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J. Biol. Chem., Vol. 282, Issue 16, 11982-11995, April 20, 2007
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-, 
-, and 
-Secretase*
1
From the
Adolf-Butenandt-Institut, Ludwig-Maximilians-University, Schillerstrasse 44, 80336 Munich, Germany and the Center for Human Genetics, Department of Molecular and Developmental Genetics, Flanders Interuniversity Institute of Biotechnology, Katholieke Universiteit Leuven, Herestraat 49, 3000 Leuven, Belgium
Received for publication, January 12, 2007 , and in revised form, February 15, 2007.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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-, 
- and 
-secretase are involved in the pathogenesis of Alzheimer disease (AD). Increased proteolytic processing and secretion of another membrane protein, the interleukin-1 receptor II (IL-1R2), have also been linked to the pathogenesis of AD. IL-1R2 is a decoy receptor that may limit detrimental effects of IL-1 in the brain. At present, the proteolytic processing of IL-1R2 remains little understood. Here we show that IL-1R2 can be proteolytically processed in a manner similar to APP. IL-1R2 expressed in human embryonic kidney 293 cells first undergoes ectodomain shedding in an -secretase-like manner, resulting in secretion of the IL-1R2 ectodomain and the generation of an IL-1R2 C-terminal fragment. This fragment undergoes further intramembrane proteolysis by -secretase, leading to the generation of the soluble intracellular domain of IL-1R2. Intramembrane cleavage of IL-1R2 was abolished by a highly specific inhibitor of -secretase and was absent in mouse embryonic fibroblasts deficient in -secretase activity. Surprisingly, the -secretase BACE1 and its homolog BACE2 increased IL-1R2 secretion resulting in C-terminal fragments nearly identical to the ones generated by the -secretase-like cleavage. This suggests that both proteases may act as alternative -secretase-like proteases. Importantly, BACE1 and BACE2 did not cleave several other membrane proteins, demonstrating that both proteases do not contribute to general membrane protein turnover but only cleave specific proteins. This study reveals a similar proteolytic processing of IL-1R2 and APP and may provide an explanation for the increased IL-1R2 secretion observed in AD. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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-site APP-cleaving enzymes 1 and 2) (for a review see Ref. 6). The subsequent intramembrane proteolysis is catalyzed by the -secretase protease complex, consisting of the essential proteins PS1 and PS2 (presenilin 1 or 2), nicastrin, Pen-2, and Aph-1 (for a review see Ref. 7). The presenilins are assumed to constitute the active site of -secretase by providing two aspartic acid residues that are critical for -secretase activity (8). Nicastrin functions as a receptor for -secretase substrates (9). As a result of -secretase cleavage, the C-terminal fragments (CTFs) of type I membrane proteins are processed to two smaller fragments. A small peptide is secreted into the extracellular space, whereas the intracellular domain is released into the cytosol. For some of these proteins, such as the cell surface receptor Notch and the cell adhesion proteins N- and E-cadherin, the liberated intracellular domain may participate in signal transduction through different mechanisms (2), whereas for other proteins, such as APP, the intracellular domain may be degraded without a prior role in signaling (10).
One of the proteins, for which RIP has been studied in much detail, is APP. In contrast to several other proteins undergoing RIP, the ectodomain cleavage of APP is not only catalyzed by one but by three different proteases, which cleave at distinct peptide bonds. Shedding of APP mainly occurs by an ADAM metalloprotease, which is also referred to as -secretase and cleaves within the A sequence (reviewed in Ref. 11). Additionally, BACE1 (also referred to as -secretase) and its homolog BACE2 cleave in the ectodomain of APP. BACE1 cleaves APP at the N terminus of the A peptide domain, thus catalyzing the first step in the generation of the A peptide (reviewed in Ref. 12), which is deposited in the brain of patients suffering from Alzheimer disease (AD) (reviewed in Ref. 13). BACE2 cleaves APP close to the -secretase cleavage site within the A domain and thus acts as an alternative -secretase (14), preventing the release of an intact A peptide.
An additional substrate for ectodomain shedding is the type II receptor for interleukin-1 (IL-1), which is widely expressed, including neurons (15). IL-1 is a potent inflammatory and immunoregulatory cytokine and is a key factor in the events leading to neurodegeneration following brain trauma, stroke, and brain inflammation. IL-1 can bind to two types of receptors (for an overview see Ref. 16). Binding to the type I receptor (IL-1R1) initiates a signaling cascade that finally leads to NF
B activation. In contrast, the type II receptor (IL-1R2) acts as a decoy receptor, which is capable of binding IL-1 but is incapable of signaling to NFB and thus acts as a "ligand sink" preventing IL-1 from binding to IL-1R1. IL-1R2 is a single-span membrane protein that contains a large extracellular ligand-binding domain, followed by a transmembrane domain and a short cytoplasmic domain of 29 amino acids. This cytoplasmic domain lacks the Toll/IL-1R domain found in the type I receptor, which would be required for the signal transduction by binding and recruiting cytosolic adaptors and kinases, such as MyD88 and IRAK. IL-1 and its signaling have been implicated in multiple ways in AD (reviewed in Ref. 17), but the underlying molecular mechanisms are not yet well understood. For example, brain trauma, which is a risk factor for AD, enhances IL-1 expression. Likewise, increased IL-1 expression has been observed in AD (reviewed in Ref. 18). In turn, overnight stimulation of astrocytes with IL-1 strongly stimulates translation of APP mRNA, leading to increased APP protein levels (19). Potentially, this may result in increased A peptide generation. However, short term treatment of neuroglioma cells with IL-1 has also been shown to stimulate -secretase cleavage of APP (20), which may prevent A generation and increase the secretion of a soluble APP form that is neuroprotective and neurotrophic (reviewed in Ref. 21). Genetic analyses have linked polymorphisms in the two IL-1 genes, IL-1 and IL-1, to an increased risk of AD. Interestingly, both polymorphisms appear to increase IL-1 production in vitro or in vivo (reviewed in Ref. 18). An additional connection between IL-1 and AD is the finding that the soluble, secreted form of IL-1R2 is elevated in cerebrospinal fluid of patients with mild to moderate AD but not in the late stages of the disease (22, 23). Given that secreted IL-1R2 binds IL-1 and acts as a sink for IL-1, the increase in IL-1R2 shedding may be a way of trying to limit detrimental consequences of increased IL-1 expression and activity in the brain. However, the molecular mechanisms governing the proteolytic processing of IL-1R2 are little understood, but the processing seems to depend at least partly on a metalloprotease activity (24, 25), which may be the ADAM protease TNF-converting enzyme (ADAM17) (26). A more detailed knowledge of IL-1R2 processing may help to better understand the role of IL-1R2 secretion and of IL-1 in the AD brain. Here we show that IL-1R2 undergoes a similar set of proteolytic cleavages as APP.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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-secretase inhibitor IV). BACE2-Fc fusion protein was kindly provided by Regina Fluhrer.
Plasmid Construction—Generation of vector peak12 expressing BACE1, BACE2, and HA-tagged alkaline phosphatase (AP) (HA-AP, soluble AP) has been described (32). The cDNAs of all shedding substrates are of human origin. Plasmid peak12/PSGL-1 3tag was described previously (32) and encodes the signal peptide of CD5, followed by an HA epitope tag and the coding sequence of PSGL-1 with an AU1 tag in the extracellular domain and a FLAG tag in the cytoplasmic domain. The PSGL-1 sequence can be cut out using an XbaI and a NotI restriction site. Peak12/HA-Xba-FLAG was generated by PCR and encodes an HA tag between the HindIII and the XbaI site and a FLAG tag between the XbaI and the NotI site. To obtain peak12/CD5-HA-Xba-FLAG, the HindIII/XbaI fragment of peak12/PSGL-1 3tag was cloned into peak12/HA-Xba-FLAG and encodes the CD5 signal peptide followed by an HA and a FLAG tag. The plasmids encoding full-length IL-1R2 or its deletion mutant (lacking part of the ectodomain) (peak12/IL-1R2, peak12/
334-IL-1R2 and peak12/322-IL-1R2) carry the CD5 signal peptide (MPMGSLQPLATLYLLGMLVASVLG), an N-terminal HA tag (YPYDVPDYA followed by the linker sequence SGGGGGLE or SGGGGGLD for the 334 mutant), and a C-terminal FLAG tag and were generated by PCR using appropriate primers. Peak12/329-IL-1R2 was generated in the same way but has no N-terminal HA tag. The PCR fragments (lacking the native signal peptide sequence of IL-1R2) were cloned into the XbaI site of peak12/CD5-HA-Xba-FLAG. Thus, the first amino acid of the IL-1R2 sequence is amino acid Gly-23 of full-length IL-1R2 (numbering corresponding to protein accession number NP_004624
[GenBank]
in the NCBI data base). Amino acid numbers of the IL-1R2 deletion mutants (322, 329, and 334) indicate that the deletions stop before the given amino acid number, which refers to its position within the HA-IL-1R2-FLAG full-length sequence (counting without the CD5 signal peptide). Vector peak12/MMP-IL-1R2 was used for retroviral generation and was obtained by cloning the Hin-dIII/NotI fragment of peak12/IL-1R2 into the HindIII/NotI sites of the peak12/MMP-KilA vector (33). An additional IRES-GFP cassette from peak12/MMP-TK-IRES-GFP (obtained from Felix Randow; IRES is of encephalomyocarditis virus origin) was cloned into the NotI site of peak12/MMP-IL-1R2 to yield peak12/MMP-IL-1R2-IRES-GFP. Vectors pMDtet.G and pMD.gagpol were described previously (34). The coding sequences of CD14 (GPI-anchored protein), CD16 (GPI-anchored protein), and P-selectin (all three lacking the native signal peptide sequence) were amplified by PCR, digested with XbaI and NotI, and cloned into plasmid peak12/PSGL-1 3tag, which was cut with XbaI and NotI. The resulting plasmids encode the CD5 signal peptide followed by an HA epitope tag and the corresponding protein sequence. As templates, pCDM12/CD14 and CD16 (obtained from Brian Seed) and pCMV/P-selectin (obtained from Denisa Wagner) were used. Peak12/pro-TGF-HA encodes human pro-TGF with an HA tag inserted between amino acids His-43 and Phe-44, which is four amino acids C-terminal to the Ala-Val propeptide cleavage site. Thus, after signal peptide and propeptide cleavage, the mature pro-TGF and the soluble TGF retain the HA tag. Peak12/HA-pro-TGF-FLAG contains an additional FLAG tag at the C terminus of pro-TGF and was generated by cloning the HindIII/XbaI-digested PCR fragment of HA-tagged pro-TGF into the HindIII and XbaI sites of peak12/CD5-HA-XBA-FLAG. TGF and TNF cDNAs were from ATCC. Peak12/FLAG-TNF-HA was created by cloning a PCR fragment of FLAG-TNF containing a 5'-HindIII site and a 3'-XbaI site that was then inserted into HindIII/XbaI peak vector, which has an HA tag between the XbaI and the NotI site. The N-terminal, cytoplasmic FLAG tag was added to TNF by PCR and suitable primers and was cloned into the HindIII/NotI sites of the peak12 vector, resulting in peak12/FLAG-TNF. For expression in neurons and glial cells IL-1R2, TGF and CD16 were cut out from the corresponding peak12 plasmids using HindIII and NotI, blunt-ended, and ligated into the SmaI site of the Semliki Forest virus (SFV) type 1. The identity of all constructs obtained by PCR was confirmed by DNA sequencing.
Cell Culture, Transfections, Western Blot—Human embryonic kidney 293-EBNA cells (HEK293) were cultured in Dulbecco's modified Eagle's medium (Invitrogen) containing 10% fetal bovine serum (Hyclone) as described (35). Cells stably expressing wild-type PS1 or its catalytically inactive mutant PS1 D385N are HEK293 cells without the EBNA gene (36). These cells were cultured as above with an additional 200 µg/ml Zeocin (Invitrogen). G418 was added to culture murine embryonic fibroblasts PS1/2-/- knock-out cells. Transfections were carried out using Lipofectamine 2000 (Invitrogen). One day after transfection, the medium was replaced with fresh medium. After overnight incubation, conditioned medium and cell lysate (in 50 mM Tris, pH 7.5, 150 mM NaCl, 1% Nonidet P-40) were collected.
To analyze the effect of PMA and TAPI-1 on shedding, cells were treated as described previously (32). For the detection of secreted and cellular APP by immunoblot, the protein concentration in the cell lysate was measured, and corresponding aliquots of lysate or conditioned medium were directly loaded onto an electrophoresis gel. For transient transfections of IL-1R2, TGF, CD14, and CD16 either together with BACE1 or BACE2, alkaline phosphatase (AP; plasmid HA-AP) was cotransfected as a transfection efficiency control. AP activity was measured as described previously (32, 37). Aliquots of the conditioned medium were treated for 30 min at 65 °C to heat-inactivate the endogenous alkaline phosphatase activity. Corresponding aliquots of lysate or conditioned medium were loaded onto the gel. Immunoblot detection was carried out using the indicated antibodies.
Infection of Primary Neurons and Glial Cells with SFV—Cortical neurons and glial cells were prepared from E14 mouse embryos from BACE1-deficient and BACE1, BACE2 double-deficient mice as described (38, 39). The BACE knock-outs were verified by Northern and Western blot detection and by functional analysis demonstrating that APP cleavage by BACE was virtually eliminated in the neurons (39).
Preparation of recombinant SFV stocks has been described previously (40). Virus was diluted 1:100 in conditioned culture medium and added to 4-day-old neurons. Two hours post-infection, cells were labeled with 100 µCi/ml [35S]methionine/cysteine for 6 h and lysed in immunoprecipitation buffer (1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS in TBS buffer). IL-1R2, TGF, and CD16 full-length and CTFs were immunoprecipitated using anti-FLAG antibody. Immunoprecipitated material was separated by SDS-PAGE, and dried gels were exposed to a PhosphorImager (Amersham Biosciences).
Retroviral Transduction—To produce retroviral supernatants (replication-deficient Moloney murine leukemia virus), plasmids pMDtet.G and pMD.gagpol and either peak12/MMP-GFP or peak12/MMP-HA-IL-1R2-FLAG-IRES-GFP were transfected into HEK293 cells by calcium phosphate precipitation. Medium was changed after 24 h, collected after 48 h, and filtered through a sterile filter. The retroviral transductions were carried out using Polybrene (Sigma).
In Vitro Generation of IL-1R2-ICD—293E cells transiently expressing full-length IL-1R2 were incubated for 4 h with PMA prior to membrane preparation. Membrane preparations were generated as described (41) and were resuspended in citrate buffer (pH 6.4, 5 mM EDTA, inhibitor mixture from Roche Applied Science) with or without 1 µM DAPT and then incubated either at 4 or 37 °C for 2 h. After incubation the supernatant and membranes were separated via ultracentrifugation. HEK293 cells (without EBNA) stably expressing wild-type PS1 or PS1 D385N and additionally transiently expressing peak12/329-IL-1R2-FLAG were directly subjected to membrane preparation without prior PMA incubation and then treated as above.
Coimmunoprecipitation of -Secretase Complex with IL-1R2 Substrate—HA-IL-1R2-FLAG was retrovirally transduced into HEK293 cells stably expressing either PS1 WT or the catalytic inactive mutant PS1 D385N. For immunoprecipitation, one 10-cm dish of each cell line was lysed in standard STE (150 mM NaCl, 50 mM Tris, 2 mM EDTA) + 1% CHAPSO buffer followed by a clarifying spin at 13,000 rpm with a Heraeus cryocentrifuge and a further purification step at 55,000 rpm in a Beckman ultracentrifuge with a TLA-55 rotor. Prior to immunoprecipitation of CTFs with FLAG M2 affinity agarose, the lysates were immunoprecipitated with HA affinity agarose for 2 h leading to a depletion of IL-1R2 full-length protein in the lysate. Subsequently, after a 2-h incubation with the lysate, FLAG M2-agarose was washed two times each with 0.1% CHAPSO wash buffer and STE buffer and afterward eluted with 100 µg of FLAG peptide and subjected to SDS-PAGE. Immunoblot detection was carried out for IL-1R2 CTF and the -secretase complex components nicastrin, presenilin 1 NTF, Aph-1, and Pen-2.
Mass Spectrometry of IL-1R2 Cleavage Sites—For analysis of -, -, and -cleavage of secreted IL-1R2 peptides, HEK293 cells were transfected with peak12/IL-1R2, peak12/334-IL-1R2, or peak12/322-IL-1R2. 48 h after transfection, fresh medium was incubated for 4 h and subsequently put on ice. In case of peak12-322-IL-1R2, medium was supplemented with 1 mmol of DAPT during incubation to prevent turnover by -secretase. Protease inhibitor mixture (Sigma) was added at a dilution of 1:100. Medium was then subjected to a clarifying spin by centrifugation. Afterward medium was subjected to immunoprecipitation with HA-agarose beads for 4 h in the case of 334-IL1R2 construct and for 2 h in the case of 322-IL1R2 construct. Bound peptides were eluted either with a mixture of 0.3% trifluoroacetic acid, 50% acetonitrile, H2O saturated with -cyano matrix, or in the case of the IL-1R2 ectodomain with HA peptide (50 µg) in 300 mM NaCl for MALDI-TOF analysis (Voyager DESTR, Applied Biosystems) and with 0.1% formic acid, 50% methanol, H2O for nanoelectrospray ionization mass spectrometry analysis (Q-STAR Applied Biosystems). MALDI-TOF spectra were recorded in the linear mode and analyzed with Data ExplorerTM (Applied Biosystems).
For determination of protein identity by mass fingerprinting, the precipitated IL-1R2 was digested with trypsin at 37 °C overnight and analyzed by MALDI-TOF. The obtained peptide fragments were compared with the NCBInr data base using MascotTM (Matrix Science). The MALDI-TOF mass spectrometer was either internally calibrated using the masses from tryptic autoproteolysis products or externally using a standard peptide mixture (Sequazyme calibration mixture III, Applied Biosystems). The sequence of the tryptic peptide corresponding to the N terminus of IL-1R2 was determined by direct nanospray infusion of the peptides derived from the tryptic digest and an MS/MS analysis of the doubly charged peptide with an m/z value of 1036.47 by tandem mass spectrometry using a hybrid quadrupole-time of flight mass spectrometer (Q-STAR, Applied Biosystems). MS/MS spectra were analyzed using BioAnalystTM.
Deglycosylation of IL-1R2 Ectodomain—The immunoprecipitated IL-1R2 ectodomain was eluted with HA peptide and subjected to deglycosylation with N-glycosidase F from the native deglycosylation kit (Calbiochem) at 37 °C overnight.
BACE2 in Vitro Assay with Synthetic Juxtamembrane Region Peptide of IL-1R2—50 pmol of synthetic peptide spanning from Val-322 to Ser-341 were digested in 50 mM Na+ acetate buffer, pH 4.4, at different temperatures (e.g. 4 or 37 °C) overnight. For the enzymatic digest, purified BACE2-Fc fusion protein was added to the buffer peptide mixture. For specific inhibition of BACE2, 2 µM C3 were added to the incubation mixture. Digested peptides were purified with C18 ZipTip according to the manufacturer's protocol and measured with an ABI Destr in positive reflector mode in a range from 750 to 3000 Da.
Immunofluorescence of BACE1 and IL-1R2—COS cells were plated on glass coverslips in 24-well dishes. The next day cells were cotransfected with IL-1R2 and BACE1. 48 h after transfection coverslips were washed two times with ice-cold PBS, fixed for 20 min in 4% paraformaldehyde/sucrose, and afterward washed again two times with PBS. To clear cells from remaining paraformaldehyde, the cells were washed with 100 mM NH4Cl for 3 min. Finally the cells were permeabilized with 1% Triton X-100 for 1 min and again washed two times with ice-cold PBS. After permeabilization cells were double-stained with a polyclonal BACE1 NT antibody and a monoclonal HA antibody (both 1:1000). Cells were washed again and afterward incubated with Alexa 488-(BACE1) and Alexa 594 (IL-1R2)-coupled secondary antibody. Cells were then washed in PBS/water and fixed on a glass specimen with Moviol. Specimens were then investigated under a Zeiss confocal microscope.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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-Secretase—Because different type I membrane proteins, including APP and Notch, are first processed by a metalloprotease and then undergo intramembrane proteolysis by -secretase, we next tested whether IL-1R2 is also processed within its transmembrane domain. If IL-1R2 is indeed a novel substrate for -secretase, its CTF should accumulate in cells lacking active -secretase and should no longer be converted to the intracellular domain (ICD) fragment. To test this possibility, three different experimental conditions of -secretase inhibition were tested using the following: (a) the well characterized -secretase inhibitor DAPT (43), (b) the dominant-negative and catalytically inactive PS1 D385N mutant (8), and (c) PS1/PS2-deficient mouse embryonic fibroblasts. First, HEK293 cells expressing IL-1R2 were treated overnight with or without DAPT. Full-length and CTFs of IL-1R2 were detected in the cell lysate by immunoblot using an antibody against the C-terminal FLAG tag. Compared with control treated cells, DAPT strongly increased the amount of the IL-1R2 sCTF without increasing full-length IL-1R2 (Fig. 2A). This finding is consistent with -secretase cleaving IL-1R2. Interestingly, DAPT did not increase the amount of the lCTF, suggesting that lCTF is either converted to the sCTF and thus does not accumulate or that the lCTF is not significantly processed by -secretase. Second, IL-1R2 was transiently transfected into wild-type kidney 293 cells (not the 293-EBNA cell variant) stably expressing wild-type presenilin 1 (PS1) or the dominant-negative PS1 D385N mutant (36). In agreement with a -secretase cleavage of IL-1R2, PS1 D385N strongly increased the amount of short IL-1R2 sCTF compared with wild-type PS1-expressing cells (Fig. 2B). As in Fig. 2A, the lCTF did not accumulate significantly but was generally more clearly detected than in the other figures, where the slightly different HEK293-EBNA cell variant (Fig. 2A) and the fibroblasts (Fig. 2C) were used. Third, compared with wild-type mouse embryonic fibroblasts, retroviral transduction of IL-1R2 into fibroblasts deficient in PS1 and PS2 resulted in accumulation of the IL-1R2 CTF (Fig. 2C, 3rd and 4th panels). Similar to Figs. 1B and 2A, mainly the sCTF was visible. The amount of full-length and secreted IL-1R2 showed no major differences between both cell types. As expected, PS1 was only detected in wild-type but not in PS1/PS2-deficient cells (Fig. 2C, bottom panel shows the PS1 CTF). Additionally, a possible interaction between the IL-1R2 CTF and the -secretase complex was tested in a coimmunoprecipitation experiment. To this aim IL-1R2 was expressed in HEK293 cells stably expressing the -secretase subunit presenilin 1 in the wild-type (WT) or in the catalytically inactive D385N form. Immunoprecipitation of the IL-1R2 C-terminal fragment led to coprecipitation of all four -secretase complex subunits nicastrin, presenilin 1, Aph-1, and Pen-2 (Fig. 2D). As expected, more -secretase subunits were precipitated in the catalytically inactive PS1 D385N cells compared with the PS1 WT cells. Together, these experiments are consistent with IL-1R2 CTF being a novel substrate for -secretase-mediated intramembrane proteolysis.
As a result of -secretase cleavage, the IL-1R2 CTF should be converted to the ICD fragment. As above, generation of this fragment should be inhibited when -secretase cleavage is blocked. Similar to what is known for the APP ICD (44), the IL-1R2 ICD may be short lived and could not be seen in the cultured cells under steady state conditions (see above Figs. 1 and 2). Thus, an established in vitro -secretase cleavage assay (41) was used for the detection of the IL-1R2 ICD. IL-1R2 was transiently transfected into HEK293 cells. The cells were additionally treated with PMA, which results in increased ectodomain shedding of IL-1R2 and in an increased amount of IL-1R2 CTFs (Fig. 1B), which can serve as substrates for subsequent -cleavage. Membranes from these cells containing full-length IL-1R2 and CTFs were incubated for 2 h at 37 °C. This resulted in the generation of the ICD of IL-1R2, which was released from the membrane and found in the supernatant after ultracentrifugation (Fig. 3A, bottom panel). Addition of the -secretase inhibitor DAPT or incubation at 4 °C prevented ICD generation (Fig. 3A). As an additional control, a further in vitro cleavage assay was carried out, in which an N-terminally truncated form of IL-1R2 was expressed. This mutant protein lacks most of the ectodomain (329-IL-1R2; Fig. 3B) and corresponds approximately to the IL-1R2 CTF generated by the PMA-inducible metalloprotease cleavage. Truncated forms of -secretase substrates, such as C99 derived from APP (45) and NotchE derived from Notch (46), can directly be cleaved by -secretase and do not need to undergo ectodomain shedding. 329-IL-1R2 was transiently transfected into HEK293 cells expressing wild-type PS1 or the dominant-negative PS1 D385N mutant. Membranes were prepared and treated in the -secretase in vitro assay as above. Generation of the IL-1R2 ICD was detected in the PS1 WT but not in the PS1 D385N-expressing cells and was also absent in the PS1 WT cells, when treated with the -secretase inhibitor DAPT or when incubated at 4 °C (Fig. 3C). 329-IL-1R2 was detected as a doublet band in the membrane fraction. Taken together, these experiments demonstrate that IL-1R2 is a novel substrate for -secretase.
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-Secretase Cleavage Site by Mass Spectrometry—Besides ICD generation, -secretase cleavage is expected to generate a soluble N-terminal peptide derived from IL-1R2, which we refer to as IL-1R2-peptide in analogy to the APP-derived A peptide and the Notch-derived N peptide (47). To determine the cleavage site of -secretase within the IL-1R2 transmembrane domain, 334-IL-1R2 was expressed in HEK293 cells. Compared with 329-IL-1R2, this mutant has an HA tag at the N terminus (Fig. 4A), which was used to immunoprecipitate the IL-1R2-peptide from the conditioned medium. MALDI-TOF mass spectrometry analysis revealed three major peptide species and several minor peptides (Fig. 4B). All peptides were generated in a -secretase-dependent manner, because the -secretase inhibitor DAPT blocked their generation. As expected, none of the peptides was visible in control transfected cells. The peptide peak at 3678 Da corresponds to a cleavage site between Ser-353 and Leu-354 in the middle of the transmembrane domain (Fig. 4, A and C). An additional fragment of 3197 Da indicates a cleavage site between Val-348 and Leu-349 in the N-terminal half of the transmembrane domain. The shortest peptide with a mass of 2507 Da corresponds to a cleavage site at the very N terminus or just outside of the IL-1R2 transmembrane domain. Alternatively, the two shorter peptides at 3197 and 2507 Da may arise through C-terminal truncation of the longer peptide at 3678 Da. The masses of the additional minor peptide peaks correspond exactly to C-terminally truncated peptides with regard to the peptide peaks at 3197 and 3678 Da. Thus, the mass spectrometric analysis shows that, like APP and other -secretase substrates, IL-1R2 is cleaved by -secretase within its transmembrane domain. A potential additional 
-cleavage at the C-terminal end of the transmembrane domain was not investigated.
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To exclude the possibility that transfection of BACE1 or BACE2 increased IL-1R2 shedding simply by activating the -secretase-like metalloprotease cleavage, we tested the following: (a) whether the metalloprotease inhibitor TAPI-1 is able to reduce the BACE1-induced increase in IL-1R2 shedding, (b) whether BACE1 and IL-1R2 colocalize in transfected cells, and (c) whether BACE2 is able to cleave in vitro a synthetic peptide encompassing the juxtamembrane domain of IL-1R2. First, HEK293 cells were transfected with IL-1R2 and either BACE1 or empty vector as a control. Treatment with the metalloprotease inhibitor TAPI-1 largely inhibited IL-1R2 shedding under control conditions (Fig. 5B, see also Fig. 1B) but did not significantly reduce the increased IL-1R2 shedding in the BACE1-transfected cells. Addition of the specific BACE inhibitor C3 (49, 50) was able to reduce the increased IL-1R2 shedding in the BACE1-transfected cells. This shows that BACE1 transfection did not indirectly increase IL-1R2 shedding by stimulating the -secretase-like cleavage. Second, immunofluorescence microscopy was used to test for a colocalization between BACE1 and IL-1R2 in transfected COS cells, which are large and adherent and thus allow good visualization of the cellular localization of both proteins. Colocalization was observed at or close to the plasma membrane, in vesicular structures, and at perinuclear sites (Fig. 5C), which agrees with the intracellular localization of BACE1 observed in other studies (48, 51, 52). Third, an Fc fusion protein of BACE2 was purified and incubated at 37 °C in vitro with a synthetic peptide encompassing the juxtamembrane domain of IL-1R2. This domain was previously shown to be required for IL-1R2 secretion (42). Generation of the cleavage products was monitored by mass spectrometry (Fig. 5D). The intact peptide incubated without BACE2 showed the expected mass of 2219.7 Da (Fig. 5D). Incubation with BACE2 resulted in the formation of two smaller fragments with masses of 916.7 and 1321 Da, revealing a cleavage between Phe-329 and Gln-330 (Fig. 5D). The fragment peaks were not observed when BACE2 was incubated in the absence of the peptide, showing the specificity of the peptide fragment peaks. Importantly, the specific BACE protease inhibitor C3 (Fig. 5D) as well as incubation at 4 °C (not shown) completely inhibited the peptide cleavage. Together, these experiments indicate that IL-1R2 can be directly cleaved by BACE1 and BACE2.
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322-IL-1R2) was used. This protein lacks most of the IL-1R2 ectodomain but retains the juxtamembrane cleavage region, which is required for secretion (42), and was used in the in vitro assay and also encodes the transmembrane and cytoplasmic domains of IL-1R2 (Fig. 6, C and D). Thus, 322-IL-1R2 is similar to N-terminally truncated APP constructs, which, like full-length APP, are cleaved by - and -secretase. 322-IL-1R2 and either control vector BACE1 or BACE2 were cotransfected into HEK293 cells. A strong increase in the formation of IL-1R2 CTFs was observed in the BACE1- and BACE2-expressing cells compared with the control cells (Fig. 6C). These CTFs had the identical apparent molecular weight as the CTFs generated from full-length IL-1R2 (not shown), revealing that cleavage of the truncated 322-IL-1R2 occurs in the same manner as for full-length IL-1R2. The secreted short ectodomain of 322-IL-1R2 was immunoprecipitated from the conditioned medium and analyzed by MALDI-TOF mass spectrometry. Cells expressing 322-IL-1R2 (but not transfected with BACE1 or BACE2) secreted a peptide with a mass of 3112.2 Da, which was not present in control transfected cells not expressing 322-IL-1R2. Because these cells were not transfected with BACE1 or BACE2, this peptide is likely to result from the metalloprotease cleavage and corresponds to a cleavage between Arg-333 and Thr-334. Additional minor peaks were observed at lower masses and correspond to C-terminally truncated peptides. They may arise through alternative proteolytic cleavages or because of the remaining low level carboxypeptidase activity. Transfection of BACE1 and BACE2 led to a strong increase in the generation of a peptide with a mass of 2614 Da, which corresponds to a cleavage between Phe-329 and Gln-330. This site is identical to the one determined in the in vitro BACE2 cleavage assay. The metalloprotease generated fragment (3112 Da) was only visible at low amounts. This analysis indicates that the metalloprotease cleavage site and the BACE1/BACE2 cleavage sites in IL-1R2 are indeed very close to each other, at a distance of four amino acids.
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(converted to soluble TGF upon shedding) and the cell adhesion protein P-selectin are type I membrane proteins, the lipopolysaccharide receptor CD14, and the Fc receptor CD16 are GPI-anchored proteins, and TNF is a type II membrane protein. Like APP and IL-1R2, all proteins undergo ectodomain shedding in a metalloprotease-dependent manner and can be found in soluble form in physiological body fluids (53, 54). The proteins were tagged with an HA epitope tag in their extracellular domain. The three type I and II membrane proteins (pro-TGF, P-selectin, and TNF) were additionally tagged with a FLAG tag in their intracellular cytoplasmic domain. Transient transfection of these constructs together with control vector, BACE1, or BACE2 was carried out as above for APP and IL-1R2. The full-length and secreted forms of all proteins were detected in the cell lysate and the conditioned medium, respectively, at their expected apparent molecular weight (Fig. 7, A–E). BACE1 and BACE2 increased the secretion and reduced the cellular levels of pro-TGF (Fig. 7A) and of CD16 (Fig. 7B) but not of P-selectin, TNF, and CD14 (Fig. 7, C–E), revealing that both proteases do not cleave all membrane proteins undergoing ectodomain shedding. Moreover, for pro-TGF an increase in the CTFs was detected in the cell lysate (Fig. 7A), which is consistent with a proteolytic cleavage very close to the membrane domain. Similar to IL-1R2, the BACE1- and BACE2-induced CTFs of pro-TGF had a very similar apparent molecular weight as the CTFs observed under control conditions (Fig. 7A). Because CD16 is a GPI-anchored protein, no CTFs were observed in the lysate. Expression of transfected BACE1 and BACE2 was verified by immunoblot (not shown). |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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-secretase-dependent intramembrane proteolysis. Among these proteins, only APP is additionally subject to ectodomain shedding by the -secretase BACE1 and its homolog BACE2. Thus, APP serves as a good reference for the study of the proteolytic processing of type I membrane proteins. Processing of APP is a key event in the pathogenesis of AD. Proteolytic processing of another membrane protein, the IL-1R2, has also been linked to AD. In particular, increased amounts of the secreted IL-1R2 ectodomain have been detected in the cerebrospinal fluid of patients with an early stage but not a late stage of AD compared with agematched controls (22, 23). In agreement with previous studies (24–26), we found that shedding of IL-1R2 shows the typical features of a protein undergoing -secretase-like ectodomain shedding by an ADAM protease. IL-1R2 shedding could be stimulated with the phorbol ester PMA and could be blocked by the metalloprotease inhibitor TAPI-1. By using mass spectrometry we found that the -secretase-like cleavage site is located in the membrane-proximal stalk region of IL-1R2. The ectodomain has proline 309 as a C-terminal residue, whereas the truncated 322-IL-1R2 has Arg-333 as a C-terminal amino acid. This could indicate that there are two different cleavage sites for the metalloprotease. However, we assume that the initial cleavage site is at Arg-333 and that the ectodomain is then trimmed, potentially by a carboxypeptidase, until proline 309. In fact, carboxypeptidases cleave inefficiently at proline residues (55) and thus may not be able to further trim the C terminus beyond proline 309. Moreover, a previous study (42) would agree well with a cleavage at amino acid 332 but not at 309. That study replaced amino acids His-324 through Ser-341 in the membrane-proximal domain of IL-1R2 by the corresponding residues of the epidermal growth factor receptor membrane-proximal domain, and they found that this mutation prevented normal secretion of the IL-1R2 ectodomain (42). Additionally, the APP -cleavage is strongly reduced by a proline mutation close to the cleavage site (56, 57), suggesting that metalloprotease-mediated ectodomain shedding occurs in helical conformations, making it unlikely that the -like cleavage in IL-1R2 occurs directly at proline 309. Interestingly, APP is cleaved C-terminally to a lysine residue and IL-1R2 C-terminally to an arginine, suggesting a preference of the -secretase-like cleavages for a positively charged residue.
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-secretase. Using different experimental approaches, including a -secretase inhibitor and cells deficient in -secretase activity, we demonstrate that IL-1R2 is a novel -secretase substrate. This fits well with the general idea that the CTFs of type I membrane proteins, which undergo ectodomain shedding, are subsequently further cleaved by -secretase, leading to the release of their ICD. -Secretase cleavage of CTFs and generation of the ICD may serve different purposes, including signal transduction and membrane protein degradation (2). Similar to APP, the physiological role of -cleavage of IL-1R2 remains unknown. It may be a degradation pathway for the IL-1R2 CTF. Alternatively, it may allow the IL-1R2 ICD to participate in a new kind of signal transduction, similar to Notch.
An additional outcome of our study is that transfection of the -secretase BACE1 and its homolog BACE2 can stimulate secretion of IL-1R2 and generation of its CTFs. As determined by mass spectrometry, the cleavage occurs C-terminally to a phenylalanine, which agrees well with the known cleavage specificity of BACE1 and BACE2. Both proteases preferentially cleave after hydrophobic residues, such as leucine in the Swedish mutant form of APP, in PSGL-1, and ST6Gal I or after methionine in wild-type APP (32, 58–60).
If IL-1R2 is also cleaved at endogenous expression levels of BACE1 and BACE2 and thus constitutes a novel substrate for both proteases, its cleavage should be reduced in BACE1 or -2 knock-out cells, which, however, was not the case. This is in contrast to other established substrates for both proteases, such as APP (39, 61–63), PSGL-1 (32), the sialyltransferase ST6Gal I (64), -subunits of voltage-gated sodium channels (65), and neuregulin-1 (50, 66). This finding could be interpreted in two ways, one being that IL-1R2 is not a physiological substrate for BACE1 and -2 and is only cleaved upon overexpression of both proteases. Although we cannot fully rule out this possibility, we consider it unlikely because of the points discussed below. First, we found that BACE1 and -2 do not simply cleave all membrane proteins tested. For example, TNF, P-selectin, and CD14 (this study) do not undergo increased shedding upon transfection of BACE1 and -2. Likewise, we previously reported that both proteases did not increase shedding of L-selectin and TNF receptor 2 (32). Thus, BACE1 and -2 clearly are specific with regard to the proteins that are cleaved and secreted in response to expression of both proteases. Second, BACE1 is expressed in all tissues, which express IL-1R2. Therefore, IL-1R2 cleavage may also occur at endogenous expression levels of BACE1. Third, we found that BACE1 and -2 induced cleavage of IL-1R2 at a peptide bond very close (four amino acids difference) to the cleavage site of the -secretase-like metalloprotease. This situation is very similar to APP, where the BACE2 cleavage site is at a distance of three to four residues from the -secretase cleavage site, such that the APP CTFs generated by both proteases (C83 through -cleavage and C79 through BACE2 cleavage) are not distinguishable by gel electrophoresis (67, 68). As a consequence, the amount of C83/C79 CTFs in BACE2 knock-out cells is not significantly different from wild-type cells (39), because C83 is still generated and presumably to an enhanced extent. Likewise, we expect that the potential decrease in IL-1R2 processing in BACE1- and BACE2-deficient cells would be compensated for by an increased metalloprotease cleavage, such that no net change in total IL-1R2 processing would be observed. If a similar situation of nearby cleavage sites by different proteases is found in other membrane proteins, it may be a particular challenge to unequivocally identify a given protein as a novel substrate for BACE proteases. In fact, a similar result was observed for the type I membrane protein TGF and the GPI-anchored protein CD16. Similar to IL-1R2, processing of both proteins was strongly enhanced upon transfection of BACE1 and -2, but their processing was not significantly altered in BACE1-deficient or BACE1 and BACE2 double-deficient cells (not shown). Both TGF and CD16 are subject to an -secretase-like cleavage in their membrane-proximal domain, which generates fragments of the same apparent molecular weight as the ones generated by transfected BACE1 and BACE2. Thus, like IL-1R2, both TGF and CD16 may be cleaved by BACE1 and -2 at a site very close to or identical to the metalloprotease cleavage site. Potentially, BACE1 and BACE2 may have a more general role as alternative -secretase-like proteases and be involved in the shedding of additional type I, type II, and GPI-anchored membrane proteins known to undergo ectodomain shedding. This hypothesis may also provide a molecular explanation for the relatively mild phenotype observed in BACE1-deficient (61–63) and in BACE1 and BACE2 double-deficient mice (39). With regard to the large number of ADAM proteases, which have at least partially redundant functions (4), BACE1 and -2 seem to be expressed at low levels. If both proteases act as alternative -secretases, their loss of expression could be compensated for by the ADAM proteases and would not necessarily lead to a similarly strong phenotype as it is observed for some of the ADAM protease knock-out mice (69, 70).
Regardless of whether or not IL-1R2 is cleaved by the endogenous BACE1, our finding that increased BACE1 expression enhances IL-1R2 shedding may provide a molecular explanation for a neuropathological change observed in AD brain. BACE1 expression is up-regulated in AD patients (71, 72) and thus may potentially be the cause of the enhanced amount of soluble IL-1R2 found in AD patients (22, 23). The increase in IL-1R2 shedding has been suggested to be a reaction of the brain to increased IL-1 concentrations found in AD brain (22). Given that IL-1 has been linked in multiple ways to AD (18), the increase in soluble IL-1R2 may be a cellular response aimed at binding the excess IL-1 and preventing detrimental effects of too much IL-1 in the brain.
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FOOTNOTES |
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1 To whom correspondence should be addressed. Tel.: 49-89-218075453; Fax: 49-89-218075415; E-mail: Stefan.Lichtenthaler{at}med.uni-muenchen.de
