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General Clinical Chemistry |
1 Institut für Biochemie, Charité-Universitätsmedizin-Berlin, Berlin, Germany.
2 Klinik für Rheumatologie und Klinische Immunologie, Charité-Universitätsmedizin-Berlin, Berlin, Germany.
aAddress correspondence to this author at: Charité-Universitätsmedizin-Berlin; Institut für Biochemie; Monbijoustr. 2, 10117 Berlin, Germany. Fax 49-30-450528916; e-mail burkhardt.dahlmann{at}charite.de.
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Abstract |
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Methods: We isolated circulating proteasomes from the plasma of 2 patients with rheumatoid arthritis and 2 with systemic lupus erythematosus and from human plasma from healthy donors. We purified the proteasomes to apparent homogeneity and then used electron microscopy for imaging and chromatography for subtype spectrum analysis. We compared subtype results with those from 20S proteasomes purified from 4 major blood cell populations. We also tested proteasomes for enzymatic activity and immunosubunit content.
Results: Circulating proteasomes from plasma of healthy donors and from patients with autoimmune disease were found to have the same size and shape as erythrocyte proteasomes, be proteolytically active, and contain standard- and immunosubunits. Chromatography revealed 6 circulating proteasome subtype peaks in healthy donor plasma and 7 in patient donor plasma. Proteasomes from erythrocytes had 3 subtype peaks and those of monocytes, T-lymphocytes, and thrombocytes each had 5 different subtype peaks.
Conclusion: Circulating proteasomes were intact and enzymatically active in plasma from healthy donors and from patients with autoimmune disease. Because the subtype patterns of circulating proteasomes clearly differ from those of proteasomes from blood cells, these cells cannot be regarded as a major source of circulating proteasomes.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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700 kDa. The 20S proteasome has a barrel-like structure composed of 4 stacked rings. Each of the 2 outer rings is built up by 7 different 
-subunits and each of the 2 central rings by 7 different ß-subunits. The -subunits form lockable pores at both ends of the barrel, allowing controlled entry of substrate proteins. Inside the proteasome, 3 of the ß-subunits of each ring expose active sites to the inner surface, thus forming a central proteolytic chamber. In addition, the -subunits can bind various proteasome regulators that are needed to built up 26S proteasomes, hybrid proteasomes, and other proteasome–regulator complexes that fulfill a variety of biological functions, such as proteolytic activation and inactivation of regulators of the cell cycle, of transcription, of differentiation, and of metabolic control and removal of proteins, including their processing to MHC class I antigens (1). All eukaryotic cells analyzed have been found to contain proteasomes. Surprisingly, 20S proteasomes, later designated circulating proteasomes, have also been detected in human serum (2)(3). Wada et al. (2) used ELISA to measured the concentrations of circulating proteasomes in sera from patients with hematologic malignancies and with various liver diseases and found that they were substantially higher than circulating proteasome in plasma of healthy patients. Similarly increased concentrations of circulating proteasomes were detected in plasma of patients with solid tumors, myeloproliferative and myelodysplastic syndromes (4), various autoimmune diseases (3), metastatic malignant melanoma or severe psoriasis (5), and critical illness with sepsis and trauma (6). Chemotherapeutic treatment of patients with acute leukemia decreased or even normalized concentrations of circulating proteasomes (2)(4). Patients with chronic lymphoid malignancies had subnormal concentrations of circulating proteasomes, except for patients with progressing disease, who had increased concentrations (4). Thus, circulating proteasomes are constitutive components of human serum and their concentration may reflect health status. Their origin is still unknown, however, as is the question of whether circulating proteasomes are intact proteolytically active 20S particles or only fragments of proteasomes. We analyzed circulating proteasomes from healthy controls and patients with autoimmune diseases and compared them with 20S proteasomes purified from 4 major blood cell populations, looking specifically at differences in subtype spectra (7).
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Materials and Methods |
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Patient SLE1 (female, age 18 years, disease duration 6 months) suffered from skin and liver involvement (SLE disease activity index, 33). Serologic tests were positive for antinuclear antibodies (titer, 1:2560) and for antibodies to dsDNA and the ribonucleoproteins Ro/SS-A and La/SS-B. Patient SLE2 (female, age 66 years, disease duration 2 years) suffered from skin and kidney involvement (SLE disease activity index, 26). Serologic tests were positive for antinuclear antibodies (titer, 1:5120) and dsDNA antibodies.
Both patients with RA had an erosive manifestation of arthritis and were seropositive for rheumatoid factor. Patient RA1 (female, age 48 years, disease duration 5 months) suffered from polyserositis [disease activity score with 28 joint counts (DAS28) 5.3], whereas patient RA2 (female, age 58 years, disease duration of 8 months) had no extraarticular organ involvement (DAS28 4.5).
Concentrates (250 mL) of human erythrocytes depleted of leukocytes and thrombocyte-apheresis concentrates depleted of leukocytes, erythrocytes, and plasma were obtained from Charité University Hospital. Monocytes and T-lymphocytes were prepared by reverse elution of T3994 leukocyte depletion filters (Fresenius HemoCare GmbH) obtained from Charité University Hospital. Human plasma was purchased from the German Red Cross (Berlin, Germany).
determination of proteasome activity
Measurements of proteasome activity were performed by use of the fluorigenic substrate Suc-LLVY-MCA (final concentration, 100 µmol/L) as described elsewhere (7).
preparation of crude extracts from blood cells
Erythrocytes were washed 3 times with a 2-fold volume of phosphate-buffered saline (PBS; 145 mM/L NaCl, 6 mM/L Na2HPO4, 2.8 mM/L NaH2PO4; rH7.4), and cells were then lysed by addition of a 3-fold volume of 1 mmol/L dithiothreitol for 30 min at 4 °C. Cell debris was pelleted by centrifugation for 1 h at 35 000g, and the supernatant was used as crude extract. Approximately 100 aliquots (each 2–3 mL, containing 1 x 1011 cells) of thrombocyte concentrates from different donors were pooled and lysed by sonification (3 x 15 s). After addition of a 4-fold volume of 1 mmol/L dithiothreitol, undissolved material was pelleted by centrifugation.
For isolation of monocytes and T-lymphocytes, T3994 filters from different blood donors were washed by reverse flow with PBS containing 1 mmol/L EDTA/2% fetal calf serum, and the cells were then concentrated by use of Ficoll-Paque PLUS (Amersham Biosciences) according to the manufacturer’s instructions. Monocytes and T-lymphocytes were diluted to 1 x 108 cells/mL and labeled with CD14- and CD8-positive selection mixture, respectively, and then separated by use of magnetic nanobead technology according to the manufacturer’s instructions (Stem Cell Technologies) yielding 2–3 x 107 cells. According to flow cytometry with C14-FITC- and CD3-FITC-conjugated antibodies (Miltenyi Biotec GmbH) for monocytes and T-lymphocytes, respectively, cell uniformity was at least 95%. Cells were washed and frozen at –80 °C, and samples were collected until at least 1 x 108 cells were isolated before a crude cell extract was prepared as described above.
purification of 20s proteasomes from blood cell extracts
Erythrocyte extract obtained from one 250-mL erythrocyte concentrate was incubated with 100 g of DEAE 52-cellulose SERVACEL (Serva) equilibrated with TEAD buffer (20 mmol/L Tris/HCl, 1 mmol/L ETDA, 1 mmol/L NaN3, 1 mmol/L dithiothreitol; pH 7.5) for 15 min at 4 °C and then washed on a Büchner funnel with 1000 mL of TEAD buffer, and bound proteins were eluted with 500 mL of 500 mmol/L NaCl dissolved in TEAD buffer. The proteasome-containing solution was concentrated by fractionated precipitation with ammonium sulfate [30%–80% saturated (NH4)2SO4], dialyzed against TEAD buffer, and then subjected to a column (total volume, 50 mL) of DEAE-Toyopearl 650S (TosoHaas) equilibrated in TEAD buffer. After the column was washed with the same buffer, proteins bound to the resin were eluted with a linear increasing gradient of 0–500 mmol/L NaCl dissolved in TEAD buffer. Proteasome-containing fractions were detected by measurement of proteolytic activity and pooled, and the enzyme was pelleted by ultracentrifugation (22 h; 100 000g). Proteasomes were dissolved in TEAD buffer and subjected to a gel filtration column (total volume, 80 mL) of Superose 6 equilibrated in the same buffer. Proteasome-containing fractions were pooled and then chromatographed on a Mono Q HR5/5 column (Amersham Biosciences). After elution with a linear increasing gradient of 0–500 mmol/L NaCl dissolved in TEAD buffer, the enzyme was dialyzed against a mixture of 20 mmol/L Tris/HCl and 1 mmol/L NaN3, pH 7.5. Finally, the enzyme was purified by affinity chromatography with an antibody to subunit 3 as ligand as described elsewhere (10). The enzyme was eluted by 2 mol/L of NaCl/Tris buffer from the affinity column and then exhaustively dialyzed against TEAD buffer.
purification of 20s proteasomes from plasma
To separate albumin and other plasma proteins from proteasomes, plasma samples were dialyzed against 20 mmol/L phosphate buffer, pH 7.1, and the enzyme solution was mixed with the same volume of Affi-Gel Blue gel (Bio-Rad) equilibrated in the same buffer. After the column was washed with the same buffer, proteasomes were eluted with 1.4 mol/L NaCl/20 mmol/L phosphate buffer, pH 7.1. The enzyme was further concentrated by fractioned ammonium sulfate precipitation as described above, and after subsequent dialysis against TEAD buffer, the enzyme was subjected to a column (150 mL) of DEAE-Sephacel (Amersham Biosciences) equilibrated in the same buffer. Proteasomes were eluted from the resin by a linear increasing gradient of 0–500 mmol/L NaCl dissolved in TEAD buffer, precipitated by saturation of the solution with 80% saturated (NH4)2SO4, and then subjected to gel filtration on Superose 6 as described above. The final purification step of 20S proteasomes from plasma was affinity chromatography with a subunit-3 antibody as ligand as described above.
quantitative determination of 20s proteasomes
Concentrations of 20S proteasomes in plasma probes were determined by the ELISA technique as described elsewhere (3).
electrophoretic methods
Electrophoresis was performed in 12.5% sodium dodecyl sulfate (SDS)-polyacrylamide gels and stained with Coomassie Brilliant Blue or blotted onto polyvinylidenfluoride membranes for immunodetection of proteasome subunits by use of subunit-specific antibodies and the enhanced chemiluminescence technique (7).
electron microscopy
Four microliters of purified 20S proteasomes were adsorbed to electron microscopy grids covered with carbon and rendered hydrophilic by 30 s exposure to a glow discharge in a plasma cleaner. Grids were washed with water and stained for 1 min with a solution of 1% uranyl acetate. Electron micrographs were recorded with an FEI FP5005 Morgagni electron microscope at an initial magnification of 40 000 and an accelerating voltage of 80 kV.
analysis of proteasome subtypes
High-resolution anion exchange chromatography (in conjunction with a SMART chromatography system; Amersham Biosciences) was used to separate 20S proteasome subtypes on a Mini Q anion exchange column equilibrated with TEAD buffer as described elsewhere (6).
purification of circulating 20s proteasomes from human plasma
In previous investigations, the concentration of proteasomes in sera from healthy donors was <400 µg/L (3), and the serum protein concentrations of proteasomes were several orders of magnitude lower than that measured in cell extracts of various tissues (11). Because of these findings and the fact that plasma contains a high concentration of anionic proteins, especially albumin, we performed an initial chromatographic step, chromatography on Cibacron Blue, to separate most of these plasma proteins from proteasomes. We then performed a conventional proteasome purification method. that included fractionation with ammonium sulfate, anion exchange chromatography on DEAE-Sephacel, gel filtration on Superose 6B, and affinity chromatography with an antibody to subunit-3 as ligand.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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10 µg purified proteasomes from 250 mL of plasma. The purity of plasma proteasome preparations was checked by SDS–polyacrylamide gel electrophoresis and revealed the typical pattern of 20S proteasome subunits, similar to that of 20S proteasomes purified from various blood cells (Fig. 1
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Inspection by electron microscopy of plasma proteasomes purified from human plasma (Fig. 2
) revealed the typical cylindrical shape composed of rings (end view), 4 of which are stacked onto each other to form the 10-nm x 15-nm barrel-like structure (side view). No obvious differences were seen between the shapes of plasma proteasomes and those of 20S proteasomes purified from human erythrocytes.
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the subtype pattern of circulating proteasomes
We used chromatography on a Mini Q anion exchange column to resolve the pattern of subtypes of circulating proteasomes. Because of the very small differences in surface charge of 20S proteasome subtypes, their reproducible resolution by the resin depended on the exactness of the salt gradient. To more easily compare chromatographic assays performed at different times with different batches of chromatography buffer, we compared circulating plasma proteasomes with human erythrocyte 20S proteasomes, which are known to be composed of 3 subtypes, designated EI–EIII (Fig. 3A
). Circulating plasma proteasomes were resolved into 2 large protein peaks, the first of which comprised 5 subtypes (P I–P V) that were not clearly separated under the experimental conditions used. The majority of circulating proteasomes eluted earlier than erythrocyte proteasomes, and only P VI eluted at approximately the same NaCl concentration as EI (Fig. 3A
), a characteristic that was also evident when both proteasome subtype patterns were compared on the basis of their chymotrypsin-like activity tested by use of the substrate Suc-LLVY-MCA (Fig. 3B
). To demonstrate that this peptide bond hydrolysis was actually catalyzed by circulating proteasomes, we repeated the assay in the presence of the proteasome inhibitor lactacystin and found that at a concentration of 1 µmol/L lactacystin inhibited the activity almost quantitatively. These experimental data clearly show that circulating proteasomes are structurally intact and proteolytically active 20S proteasomes.
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circulating proteasomes from patients with autoimmune diseases
The finding that the concentration of circulating proteasomes increases dramatically in patients suffering from various diseases including autoimmune diseases (3)(4) suggests that an increased amount of circulating proteasomes can be regarded as a marker of cells damaged under these pathologic conditions. We compared the pattern of proteasome subtypes in circulating proteasomes for patients with autoimmune disease with that found in healthy controls. To ensure that the purification procedure yielded circulating proteasomes in an amount allowing proteasome subtype analysis, we pooled plasma samples (50 mL) of the 2 SLE patients and of the 2 RA patients. The concentrations of circulating proteasomes in the 2 SLE patients were 3367 and 1184 µg/L plasma and in the 2 RA patients were 312 and 225 µg/L plasma. The subtype pattern obtained with purified proteasomes from the 2 pooled plasma samples is shown in Fig. 4A
. The separation of subtypes equivalent to P I–P V from subtype P VI was as distinct in both groups of patients as in healthy donors, and subtype P VI contained a small peak at its descending shoulder. Immunoblotting of aliquots of the fractions obtained by chromatography on a Mini Q column (Fig. 4A
) revealed that circulating proteasomes from both patient groups contained immunosubunits LMP2 (ß2i) and LMP7 (ß5i), as previously reported for circulating proteasomes from healthy donors (3). In contrast, proteasomes from erythrocytes contain standard proteasome subunits only (12). These data suggest that circulating proteasomes do not originate from erythrocytes.
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Therefore, we compared the pattern of subtypes of circulating proteasomes with those of proteasomes from other human major blood cells, thrombocytes, monocytes, and T-lymphocytes. As shown in Fig. 5
, proteasomes from all 3 types of leukocytes have characteristic proteasome subtype spectra, none of which are identical or even similar to spectra of circulating proteasomes. This finding suggests that circulating proteasomes do not originate from any of these cell types.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Egerer et al. (3) detected proteasomal antigens and hydrolytic activity toward fluorogenic peptides in sucrose gradient fractions containing high–molecular mass proteins from serum of patients with SLE and primary Sjögren syndrome, suggesting that these sera contain proteasomes as intact particles. Egerer et al. failed to detect these signals in equivalent fractions obtained from serum of healthy control donors.
Our present experiments with plasma from healthy donors reveal that plasma from healthy persons also contains 20S proteasomes, which, once purified to apparent homogeneity, exhibit the same size and shape as intracellular 20S proteasomes and also catalyze the hydrolysis of the fluorigenic tetrapeptide Suc-LLVY-MCA, an activity that can be inhibited by the proteasome inhibitor lactacystin. Thus, ELISA-based detection of proteasomal antigens in sera from healthy donors actually measures these small amounts of circulating proteasomes. Findings of a wide range of proteasomal antigen concentrations in healthy plasma (<400 to 2400 µg/L) may be attributable to the use of different antibodies and ELISA techniques by different investigators (2)(3)(4)(19).
20S proteasomes can be divided into 2 large subpopulations, standard proteasomes and immunoproteasomes. These subpopulations differ in regard to the active site subunits ß1, ß2, and ß5 in standard proteasomes, which are replaced during de novo biogenesis of immunoproteasomes by immunosubunits ß1i, ß2i, and ß5i, the transcription of which is induced by several cytokines, such as interferon-
(20). These 2 subpopulations and their intermediate forms can be separated by high-resolution anion exchange chromatography. By use of this technique we found that 20S proteasomes from different tissues have specific patterns of proteasome subtypes (7) and showed that 20S proteasomes from 4 major blood cell types, erythrocytes, thrombocytes, monocytes, and T-lymphocytes, can also be differentiated by specific subtype patterns with distinct numbers and proportion of peaks. We found that rather than exhibiting subtype patterns identical or even very similar to those of the 4 blood cell types, circulating proteasomes exhibited their own subtype pattern, and this pattern was identical in plasma samples from 3 different healthy donors. Therefore, we concluded that the origin of these circulating proteasomes, at least under nonpathologic conditions, was identical and not caused by cytolysis of any of the 4 types of blood cells investigated here. Because other investigators have found a poor or even absent correlation between concentrations of plasma lactate dehydrogenase and circulating proteasomes (4)(5), our data call into question the supposition that proteasomes are released into the plasma because of cytolysis or defective apoptosis (21). Our finding that the subtype patterns of circulating proteasomes in patient samples differ slightly from those in healthy donor samples indicates that the origin of circulating proteasomes may vary and may somehow reflect the health status of the sample donor. On the other hand, the subtype patterns in both groups of autoimmune patients were very similar, irrespective of the clearly different concentrations of circulating proteasomes found in both groups of patients and their different types of autoimmune diseases.
Because circulating proteasomes from autoimmune patients were found to contain the immune subunit ß5i (LMP7), it was suggested (3) that they originate primarily from immunocompetent cells such as monocytes and lymphocytes as well as from nonprofessional antigen-presenting cells. This suggestion is not unequivocally sustained by our data, which show proteasome subtype patterns in sera of patients with autoimmune disease that are clearly different from those in monocytes and T-lymphocytes. Considering that circulating proteasomes from control donors also contain immunosubunits ß1i (LMP2) and ß5i (LMP7) and that their subtype patterns differ from those of monocytes, T-lymphocytes, thrombocytes, and erythrocytes to the same degree as those of autoimmune patients, these 4 blood cell types are unlikely to be the origin of the circulating proteasomes characterized in the present study.
Two additional cell types have been proposed as origins of extracellular, noncirculating proteasomes. Human spermatocytes seem to contain 85% of their proteasomes on their surface and may play a role in the acrosome reaction (22). Sperm membrane autoantibodies from infertile men have been found to recognize proteasome subunits 5 and 6 (23), corroborating the finding that proteasomes are exposed at the spermatocyte surface. Proteasomes have also been found in bronchoalveolar lavage specimens of the epithelial lining fluid of the lung (24). Because no clear correlation exists between concentrations of lactate dehydrogenase and of proteasomes within the extracellular fluid, damage to epithelial cells does not seem to trigger enzyme released. Thus, the origin, the cellular extrusion mechanism(s), and the functions of extracellular, circulating proteasomes remain enigmatic. Nevertheless, their concentration is a useful diagnostic criterion for disease activity in patients with various illnesses (2)(3)(4)(5)(6). Our investigation showed that the pattern of subtypes of circulating proteasomes is changed in patients with SLE and RA compared with controls, and this change may somehow reflect the disease state of the donors and may be the basis for development of a new diagnostic test system to detect disease and monitor therapy.
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Acknowledgments |
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Footnotes |
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References |
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-induced immune adaptation of the proteasome system is an accelerated and transient response. Proc Natl Acad Sci U S A 2005;102:9241-9246.The following articles in journals at HighWire Press have cited this article:
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  M. Majetschak, M. Perez, L. T. Sorell, J. Lam, M. E. Maldonado, and R. W. Hoffman Circulating 20S Proteasome Levels in Patients with Mixed Connective Tissue Disease and Systemic Lupus Erythematosus Clin. Vaccine Immunol., September 1, 2008; 15(9): 1489 - 1493. [Abstract] [Full Text] [PDF]
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 S. Scheffler, U. Kuckelkorn, K. Egerer, T. Dorner, K. Reiter, A. Soza, G.-R. Burmester, and E. Feist Autoimmune reactivity against the 20S-proteasome includes immunosubunits LMP2 ({beta}1i), MECL1 ({beta}2i) and LMP7 ({beta}5i) Rheumatology, May 1, 2008; 47(5): 622 - 626. [Abstract] [Full Text] [PDF]
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 T. Szerafin, K. Hoetzenecker, S. Hacker, A. Horvath, A. Pollreisz, P. Arpad, A. Mangold, T. Wliszczak, M. Dworschak, R. Seitelberger, et al. Heat Shock Proteins 27, 60, 70, 90{alpha}, and 20S Proteasome in On-Pump Versus Off-Pump Coronary Artery Bypass Graft Patients Ann. Thorac. Surg., January 1, 2008; 85(1): 80 - 87. [Abstract] [Full Text] [PDF]
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 S. U. Sixt, M. Beiderlinden, H. P. Jennissen, and J. Peters Extracellular proteasome in the human alveolar space: a new housekeeping enzyme? Am J Physiol Lung Cell Mol Physiol, May 1, 2007; 292(5): L1280 - L1288. [Abstract] [Full Text] [PDF]
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
) and presence (
) of 1 µmol/L proteasome inhibitor lactacystin. For comparison, Suc-LLVY-MCA hydrolyzing activity was measured in chromatographic fractions obtained with erythrocyte proteasomes (
).
