeting of NPP1 and NPP2 to the plasma membrane or the culture medium, respectively. The N-terminal domain of NPP1 and NPP2 consists consecutively of a polar, a hydrophobic and two somatomedin-B-like subdomains (Fig. 2A). Swapping of the hydrophobic subdomain between NPP1 and NPP2, with or without the N-terminal polar subdomain, was sufficient to retarget these isozymes (Fig. 2C). This finding strongly suggests that the hydrophobic subdomain determines the trafficking pathway of NPP1 and NPP2. Finally, we have generated six NPP2 mutants by replacing the residues of the hydrophobic subdomain four by four by the corresponding residues of NPP1 (Fig. 2D). Surprisingly, these mutants accumulated in the medium to a similar extent as did NPP2, suggesting that the targeting of NPP2 is not determined by a sequence motif within its hydrophobic subdomain.
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NPP1 and NPP2 harbour a signal anchor and signal peptide, respectively
In further agreement with the view that the trafficking pathways of NPP1 and NPP2 are determined by their N-terminal hydrophobic subdomain, we observed that NPP1 with the hydrophobic subdomain of NPP2 (NPP1-212-30-1) had a subcellular localization similar to that of NPP2 (Fig. 3A). Conversely, NPP2 with the hydrophobic subdomain of NPP1 (NPP2-159-79-2) was targeted to the plasma membrane. Collectively, our data are consistent with the notion that the hydrophobic subdomain of NPP1 is part of a classical `signal anchor' that mediates the uptake of NPP1 in the endoplasmic reticulum and that anchors NPP1 as a type-II transmembrane protein. The accumulation of NPP2 and NPP1-212-30-1 in the medium could then be the result of intramembrane proteolysis, ectodomain shedding or processing of the N-terminal region as a `signal peptide'. To differentiate between these possibilities we have examined whether there exists a cellular pool of NPP2 with an intact N-terminus, which would be expected to accumulate if NPP2 were solubilized by intramembrane proteolysis or ectodomain shedding during its later stages of maturation. By contrast, if NPP2 were synthesized as a pre-protein, the N-terminal signal peptide would be expected to be removed during translation. In Fig. 3B it is shown that NPP1 and NPP2-159-79-2 in cell lysates retained HA-immunoreactivity and thus had an intact N-terminus, whereas the N-terminus of NPP2 and NPP1-212-30-1 lost its N-terminus, in accordance with the view that NPP2 possesses an N-terminal signal peptide.
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The Signal-P program (Nielsen et al., 1997) predicts with 99% certainty that the N-terminus of NPP1 functions as a signal anchor (not shown), whereas the N-terminal 27 residues of NPP2 are identified with 93% certainty as a cleavable signal sequence (Fig. 4A). Interestingly, the mutations of the hydrophobic region of NPP2 that are described in Fig. 2D and that did not affect the secretion of NPP2, were also not predicted by the Signal-P program to affect the functionality of the signal sequence. The predicted signal sequence of NPP2 consists of an N-terminal polar region with a net positive charge, a hydrophobic core of 10 residues and a C-terminal polar region with a small residue at position 1 (Gly) and an uncharged residue at position 3 (Cys), all in close accordance with the properties of established signal sequences (Martoglio and Dobberstein, 1998
; Stroud and Walter, 1999
). To explore whether NPP2 is indeed intracellularly hydrolysed after Gly27, as predicted by the Signal-P program, we made use of anti-Flag antibodies that either recognize the Flag-epitope only when it contains a free aminoterminus (M1-antibody) or that recognize both an N-terminally free as well as an internal Flag-epitope (M2-antibody) (Stroud and Walter, 1999
). We generated fusions of NPP2 with a Flag-tag inserted after either Gly27 or Phe28. The M2 antibodies recognized both fusions in the cell lysates and the medium, whereas the M1 antibodies only recognized the fusion with the Flag-tag after Gly27, consistent with the prediction that NPP2 is proteolyzed after Gly27 (Fig. 4). As a further support for this conclusion, we expressed HA-NPP2-Myc in HEK293 cells, affinity-purified the NPP2 fusion from the cell lysates after 24 hours and identified by N-terminal sequencing Phe28 of NPP2 as the first residue (not illustrated). Following the removal of the pre-peptide, pre-proteins follow the classical secretory pathway. That this also holds true for NPP2 is indicated by our observation (not illustrated) that the accumulation of NPP2 in the culture medium was largely abolished by brefeldin A, an inhibitor of protein transport from the endoplasmic reticulum to the Golgi apparatus (Klausner et al., 1992).
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NPP2 is also processed by proprotein convertases
C-terminal to the signal peptidase cleavage site, NPP2 contains the sequence (residues 32-35) Arg-Ile-Lys-Arg (Fig. 5A), a consensus site for cleavage by furin and other members of the family of proprotein convertases (PCs) (Thomas, 2002; Taylor et al., 2003
; Duckert et al., 2004
). To examine whether NPP2 is indeed processed by PCs, we introduced a Flag epitope after Arg35. Immunoblotting with M1 and M2 antibodies confirmed that NPP2 was cleaved after Arg35, both intracellularly and in the medium (Fig. 5B). However, the M1/M2 signal ratio was much higher in the medium than in the cell lysate, indicating that proteolysis between Arg35 and Ala36 only occurred just before or after secretion. Cleavage of pro-NPP2 was not detectably affected by the addition of a cell-impermeable inhibitor (0.1 mM hexa-D-arginine) of furin(-like) endoproteases to the culture medium (Cameron et al., 2000
), indicating that processing by furins occurred either primarily intracellularly or by a PC that is insensitive to hexa-D-arginine (not illustrated). Conversely, the proteolysis between Arg35 and Ala36, as detected with the M1 antibodies, was blocked by the co-expression of
1-antitrypsin Portland (
1-PDX) (Fig. 5C), which inhibits most PCs under these conditions (Jean et al., 1998
; Benjannet et al., 1997
). Moreover, following the RNAi-mediated knockdown of furin or PACE4 in mouse insulinoma ß-TC3 cells, the removal of the pro-peptide was hampered (Fig. 5D). Cleavage of NPP2 between Arg35 and Ala36 could also be confirmed by N-terminal sequencing of NPP2 that was affinity-purified from the medium (not shown). Moreover, NPP2 that was affinity-purified from the culture medium after the co-expression with
1-PDX started with Phe28, the first residue after hydrolysis by the signal peptidase.
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Comparison of pro-NPP2 and NPP2
The expression of HA-NPP2-Myc in the presence or absence of 1-PDX enabled us to compare the properties of pro-NPP2 and NPP2. Neither immunoblotting (Fig. 6A) nor pulse-chase experiments (not shown) provided any evidence for different rates of synthesis, secretion and turnover of pro-NPP2 and NPP2. However, affinity-purified NPP2 consistently showed a 30% higher specific lysophospholipase-D activity than did pro-NPP2, assayed with either lysophosphatidylcholine (Fig. 6B) or sphingosylphosphorylcholine (not shown) as substrates. We have also considered the possibility that the pro-peptide is secreted together with NPP2 and regulates its catalytic activity. However, up to 10 µM of the synthetic pro-peptide (FTASRIKR) did not affect the lysophospholipase-D activity of NPP2 (not shown). Moreover, neither the pre-peptide nor the pro-peptide could be detected by immunofluorescence microscopy with antibodies against the N-terminal HA-tag or Flag-tag, respectively (not shown), indicating that these peptides are rapidly targeted for degradation and do not have an additional biological function.
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Secreted and cell-associated NPP2 are differently glycosylated
Compared with the cell-associated pool of NPP2, secreted NPP2 consistently migrated slower during SDS-PAGE (Fig. 2, Fig. 5B, Fig. 7). This difference cannot be explained by the removal of the pro-peptide since pro-NPP2, if anything, migrated slower than NPP2 (Fig. 7, last two lanes; see also Fig. 6). Neither did the distinct migration of cell-associated and secreted NPP2 reflect a different sensitivity to reducing agents, since their mobility remained distinct in the absence of reducing agents. However, after a pretreatment of the cellular and secreted pool of NPP2 with N-glycosidase F they migrated identically, showing that the composition of their N-linked oligosaccharides is different.
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Tokumura et al. previously speculated that NPP2 is a heterodimer of full-length NPP2 and a C-terminal 30-kDa fragment, roughly corresponding to the nuclease-like domain (Tokumura, 2004; Tokumura et al., 2002
). However, we did not detect lower-molecular-mass bands in crude or purified preparations of NPP2 with antibodies against the C-terminal Myc-tag.
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Discussion |
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We have found that cleavage of pro-NPP2 by PCs is associated with a moderate activation of NPP2 as a lysophospholipase-D (Fig. 6B), similar to the activation of BACE, the ß-secretase involved in the processing of the amyloid precursor protein (Benjannet et al., 2001; Creemers et al., 2001
). However, the removal of the pro-peptide does not appear to play a role in the secretion or stability of NPP2 since an inhibition of cleavage by furins did not affect the extracellular accumulation of NPP2 (Fig. 6A). In further agreement with this view we found that NPP1 with the signal peptide of NPP2, but lacking the pro-peptide of NPP2, was also efficiently secreted (Fig. 2C). PCs belong to the seven member family of subtilisin-like proprotein convertases that are implicated in the proteolysis and activation of a variety of substrates including neuropeptides, peptide hormones, growth factors, enzymes, coagulation factors, viral coat proteins and bacterial toxins. Furin is expressed in all cells and represents the workhorse of the family but most cell types also express other isozymes that have (partially) overlapping substrate specificity (Thomas, 2002
; Taylor et al., 2003
). Our data suggest that pro-NPP2 is a substrate not only for furin but also for at least one other isozyme, namely PACE4 (Fig. 5D).
NPP2 is an attractive target for the treatment of cancer because it acts extracellularly and promotes tumour development at various levels (see Introduction). In principle, inhibitors of NPP2 function could be directed towards its synthesis, maturation and/or its catalytic activity. One feasible target would be the inhibition of NPP2 processing by PCs. Interestingly, PCs themselves have been implicated in tumorigenesis, and clinical trials with furin inhibitors as an anticancer treatment are underway (Thomas, 2002; Taylor et al., 2003
). It is possible that the efficiency of furin-inhibitors as anticancer agents stems, at least in part, from their effect on the maturation of NPP2. Conversely, our current work suggests that the inhibition of maturation of NPP2 with furin inhibitors would only moderately decrease the lysophospholipase-D activity of NPP2. Therefore, the development of inhibitors of the transcription or the catalytic activity of NPP2 emerges as a more attractive therapeutic goal.
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Acknowledgments |
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Footnotes |
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