Chen et al. 10.1073/pnas.0709805104. |
Fig. 6. Pulse-chase experiment of KL processing. After transfection with the KL plasmid, cells were pulsed with [35S]methionine and cysteine and chased as indicated. The KL proteins in the medium and lysate were immunoprecipitated with monoclonal rat anti-KL antibodies and analyzed by SDS/PAGE and autoradiography. Arrows indicate the KL fragments in the medium.
Fig. 7. Processing of KL constructs and searching for cleavage sites. (A) Expression of KL, KL V5, and KL GFP constructs in COS-7 cells. The transfection and sample preparation methods were as described in Fig. 1. Similar processed fragments of KL were detected in the medium for all three constructs (lanes 4-6). (B) Western blotting of KL V5 and KL GFP constructs by using anti-V5 and anti-GFP antibodies. The V5 and GFP antibodies recognized the intracellular region of KL, and thus no signal was detected in the proteins from the medium because the tag was removed by the sheddase (M, lanes 2 and 4). The estimated sizes of the KL V5 and KL GFP processing fragments in the cells are indicated. (C) Schematic diagram of the KL V5 construct. The anti-V5 antibody recognition site is indicated. The a- and b-cleavage sites and the estimated molecular mass of the KL V5 fragments are illustrated. (D) Silver staining of the SDS/PAGE gel from the immunoprecipitated samples of the mock transfected (control) and KL GFP transfected (KL GFP) by using anti-GFP antibody. The bands (arrowheads 1, 2, and 3) corresponding to the KL GFP fragments shown in B and C were isolated and prepared for MS analysis as described in Materials and Methods. (E) The sequence of KL GFP with the signal sequence removed. Red, amino acids identified by MS from the 148-kDa band; blue, amino acids identified by MS from both the 98-kDa band; green, amino acids identified by MS from 36-kDa band. The proposed cleavage sites, QKL and KKRK, and the putative N-glycosylation sites (bold, N) are underlined.
Fig. 8. Statistical analysis of the densitometric results of the KL fragments of 130- and 68-kDa bands in Fig. 3A from three independent transfections. Significance of results: *, P < 0.05; **P < 0.005.
Fig. 9. Statistical analysis of the results from Fig. 4D. The intensities of the 130- and 68-kDa bands and ADAM10 (compare lanes 8 and 7, arrowhead 2 in Fig. 4D) and ADAM17 protein (compare lanes 12 and 10 in Fig. 4D) were analyzed densitometrically by using the average intensity of controls from three independent experiments as 100%. Significance of results comparing with its own group (no insulin or with insulin): *, P < 0.05; **, P < 0.005.
Fig. 10. (A) Statistical analysis of the results from Fig. 5A. The intensities of the 130- and 68-kDa bands (lanes 3 and 4), ADAM10 and ADAM17 (lanes 5-8), and sAPP-a were analyzed by using the average intensity of the controls as 100% from three independent experiments. Significance of results: *, P < 0.05; **, P < 0.005. M, medium; L, lysate. (B) Statistical analysis of the results from Fig. 5B from three independent experiments.
SI Text
Materials and Methods
Plasmid Construction.
The KL cDNA in pcDNA3.1 vector was kindly provided by Hal Dietz (Johns Hopkins University School of Medicine, Baltimore). The KL V5 plasmid was prepared by PCR amplification and ligation into pcDNA3.1 V5-His TOPO vector (Invitrogen) by using the following primers: 5-'CACCATGCCCGCCAGCGCCCCGCC-3' and 5'-TTTGTAACTTCTTCTGCCTTTC-3'. To construct the truncated KL980 plasmid with the TM and cytoplasmic regions removed, a XhoI site was introduced into the KL/pcDNA3.1 plasmid by using a QuikChange Site-Directed mutagenesis kit (Stratagene) with the following sense and antisense primers: 5'-TTTCACACCCGAAAGCGCTCGAGGGGCTTTCATAGCTTTTC-3' and 5'-GAAAAGCTATGAAAGCCCCTCGAGCGCTTTCGGGTGTGAAA-3'. The mutated KL/pcDNA3.1 with an XhoI site right after K980 of KL cDNA sequence was digested with XhoI, which removed the KL cDNA sequence-encoding amino acid residues 981-1012 and self-ligated. To construct the KL GFP plasmid, the XhoI-AgeI fragment containing GFP cDNA was ligated into KL/pcDNA3.1 vector digested with XhoI and AgeI. All of the constructs were confirmed by DNA sequencing. The cDNA of ADAM10 with HA tag was kindly provided by Falk Fahrenholz (Johannes Gutenberg-Universität, Mainz, Germany) (1). The cDNAs of human Timp-1, Timp-2, Timp-3, and mouse ADAM17 were purchased from Open Biosystems. Timp-1 cDNA was in pSPORT6 mammalian expression vector. The same method was used to construct Timp-2, Timp-3, and ADAM17 with V5-tag by using pcDNA3.1 V5-His TOPO kit with the following primers: 5'-CACCATGGGCGCCGCGGCCCGC-3' (Timp-2 5'-primer), 5'- TGGGTCCTCGATGTCGAGAAAC-3' (Timp-2 3'-primer), 5'- CACCATGACCCCTTGGCTCGGGCTC-3' (Timp-3 5'-primer), 5'-GGGGTCTGTGGCATTGATGA-3' (Timp-3 3'-primer), 5'-CACCATGAGGCGGCGTCTCCTCATC-3' (ADAM17 5'-primer), 5'-GCACTCTGTCTCTTTGCTGTC-3' (ADAM17 3'-primer).Cell Culture, Transfection, and Protein Sample Collection
. COS-7 cells were maintained in DMEM supplemented with 10% FBS and antibiotics at 37°C and 5% CO2. Cells grown on six-well plates (BD Falcon) were transfected with 1 mg of the expression vectors indicated in each experiment by using Lipofectamine Plus reagent (Invitrogen). The same total amount of DNA (1 mg) was used for each cotransfection. Forty-eight hours after transfection, cells were washed twice with HBSS (Invitrogen) and incubated with serum-free DMEM with or without proteinase inhibitors at 37°C for 1-6 h. The medium was collected and centrifuged at 16,000 ´ g for 1 min to remove the detached cells. Ten micrograms/ml BSA (final concentration) was added to the conditioned medium as a carrier protein and as a control for precipitation efficiency. The samples were then precipitated with 25% TCA (final concentration) and kept at -20°C for 5 min, on ice for at least 1 h, and then centrifuged for 15 min at 16,000 ´ g. The protein pellet was then washed with ice-cold acetone and centrifuged three times for 5 min at 16,000 ´ g. The final protein pellets were dried in a 100°C water bath for 10 min and dissolved in 2X Laemmli sample buffer at 100°C for 10 min. The cells on the plate were washed twice with ice-cold PBS and lysed with RIPA buffer [150 mM NaCl, 1% Triton X-100, 0.5% deoxycholate, 0.1% SDS, 50 mM Tris (pH 7.5)] and 1 mM PMSF. The cell lysate was centrifuged at 16,000 ´ g for 15 min, and the supernatant was collected for SDS/PAGE.Tissue Preparation.
Mouse brain or kidney tissues were perfused with KrebsHeinseleit (6.41 mM Na2HPO4, 1.67 mM NaH2CO3, 137 mM NaCl, 2.68 mM KCl, 5.55 mM glucose, 0.34 mM CaCl2, 2.14 mM MgCl2) and homogenized in 10× (vol/wt) ice-cold RIPA buffer [50 mM Tris·HCl (pH 7.4), 1% Triton X-100, 150 mM NaCl, 0.1% SDS] with 1 mM EDTA, 1 mM PMSF, and 5 mg/ml aprotinin, leupeptin, and pepstatin. Tissues were homogenized with five to six pulses at 60 rps in a Talboys Instrument 101 mechanical homogenizer. Genomic DNA was sheered by passing homogenates through a 25-gauge needle five times. Tissue was then spun at 10,000 ´ g for 30 min, and the supernatant was collected for SDS/PAGE and Western blot analysis.Rat kidney was sliced into 500-mm-thick slices with a Vibratome fresh tissue sectioning system and kept in DMEM bubbling with 95% O2 and 5% CO2 until all of the slices were collected. The kidney slices were washed three times with 50 ml of ice-cold DMEM and incubated in serum-free DMEM in six-well plates (10-12 slices per well) in a tissue culture incubator for 4 h. The conditioned medium was collected and the proteins were precipitated with TCA as described above. The kidney slices were collected and homogenized in RIPA buffer as described. The KL in the tissue lysates and medium was analyzed by SDS/PAGE and Western blot.
Western Blotting.
Protein concentrations were measured by using the Micro BCA Protein Assay Reagent Kit according to the manufacturer's protocol. For SDS/PAGE, cell lysates containing the same amount of total protein were boiled for 5 min and loaded on a 4-20% precast Tris·HCl gels (Bio-Rad). For protein collected from the medium, the whole samples from the TCA precipitation were loaded. Proteins were transferred to nitrocellulose membranes (Millipore). All antibodies were diluted in TBST [50 mM Tris (pH 8.0), 150 mM NaCl, and 0.1% (vol/vol) Tween 20] containing 5% (wt/vol) nonfat dry milk (Carnation). Secondary antibodies were horseradish peroxidase-conjugated goat anti-mouse, anti-rat or anti-rabbit (1:5,000; Kirkegaard and Perry Laboratories). Enhanced chemiluminescence (ECL) was detected by using SuperSignal West Pico Chemiluminescent Substrate (Pierce). Autoradiography was done by using Kodak Scientific Imaging Film X-Omat AR (Eastman Kodak). In addition to the KL antibody, the following primary antibodies were used: anti-V5 rabbit polyclonal antibody (1:5,000; Abcam), anti-ADAM17 rabbit polyclonal antibody (1:1,000; QED Bioscience), anti-Timp-1 rabbit polyclonal antibody (1:1,000; Sigma-Aldrich), anti-HA monoclonal antibody (1:1,000; Sigma-Aldrich), anti-GFP polyclonal antibody (1:1,000; Invitrogen/Molecular Probes), and anti-b-tubulin monoclonal antibody (1:1,000; Zymed/Invitrogen).Pulse-Chase Biosynthetic Labeling and Immunoprecipitation.
Forty-eight hours after transfection, cells were incubated for 30 min in methionine-free MEM. Cells were radiolabeled for 15 min with 60 mCi of Easy Tag Express [35S] label (Perkin-Elmer and chased for 90 min in DMEM. To terminate intracellular transport and secretion, cells were rapidly transferred to ice, the medium was removed and collected, and the cells were washed twice with PBS and lysed in RIPA buffer [0.1% SDS, 1% Triton X-100, 1% sodium deoxycholate, 10 mM Tris·HCl (pH 7.4), 1 mM EDTA, 150 mM NaCl, 1 mM PMSF]. KL proteins were subsequently immunoprecipitated by using a monoclonal rat anti-KL antibody and protein-G agarose. Samples were analyzed by SDS/PAGE and visualized by autoradiography.Immunoprecipitation.
Cell lysates from 1-4 ´ 100-mm plates of cells transfected with Mock or KL GFP were incubated with 5 mg of anti-GFP polyclonal antibody at 4°C for 1 h and then 50 ml of protein-A agarose beads in 50% aqueous slurry for overnight at 4°C. The protein-bound antibody-agarose pellets were washed twice with ice-cold RIPA buffer and twice with ice-cold TSA [10 mM Tris (pH 8), 140 mM NaCl]. The purified proteins were eluted from the antibody-agarose beads by boiling for 5 min in 2× Laemmli sample buffer. The eluted protein samples were loaded onto 4-20% Tris·HCl gels and analyzed by silver staining according to Yan et al. (2).RT-PCR
. Total RNA was isolated by using RNeasy kit (Qiagen) following the manufacturer's protocol. Yields of RNA were determined spectrophotometrically. A reverse transcription was performed with 2 mg of total RNA from each sample. PCRs were performed on 1/10 of each RT sample by using Advantage 2 polymerase (Clontech) with the following condition: 94°C for 3 min, followed by 20 repeating cycles of 94°C (30 s), 55°C (30 s), and 68°C (60 s). The PCR products were analyzed by 1.5% agarose gel electrophoresis. The primers used for PCR were: Timp1 (5'-ATGGCCCCCTTTGAGCCCC-3', 5'-GGCTATCTGGGACCGCAGGG-3'), Timp3 (5'-ATGACCCCTTGGCTCGGGCTC-3', 5'-GGGGTCTGTGGCATTGATGA-3'), ADAM10 (5'-CAGTGGTCGAACCATCACCCTGCAACCTGG-3', 5'-GATAACTCTCTCGGGGCCGCTGACGCTGGG-3'), ADAM17 (5'-CCCAGTAACGTCGAAATGCTGAGCAGCATG-3', 5'-GCTGTCAACACGATTCTGACGCTGCAGTTT-3'), and GAPDH (5'-GCATCCTGGGCTACACTGAG-3', 5'-CTTTACTCCTTGGAGGCCATG-3').Mass Spectrometry
Sample Preparation.
Visualized silver-stained bands from SDS/PAGE gels were diced and washed extensively with 100 mM ammonium bicarbonate (3). Proteins in the gel pieces were then reduced with 50 mM DTT, alkylated with 100 mM iodoacetamide, washed with 100 mM ammonium bicarbonate with 50% acetonitrile (ACN), washed with 100% ACN, and digested in-gel (4) with mass spectrometry-grade trypsin (Promega) in 25 mM ammonium bicarbonate by using a 1:50 enzyme/substrate ratio. Peptides were eluted with 25 mM ammonium bicarbonate (pH 8), 50% ACN/1% TFA, and, finally, 100% ACN. Eluates were dried down and resuspended in 0.1% TFA. Peptides were desalted by using C18 ZipTips (Millipore) and eluted with 70% ACN/0.1% TFA with a-cyano hydroxycinnamic acid (CHCA) on to plate.MALDI-TOF MS.
Proteolyzed, desalted samples were analyzed by using an Applied Biosystems Voyager DE-STR matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) MS in positive ion, reflectron mode, with irradiation from a 337-nm nitrogen laser (5). Peptides were spotted onto a MALDI target by using the dried droplet technique using CHCA matrix. Spectra were acquired by using 1,650-1,750 laser power summing 200 laser shots. Both external (Applied Biosystems) and internal (enzyme autolysis) standards were used for spectrum calibration. Acquired MS data were analyzed by using Data Explorer software (Applied Biosystems), and experimental mass values were compared with theoretical values generated by the sequence analysis software, Protein Prospector (University of California, San Francisco).Results and Discussion
The Kinetics of KL Shedding.
To study the kinetics of KL processing, we performed pulse-chase experiments with KL-transfected COS-7 cells. The KL antibody immunoprecipitated two bands from the medium with approximately twice the size of KL 130- and 68-kDa fragments (SI Fig. 6, arrows). We believe that the observed secreted bands are either KL dimers, as we have seen in our Western blot experiments (see Fig. 3 A and B). Dimers of secreted KL have been reported (6). Alternatively, the higher molecular mass bands could represent a KL-containing complex. The upper band appeared slightly earlier than the lower band at 30- and 60-min chase points, suggesting that a-cut preceded b-cut, assuming these bands represent dimerized KL fragments (SI Fig. 6 Right). Interestingly, we did not detect a 68-kDa KL fragment in the cell lysate at all chase points likely because the 68-kDa fragment was below detection levels. This finding is consistent with the result that a-cut preceded b-cut (SI Fig. 6 Left). In contrast, KL processing in the cell lysate, as seen in Fig. 1A, represents the steady-state levels of KL and the 68-kDa fragment is observed.Searching for the Cleavage Sites in KL.
To search for ADAM10- and ADAM17-induced cleavage sites in KL by using mass spectrometry, we constructed a KL V5 fusion protein with V5 at the C terminus of KL. When expressed in COS-7 cells, the tagged proteins have the same digestion pattern as the WT-KL (SI Fig. 7A, lanes 4-6). We detected the same KL fragments in the medium for KL, KL V5, and KL GFP constructs, and we also were able to detect the 68-kDa band in the cell lysate with the anti-KL antibody (SI Fig. 7A). Using the anti-V5 antibody, we detected 140-, 64-, and 15-kDa bands corresponding to full-length, b-cut, and a-cut fragments, respectively (SI Fig. 7 B and C). The pattern of KL GFP processing was corresponding to KL and KL V5 (SI Fig. 7B and Fig. 4 B and C). To identify the amino acid sequence of the cleavage sites, we immunoprecipitated the KL GFP protein from the cell lysate, isolated the 164-, 98-, and 36-kDa fragments, and analyzed them by MS. SI Fig. 7 D and E shows the silver-stained immunoprecipitated KL GFP fragments and the corresponding MS results. We were able to identify the trypsin-digested fragments of the predicted KL GFP fusion protein with ≈40% coverage (SI Fig. 7E, red letters). The trypsin-digested fragments from the 98-kDa band matched to the C-terminal half of KL and GFP sequence, and the closest fragment to the predicted N terminus of the 98-kDa band we identified was CMASELVR starting at amino acid C621 (SI Fig. 7E, blue letters). The predicted molecular mass for amino acids 29-620 of KL is 68 kDa, close to the band we observed in the KL fragment from the b-cleavage. We were not able to identify the semitrypsinated peptide (i.e., not ending with a K or an R because of cleavage by the sheddase) from L500 to R620 possibly because of glycosylation or other potential posttranslational modifications. The putative N-glycosylation sites are shown in SI Fig. 7E. We were able to identify the trypsin-digested fragments matching GFP from the MS analysis of the 36-kDa fragment (SI Fig. 7E, green letters). Again, we were not able to identify the semitrypsinated peptide close to the juxtamembrane region of KL for the a-cleavage site.KL is a glycoprotein. Using water-soluble biotin labeling of cell surface proteins, Imura et al. (6) reported that the 135-kDa protein is on the cell surface, whereas the 130 kDa is found intracellularly. Both the 135- and 130-kDa bands became 110 kDa with the treatment of N-glycosidase, suggesting both bands are N-glycosylated (6). In our MS studies, we were not able to identify the semitrypsinized peptides, which would be identified as the sheddase cleavage sites after the tryptic digest of purified KL possibly because of glycosylation or other posttranslational modifications. Nevertheless, the precise proteolytic site of the b-cleavage should be close to the trypsin-digested peptide we identified starting at C621 judging from the molecular mass of the KL fragment (SI Fig. 7). There are two putative glycosylation sites between the proposed cleavage motif, KKRK and C621 (SI Fig. 7). Another possible cleavage site is the QKL motif in KL, which also is the cleavage sequence of ADAM17 in APP (7). However, it is well documented that the ADAM17-mediated cleavage is not sequence-specific, and mutations within the cleavage region of substrates do not affect cleavage (8-11). Substrates belonging to the EGF family ligand proteins are cleaved by ADAM17 outside of the transmembrane domain, leaving behind a juxtamembrane stalk with 7-14 amino acid residues (12). For APP, the juxtamembrane stalk is 12 amino acid residues, with the cut occurring between 668K-L669 (7, 13, 14). It is likely that ADAM17 also cleaves KL at the juxtamembrane region judging from the size of the KL fragment released after a-cleavage.
1. Lammich S, Kojro E, Postina R, Gilbert S, Pfeiffer R, Jasionowski M, Haass C, Fahrenholz F (1999) Proc Natl Acad Sci USA 96:3922-3927.
2. Yan JX, Wait R, Berkelman T, Harry RA, Westbrook JA, Wheeler CH, Dunn MJ (2000) Electrophoresis 21:3666-3672.
2. Gharahdaghi F, Weinberg CR, Meagher DA, Imai BS, Mische SM (1999) Electrophoresis 20:601-605.
3. Shevchenko A, Wilm M, Vorm O, Mann M (1996) Anal Chem 68:850-858.
4. Ratts R, Zeng H, Berg EA, Blue C, McComb ME, Costello CE, vanderSpek JC, Murphy JR (2003) J Cell Biol 160:1139-1150.
5. Imura A, Iwano A, Tohyama O, Tsuji Y, Nozaki K, Hashimoto N, Fujimori T, Nabeshima Y (2004) FEBS Lett 565:143-147.
6. Buxbaum JD, Liu KN, Luo Y, Slack JL, Stocking KL, Peschon JJ, Johnson RS, Castner BJ, Cerretti DP, Black RA (1998) J Biol Chem 273:27765-27767.
8. Tsakadze NL, Sithu SD, Sen U, English WR, Murphy G, D'Souza SE (2006) J Biol Chem 281:3157-3164.
9. Hooper NM, Karran EH, Turner AJ (1997) Biochem J 321:265-279.
7. Arribas J, Lopez-Casillas F, Massague J (1997) J Biol Chem 272:17160-17165.
8. Black RA, Doedens JR, Mahimkar R, Johnson R, Guo L, Wallace A, Virca D, Eisenman J, Slack J, Castner B, et al. (2003) Biochem Soc Symp 70:39-52.
9. Hinkle CL, Sunnarborg SW, Loiselle D, Parker CE, Stevenson M, Russell WE, Lee DC (2004) J Biol Chem 279:24179-24188.
10. Sisodia SS (1992) Proc Natl Acad Sci USA 89:6075-6079.
11. Slack BE, Ma LK, Seah CC (2001) Biochem J 357:787-794.