VII.3 Possible tetramerization of the proteasome maturation factor
VII.3.3 Materials and methods
K-562, Dakiki, BJA/B, Ramos, Daudi, Raji, Jurkat, CEM, HEK293, and HepG2 cells were cultured in Iscove`s Modified Dulbecco`s Medium (IMDM) supplemented with 1% UltraGlutamine-I, penicillin/streptomycin (100 U/ml/
100 µg/ml) (all Cambrex, Verviers, Belgium) and 10% fetal calf serum (FCS) (Linaris, Wertheim-Bettingen, Germany). Cells were maintained at 37°C with 7.5% CO2. Neutrophils were isolated by Percoll (Amersham Biosciences, Uppsala, Sweden) density gradient centrifugation and monocytes (CD14+), B-cells (CD19+), natural killer cells (CD16/56+), CD4+- and CD8+-T-cells by cell sorting with a FACS Vantage SE cell sorter (BD Biosciences, Allschwil, Switzerland) from blood of healthy volunteers. Purity of the isolated cells was checked by flow cytometry and was always above 90%. Peripheral blood mononuclear cells (PBMC) were isolated from EDTA blood by Ficoll (Biochrom, Berlin, Germany) density gradient centrifugation.
VII.3.3.2 Quantitative real-time RT-PCR analysis
Total RNA from the cells was prepared with the Qiagen RNeasy kit with additional DNase digestion (Qiagen, Basel, Switzerland) and reverse transcribed with random hexamer primers using the TaqMan reverse transcription reagents kit from Applied Biosystems (Rotkreuz, Switzerland). To verify that the primers did not amplify genomic DNA, some RNA samples were diluted and incubated as the normal samples, but did not receive reverse transcriptase (-RT samples). POMP mRNA levels were detected by SYBR green real-time PCR using the following primers for POMP (GenBank:
AF262975): TG-84: 5´-TCG ACG AGC TGC GGA AGA TG-3´ (exon 1) and TG-85: 5´-CAG TAC AAA GAT GAC TAT AAC CAA GAT GCA G-3´ (exon 6) on an ABI Prism 7700 TaqMan (Applied Biosciences) with the QuantiTect SYBR green PCR mastermix (Qiagen) using a three-step cycling protocol (15 min 95°C, 45 cycles: 15 sec 94°C, 30 sec 55°C, 30 sec 72°C).
Housekeeping genes used for normalization were ubiquitin C (UBC; TG-142:
5´-ATT TGG GTC GCG GTT CTT G-3´ and TG-143: 5´-TGC CTT GAC ATT
CTC GAT GGT-3´) and β2-microglobulin (B2M; TG-136: 5´-GCT ATC CAG CGT ACT CCA AAG ATT C-3´ and TG-137: 5´-CAA CTT CAA TGT CGG ATG GAT GA-3´) with the SYBR green PCR mastermix (Applied Biosciences). Cycling conditions for the housekeeping genes were (two-step protocol): 2 min 50°C, 10 min 95°C, 40 cycles: 15 sec 95°C, 1 min 60°C.
Relative mRNA expression was calculated with the comparative Ct method (ΔΔ Ct) using PBMC as reference cells. Statistical analysis was done with GraphPad Instat (Instat Statistics, GraphPad Software, San Diego, USA).
One-way analysis of variance with unpaired parametric testing using the Tukey-Kramer Multiple Comparisons Test was performed for comparing PBMC with the different leukocyte subsets. (*) was considered significant (p<0.05) and (**) very significant (p<0.01).
VII.3.3.3 Purification of recombinant POMP and production of a polyclonal antibody against POMP
The following POMP sequence representing the full length mRNA was used for cloning: GenBank accession number AF262975 (Homo sapiens voltage-gated potassium channel β subunit 4.1, K+vβ4.1/KCNA4B, (271)), which is identical within the coding sequence to AF077200 (HSPC014) that was described as proteassemblin (223), as well as to AF125097 (HSPC036) that was described as hUmp1 (222) and POMP (224). The cDNA was cloned into the pGEX-4T-3 bacterial expression vector (Amersham Biosciences, Freiburg, Germany) using the following primers for PCR amplification: TG-104 (Bam HI): 5´-GCG GGA TCC CGG ATG AAT GCC AGA GGA CTT GGA-3´
and TG-105 (Xho I): 5´-CCG CTC GAG CCT ATT ACA GTA AAC CAA GTT TAT ATT CCA CCA T-3´. The glutathione S-transferase (GST)-POMP fusion protein was expressed in E. coli BL21 and was partly soluble and partly insoluble in inclusion bodies. Therefore, the protein was purified either from the supernatant, and/or the pellet was dissolved in a small volume of urea buffer (8 M urea, 100 mM NaH2PO4, 10 mM Tris-HCl, pH 8.0) and quickly injected into a large volume of PBS supplemented with complete protease inhibitor cocktail (Roche, Rotkreuz, Switzerland), and subsequently purified over a glutathione sepharose column according to the manufacturer’s protocol
Fast Flow). The GST-tag was removed by on-column digestion with thrombin (Amersham Biosciences) and the resulting 16 kDa protein was used for immunization of a Chinchilla Bastard rabbit (Charles River, Sulzfeld, Germany). From the rabbit serum polyclonal antibody was purified over a protein G column (HiTrap G HP, Amersham Biosciences).
VII.3.3.4 6xHis-POMP purification
The AF262975 sequence was cloned into the pQE-32 expression vector (Qiagen), adding a 6x histidine-tag to the N-terminus of the POMP sequence.
The following primers were used for amplification: TG-145 (Bam HI): 5´-GCG GGA TCC CGA TGA ATG CCA GAG GAC TTG GA-3´ and TG-146 (Pst I): 5´-CCG CTG CAG CCT ATT ACA GTA AAC CAA GTT TAT ATT CCA CCA T-3´. 6xHis-POMP was purified from E. coli SG13009[pREP4] under denaturing conditions with Ni-NTA agarose (both Qiagen). All buffers used were 8 M urea, 100 mM NaH2PO4, 10 mM Tris-HCl at different pH; pH 8.0 for lysis, pH 6.3 for washing, pH 5.9 for the first elution, pH 4.5 for the second elution step.
VII.3.3.5 Gel-filtration chromatography of POMP
Gel-filtration chromatography of POMP (after removal of the GST-tag by thrombin cleavage) was performed on a Superdex 75 pg HiLoad 16/60 column (Amersham Biosciences) equilibrated in TN buffer (50 mM Tris-HCl, 150 mM NaCl, pH 8.0). The column was run with an ÄKTAPrime chromatography system (Amersham Biosciences) at 0.8 ml/min at RT.
Calibration proteins were the cytoplasmic domain of CD21 (CD21cyto, 4.4 kDa, (272)), ribonuclease A (13.7 kDa) and ovalbumin (43 kDa) (LMW gel-filtration calibration kit, Amersham Biosciences). Eluted fractions were collected and analyzed on a 4-12% BisTris NuPage SDS gel (Invitrogen AG, Basel, Switzerland) under reducing conditions and blotted onto Protran nitrocellulose membranes (Schleicher & Schuell GmbH, Dassel, Germany).
Immunoblots were developed as described below.
VII.3.3.6 Immunoprecipitation, SDS-PAGE and immunoblotting
Cells were collected by centrifugation and lysed in PBS supplemented with complete protease inhibitor cocktail by one freeze-thaw cycle. After centrifugation, the supernatant was used as cytosolic extract and the pellet was lysed for 30 min on ice in lysis buffer (1% NP-40, 50 mM NaCl, 50 mM NaF, 30 mM Na4P2O7, 5 mM EDTA, 1 mM Na3VO4 and complete protease inhibitor cocktail). After centrifugation, the supernatant was used as membrane enriched lysate. Immunoprecipitations were performed with the polyclonal POMP antibody and Protein A Sepharose (Sigma). Lysates or immunoprecipitations were then loaded on 12% or 4-12% Bis/Tris NuPAGE SDS gels (Invitrogen) and blotted onto Protran nitrocellulose membranes (Schleicher & Schuell GmbH). To verify transfer efficiency, the membranes were stained with Ponceau S red (Sigma) (0.5% (w/v) in 5% acetic acid). The same results were achieved with the MemCode Reversible Protein Stain for nitrocellulose membranes from Pierce (data not shown), which was described to have a detection limit of 25 ng (250 ng for Ponceau S red). For immunoprecipitation and immunoblotting, the polyclonal POMP antibody was purified over a protein G column (Amersham Biosciences), except for Figure 23C and Figure 26A where the rabbit serum was used without further purification. The blots were developed with anti-POMP or a 5xHis-specific mAb (Qiagen) and goat anti-rabbit or goat anti-mouse POD-labeled secondary antibody (both Sigma), and the chemiluminescent substrate SuperSignal West Pico from Pierce (Pierce/Perbio Science, Lausanne, Switzerland).
VII.3.3.7 SDS-PAGE Coomassie- and zinc-stainings
For Coomassie-stainings, the gels were fixed and stained at the same time with 0.2% (w/v) Coomassie Brilliant Blue R250 (Roth, Reinach, Switzerland) in 50% methanol, 10% acetic acid and destained with 20% methanol, 10%
acetic acid. Zinc-stainings were done with the imidazol-based zinc-staining from Roth (Roti-White-staining). In some cases (shown in Figure 23D), the gels were stained a second time after the first staining vanished (usually after one to two weeks when the gels were stored in ddH2O at RT), which greatly
reduced the background. In Figure 24D, the Coomassie-stained gel was stained in a second step with a zinc-staining according to the protocol of Fernandez-Patron et al. (273), but using the commercial zinc-staining from Roth. For documentation, zinc-stained gels were scanned with a black background and the resulting images were further processed with the Adobe PhotoShop program, using the “auto-contrast” or “auto-levels” function. The sensitivity for this staining was given to be below 15 ng.
VII.3.3.8 Immunofluorescence stainings and confocal microscopy
HEK293 cells were grown over night to a cell density of approximately 50% on poly-L-lysine (Sigma) coated cover slips. Daudi cells were washed twice with serum-free IMDM and then allowed to adhere on poly-L-lysine coated cover slips for 1 h at 37°C. Then, the cells were washed twice with PBS, fixed with 4% paraformaldehyde and permeabilised with 0.5% Triton X-100 (HEK293) or with 0.5% saponin (Daudi). Cells were stained with the purified anti-POMP antibody and Alexa Fluor 488-labelled secondary goat anti-rabbit F(ab)2 and nuclei were stained with DAPI (both Molecular Probes/Juro, Lucerne, Switzerland) using a standard protocol. The stained cells were mounted with mowiol on microscope slides and examined with a Zeiss LSM 510 Meta confocal microscope (Zeiss, Jena, Germany), using a Plan-Apochromat 63x/1.4 oil DIC objective. Deconvolved microscope images were obtained by collecting z-stack images with 0.2 μm thickness of the optical sections (10.8 μm in total) and by using AutoDeblur software (AutoQuant, Troy, USA) for deconvolution. 3D reconstructions were made with the ImarisXT software (Bitplane AG, Zurich, Switzerland) using the “surpass” function with maximum intensity projection (MIP).
VII.3.4 Results
VII.3.4.1 POMP-expression in primary leukocyte subsets and human cell lines
To investigate the expression levels of POMP in different leukocyte populations, quantitative real-time RT-PCR analysis with specific primers for POMP and the two housekeeping genes UBC and B2M was performed. B-cells, neutrophils, monocytes, and natural killer (NK) cells revealed slightly increased POMP levels compared to helper and cytotoxic T-cells and the reference cells for this PCR, peripheral blood mononuclear cells (PBMC) (Figure 22A). In addition, POMP mRNA levels were examined in 10 different human lymphoid and non-lymphoid human cell lines (Figure 22B). The bone marrow-derived chronic myelogenous leukemia line K-562 expressed more than 3 times more POMP than the control cells (PBMC). The mature B-cell lines BJA/B, Daudi and Raji, all non-Hodgkin’s lymphomas of the Burkitt’s lymphoma type, did show increased levels as well. The T-cell derived lines Jurkat (acute T-cell leukemia) and CEM (T-lymphoblast, acute lymphoblastic leukemia) revealed 4 and 3 times greater POMP levels compared to PBMC, respectively. Furthermore, two non-lymphoid epithelial cell lines, HEK293 (human embryonic kidney) and HepG2 (hepatocellular carcinoma) showed the highest POMP levels with 5 and 11 times more POMP mRNA than PBMC, respectively. In summary, transformed cell lines showed higher POMP expression levels in comparison to primary leukocytes.
PBM
relative expression of POMP (ΔΔCt +SEM)
PBMC
relative expression of POMP (ΔΔ Ct)
n=11
relative expression of POMP (ΔΔCt +SEM)
PBMC
relative expression of POMP (ΔΔ Ct)
n=11
relative expression of POMP (ΔΔ Ct)
n=11
POMP is differently expressed in leukocyte subsets and various cell lines Quantitative real-time RT-PCR analysis of POMP-expression in human leukocyte subsets (A) and different human lymphoid and non-lymphoid cell lines (B). Peripheral blood mononuclear cells (PBMC) were used as reference cells and the housekeeping genes UBC and B2M were used for normalization. The results of two independent RNA preparations are shown and calculations were performed with the comparative Ct method (ΔΔ Ct).
VII.3.4.2 POMP is only weakly stained by Coomassie blue
POMP was purified as a GST- as well as a 6xHis-tagged fusion protein from bacterial expression systems. GST-POMP was purified under native conditions and the GST-tag was removed through cleavage with thrombin.
Kinetics of the digest were determined by incubating GST-POMP with thrombin for 5 sec up to 22 h and complete cleavage was achieved after 10 min (data not shown). The eluted POMP protein (15.8 kDa) was hardly detectable when the SDS gel was stained with Coomassie blue, while the GST-tag (26 kDa) that was eluted from the column after POMP recovery was visible as well as the GST-POMP-fusion protein (42 kDa) (Figure 23A). The identity of POMP and GST was confirmed by peptide mass fingerprinting (data not shown). Staining of an immunoblot membrane with Ponceau red (Figure 23B) or a commercially available turquoise blue stain (see material and methods, data not shown) did not result in visualization of POMP. The blot was incubated with polyclonal serum raised against POMP and here, both the fusion- and the cleaved protein were detectable (Figure 23C). The question then arose why was POMP visible as GST-fusion protein, but not after tag-removal. To answer this question, different approaches to determine the protein concentration of the eluted POMP were undertaken. The conventional colorimetric methods of protein concentration determination mostly rely on the Coomassie dye (e.g. Bradford assay) or on the presence of aromatic amino acids and cystines (e.g. bicinchoninic acid (BCA) and Lowry assay), or additionally on high protein concentrations (Biuret assay). All these methods could not be applied to POMP protein concentration determination since it contains one tyrosine and, one cysteine only, and possesses no tryptophans. Therefore, an alternative approach was used. In reverse zinc-stainings proteins appear as unstained, transparent bands and only the background is stained white through selective precipitation of the metal cations on the gel matrix. One third of the elution fractions E1 (lane 8) and E2 (lane 9) shown in Figure 23A were loaded on a zinc-stained gel and the amounts were calculated as 13 and 2.4 μg, respectively (Figure 23D).
The protein concentration was determined spectroscopically by absorbance at 280 nm, using the molar extinction coefficient (ε) of 1490 M-1cm-1 for a folded
protein according to Pace and co-workers (274) (Table 3) and the Lambert-Beer law (c=A280*Mw*ε-1*d-1). Interestingly, with this staining, elution fraction E1 appeared as 1-2 μg and elution E2 as ~0.5 μg when compared to BSA.
The detection limits for Coomassie- and Ponceau-stainings are given as 0.5-0.05 μg (273,275) and 0.25 μg (275), respectively. According to these guidelines, POMP should have been stained much better by Coomassie blue and Ponceau red. It can therefore be concluded that POMP has very weak staining properties with these two dyes.
Purification of POMP
(A) GST-POMP-expression was induced in BL21 E. coli for 3 h with 1 mM IPTG and the protein was purified over a GST-column and removal of the GST-tag with thrombin cleavage occurred directly on the column for 7 h at 30°C. The SDS gel was stained with Coomassie blue. M=molecular weight marker, 1=lysate, 2=cleared lysate, 3=flowthrough, 4=wash 1, 5=wash 5, 6=wash 10, 7=flowthrough/ thrombin load, 8=eluate 1 (POMP), 9=eluate 2 (POMP), 10=eluate 1 (GST), 11=eluate 2 (GST). (B) and (C): Another purification of GST-POMP (thrombin digest for 10 min at RT), the blotted membrane was stained with Ponceau red (B) and the same blot developed with the polyclonal anti-POMP serum (diluted 1:10,000, not protein G-purified) (C). M=molecular weight marker, 1=uninduced culture lysate, 2=induced culture lysate (3 h, 1 mM IPTG), 3=flowthrough, 4=lysate (load), 5=flowthrough, 6=wash 1, 7=wash 10, 8=eluate 1 (POMP), 9=eluate 2 (POMP), 10=eluate 3 (POMP), 11=eluate 2 (GST). (D) Quantification of POMP with BSA on a zinc-stained SDS-PAGE (second zinc-staining; see material and methods), defined amounts of bovine serum albumin (BSA) were loaded and the calculated amounts of POMP are given in the last two lanes.
Table 3: Physicochemical properties of POMP and Ump1
Because of the problems described above, a second approach to purify POMP was applied. POMP was cloned into the pQE-32 vector for His-tag based affinity protein purification. 6xHis-POMP was purified under denaturing conditions using 8 M urea buffer and elution occurred due to lowering the pH.
Again, the protein was not detectable by Coomassie-staining (Figure 24A).
Purification of 6xHis-POMP
6xHis-POMP was expressed in E. coli SG13009[pREP4] and purified on Ni-NTA agarose under denaturing conditions. Coomassie-staining (A), zinc-staining (B), and immunoblot with a His-tag-specific antibody (C). M=molecular weight marker, 1=uninduced culture lysate, 2=induced culture lysate (2 h, 1 mM IPTG), 3=lysate (8 M urea, pH 8.0), 4=load (8 M urea, pH 8.0), 5=flowthrough, 6=wash 1 (8 M urea, pH 6.3), 7=wash 5 (8 M urea, pH 6.3), 8=6xHis-POMP elution 1 (8 M urea, pH 5.9), 9=6xHis-POMP elution 2 (8 M urea, pH 5.9), 10=6xHis-POMP elution 3 (8 M urea, pH 4.5), 11=6xHis-POMP elution 4 (8 M urea, pH 4.5). In (B) only one third of the protein amounts loaded in (A) and (C) were loaded per lane. (D) Quantification of 6xHis-POMP with defined amounts of BSA and CD21cyto. In the first step, the SDS-PAGE was stained with Coomassie blue 1, and in a second step the same gel was stained by a reversible zinc-staining 2.
The amount of 6xHis-POMP protein loaded was calculated with the ε=1280 M
-1cm-1 for an unfolded protein according to the method of Edelhoch (276) as 6.7 μg for elution fraction 3 (lane 10) and 8.1 μg for elution 4 (lane 11). In a reverse zinc-staining however, 6xHis-POMP was easily detectable as inferred from lanes 7-11, although only one third of the protein amounts were loaded per lane (i.e. 2.3 μg in lane 10, 2.7 μg in lane 11) (Figure 24B) compared to the Coomassie-stained gel (Figure 24A). Moreover, immunodetection with a His-tag specific antibody showed that the protein was present and not degraded (Figure 24C). To verify the results obtained from the zinc-staining in Figure 23D where the protein concentration was determined for a folded protein (274), two different amounts of 6xHis-POMP calculated by the Edelhoch method (276) were loaded next to defined quantities of BSA as well as to GST-purified CD21cyto, a 4.4 kDa protein (after GST-tag removal;
(272)). The SDS-PAGE was Coomassie blue stained in the first step where 6xHis-POMP remained undetectable, and a zinc-staining was applied on top of the first staining (273). Here, 6xHis-POMP could easily be visualized (Figure 24D) thus confirming the results seen in Figure 23D.
VII.3.4.3 Tetramerization of POMP
Gel-filtration of GST-purified POMP under non-denaturing conditions at pH 8.0 resulted in a single peak consisting of POMP and corresponding to a molecular mass of ~64 kDa, reminiscent of tetramers (Figure 25A). The range of the Superdex 75 pg chromatography column was given to be from 3 to 70 kDa and the void volume (V0) was 45 ml. Surprisingly, POMP was eluted
very early from the column with an elution volume of 50 ml instead of the expected 88 ml. Analysis of the collected fractions with SDS-PAGE under reducing conditions yielded a band of ~16 kDa in Western blot analysis (Figure 25B).
Figure 25
Gel-filtration chromatography of POMP
(A) Purified POMP was applied on a Sephadex 75 pg chromatography column under native conditions (50 mM Tris-HCl, 150 mM NaCl, pH 8.0). Ovalbumin (43 kDa), ribonuclease A (13.5 kDa) and CD21cyto (4.4 kDa) served as calibration proteins.
(B) Western blot analysis of the POMP-containing fractions; L=filtered sample (load), fractions 1+2, and 5-9. Asterisks indicate POMP dimers and tetramers. (C) SDS-PAGE under non-reducing and reducing conditions and immunoblot analysis of 6xHis-POMP with purified POMP-specific antibody.
In the loaded sample as well as in the eluted fractions, POMP dimers and tetramers were also faintly visible above 28 kDa and above 62 kDa (indicated with an asterisk). 6xHis-POMP was further analyzed on non-reducing gels, revealing two single bands (17-18 kDa and around 38 kDa), suggesting that POMP can dimerize under these conditions, presumably through formation of disulfide bridges with the single cysteine (Cys37) present in the POMP sequence (Figure 25C). However, since the environment is reducing in the cytosol, it seems unlikely that this type of interaction is essential for POMP multimerization. Whole cell lysates run on non-reducing gels showed a single band at the predicted molecular weight of approximately 16 kDa (not shown).
VII.3.4.4 Cellular distribution of POMP
First, specificity of the polyclonal anti-POMP antibody was tested by comparing the staining properties of the polyclonal serum with the pre-immune serum (Figure 26A). In addition to monomeric POMP, the polyclonal serum also recognized bands which could correspond to POMP dimers and tetramers (indicated by asterisks), while the pre-immune serum did not show a specific staining of the purified POMP, GST or 6xHis-POMP protein. The cellular localization of POMP was determined by using purified anti-POMP antibody for immunoprecipitations from cytosolic- and membrane-enriched lysates of the two B-lymphoid cell lines, Raji and Daudi.
Figure 26
Localization of POMP
(A) Specificity of the generated rabbit polyclonal POMP antibody: Purified POMP, GST and 6xHis-POMP were separated on a 12% SDS-PAGE, blotted onto a membrane and the blots developed with rabbit serum collected prior to immunization, and with serum collected after 3 injections of purified POMP. Asterisks indicate POMP dimers and tetramers. (B) Immunoprecipitation of POMP from cytosolic- (C) and membrane-enriched (M) lysates of (107) Raji and (107) Daudi cells and immunoblot analysis with purified POMP-specific antibody.
The majority of the protein was not membrane-associated, but cytosolic (Figure 26B). To further investigate the cellular localization of POMP, the Daudi B-lymphoid cell line (Figure 27A-C) and the non-lymphoid HEK293 cells (Figure 27D-F) were immunostained with purified POMP antibody.
POMP DAPI merge
A B C
D E F
Daudi HE K29 3
G
H I
POMP DAPI merge
A B C
D E F
Daudi HE K29 3
POMP DAPI merge
A B C
D E F
Daudi HE K29 3
POMP DAPI merge
POMP DAPI merge
A B C
A B C
D E F
D E F
Daudi HE K29 3
G
H I G
H I
Figure 27
Immunofluorescence stainings of POMP
Immunofluorescence stainings of POMP (Alexa Fluor 488, green) and nuclei (DAPI, blue) in Daudi (A-C) and HEK293 cells (D-F). Deconvolved pictures of POMP and DAPI stained HEK293 cells from different perspectives. (G) XY-view from the top of one HEK293 cell, (H) 3D-reconstruction with isosurface around the nucleus (DAPI) and clipping of the nucleus, (I) XZ-view from the side of the cell shown in G. Original magnification was x630 for all images. Scale bars: 20 μm.
In both cell types, it was evident that POMP localizes to the cytosol as well as to the nucleus. The control staining with unspecific rabbit antibody and the secondary antibody did not show the specific cytosolic and nuclear staining (not shown). Since the nuclear POMP staining showed discrete spots where the staining was more intense, we further processed z-stack images of HEK293 cells by deconvolution and 3D reconstructions (Figure 27G-I), confirming that the spots were localized within the nucleus. In Daudi B-cells this strong staining in dots was not present (Figure 27A-C).
Immunofluorescence staining of proteins within PML-NBs is dependent on the
Immunofluorescence staining of proteins within PML-NBs is dependent on the