RNase L Proteolysis in Chronic Fatigue Syndrome: A Diagnostic Marker?
July 31, 2002
By E. Demettre(1)‡, L. Bastide(1,2)‡, A. D’Haese(2)‡, K. De Smet(2), K. De Meirleir(3), K. P. Tiev(4), P. Englebienne(2,5) and B. Lebleu(1)*
(1) UMR 5124 CNRS, Université Montpellier 2, Montpellier, France, (2) R.E.D Laboratories, Zellik, Belgium, (3) Department of Human Physiology and Medicine, Vrije Universiteit Brussel, Brussels, Belgium, (4) Service de Médecine Interne, Hôpital Saint Antoine, Paris, France, (5) Dept. Nuclear Medicine, Université Libre de Bruxelles, Brussels, Belgium. (‡ The three first authors contributed equally to this paper.)
*To whom correspondence should be addressed (telephone: 33 467 613 662, fax: 33 467 040 231, E-mail: email@example.com)
Footnote: These studies were financed by the Centre National de la Recherche Scientifique, by the CFIDS Association of America and by R.E.D. Laboratories. The authors thank Dr. J. Derencourt for his help with the N-terminal sequencing, Prof. R. J. Suhadolnik (Temple University) for communicating unpublished data and for valuable discussions, and Dr. I. Robbins for editing the manuscript.
A 37 kDa binding polypeptide accumulates in peripheral blood mononuclear cells (PBMC) extracts from Chronic Fatigue Syndrome (CFS) patients and is being considered as a potential diagnostic marker (1). We establish here that this low molecular weight 2-5A binding polypeptide is a truncated form of the native 2-5A dependent ribonuclease L (RNase L), generated by an increased proteolytic activity in CFS PBMC extracts. RNase L proteolysis in CFS PBMC extracts can be mimicked in a model system in which recombinant RNase L is treated with human leukocyte elastase (HLE). RNase L proteolysis leads to the accumulation of two major fragments with molecular weights of 37 and 30 kDa. The 37 kDa fragment includes the 2-5A binding site and the N-terminal end of native RNase L. The 30 kDa fragment includes the catalytic site in the C-terminal part of RNase L.
Interestingly, RNase L remains active and 2-5A dependent when degraded into its 30 kDa and 37 kDa fragments by proteases of CFS PBMC extract or by purified HLE. The 2-5A dependent nuclease activity of the truncated RNase L could result from the association of these digestion products, as suggested in pull down experiments.
Chronic fatigue syndrome (CFS) is characterized by long-lasting and debilitating fatigue, myalgia, impairment of neurocognitive functions, and flu-like symptoms, that are often severely worsened after physical exercise. A case definition has been proposed by the Center for Disease Control in Atlanta under the name of Holmes (2) and Fukuda (3) criteria. Most of these symptoms are unfortunately common to other diseases thus complicating a diagnosis, which still relies on extensive clinical testing to exclude other pathologies (4). A large proportion of the patients report an infectious episode at the onset of their chronic fatigue. No single agent has been conclusively associated with the disease although several candidates including HTLV-1, HHV-6, enterovirus or mycoplasma have been proposed (5-8).
Dysregulation of immune functions has also been suggested and natural killer (NK) cell cytotoxicity was significantly diminished in patients (when compared with normal controls) (9).
These observations have prompted studies of possible dysfunctioning of interferon-induced responses. An upregulation of the 2-5A / RNase L antiviral pathway in PBMC cells of CFS patients has been described (10). RNase L is the terminal enzyme in the 2-5A synthetase/RNase L antiviral pathway and plays an essential role in the elimination of viral mRNAs (11) (for a recent review).
Activation of RNase L requires the binding of a small 2’,5’ linked oligoadenylate (2-5A). Intriguingly, a low molecular weight 2-5A binding polypeptide has been observed in a subset of patients diagnosed for CFS (12).
Similar observations have been made in a larger study including CFS patients and control groups (1). In the latter study the presence of a 37 kDa 2-5A binding polypeptide was found in a significant proportion of the CFS population while absent in most controls, thus providing the basis for a potential biochemical marker of CFS.
This low molecular weight 2-5A binding polypeptide is related to RNase L since it is recognized by polyclonal antibodies raised against RNase L (12). Moreover, an increased nuclease activity has been reported in a subset of CFS patients whose PBMC extracts did not contain any full size 83 kDa RNase L (13). This observation has remained puzzling since the P-loop region (which is responsible for 2-5A binding) and the C-terminal end (which contains the catalytic site of RNase L) are far apart and cannot be fitted within a single 37 kDa polypeptide.
In the present study, we establish that an increased proteolytic activity in PBMC extracts from CFS patients is responsible for the accumulation of this truncated form of RNase L and that a 2-5A dependent nucleolytic activity is maintained in the absence of intact RNase L. Moreover, we demonstrate that proteolysis of RNase L by proteases in CFS PBMC extract or by purified HLE gives rise to two major fragments with molecular weights of 37 and 30 kDa. The 37 kDa fragment includes the 2-5A binding site and the N-terminal end of full size RNase L. The 30 kDa fragment starts in the second half of the protein kinase homology domain and includes the catalytic site in the C-terminal part of RNase L.
Production of recombinant human RNase L (recombinant RNase L) protein
Recombinant RNase L was cloned and expressed in baculovirus infected Sf21 insect cells by ATG Laboratories (Eden Prairie, MN). An 6xHis-tag was inserted at the amino terminus of the protein which was purified to 90-95% homogeneity by metal chelate chromatography on Nickelnitrilotriacetic acid (Ni-NTA) agarose.
Isolation of peripheral blood mononuclear cells (PBMC)
Venous blood samples were drawn from patients who fulfilled the Holmes and Fukuda criteria for CFS (2,3) or from healthy individuals. Patients with CFS and healthy individuals were recruited from the Free University of Brussels (Brussels, Belgium) and from the Saint Antoine Hospital (Paris, France). The procedure for isolating PBMC was started within 2 h of sampling. Heparinized whole blood was diluted 1:1 with phosphate buffered saline. Two volumes of diluted blood were overlaid on one volume of Ficoll Hypaque® (Sigma-Aldrich) (density of 1.080) and centrifuged at 20°C at 500g for 30 min. The PBMC layer was removed, washed with phosphate buffered saline and centrifuged. The isolated PBMC pellets were resuspended in 5 ml red blood cell lysis buffer (155 mM NH4Cl, 10 mM NaHCO3, pH 7.4, 0.1 mM EDTA), kept on ice for 5
min, and centrifuged (20°C, 500g, 10 min). The PBMC were washed with phosphate buffered saline and centrifuged again. The pellets were frozen at -80°C until use.
PBMC and Daudi cells extracts
PBMC from six CFS patients and from three healthy individuals were pooled to create respectively CFS PBMC pellets and control PBMC pellets.
The cells were resuspended in 2 volumes of hypotonic buffer (20 mM Hepes pH 7.5, 10 mM K acetate, 1.5 mM Mg acetate, 0.5 % [v/v ethylphenylpolyethylenglycol [Nonidet P 40] detergent) by repeated pipetting and centrifuged at 10,000 g for 15 min at 4°C. The protein concentration in the supernatant was determined by spectrophotometry (14).
Synthesis and labeling of the 2-5A probe
Chemically synthesized 5'-monophosphorylated 2’,5’ linked oligoadenylate tetramer (2-5A) was generously given by Prof. W. Pfleiderer (University of Konstanz, Germany). The 2-5A probe was labeled by ligation of [32P] pCp (specific activity : 3,000 Ci/mmole, ICN Pharmaceuticals) to the 3'-terminus of 2-5A with T4 RNA ligase (Amersham Pharmacia Biotech) and purified by highperformance liquid chromatography on a Hypersil ODS 5 µm C18 column. The terminal 3'- phosphate group was removed by treatment with T4 polynucleotide kinase (Gibco BRL) thanks to its 3'-phosphorylase activity. The pH was adjusted to 4.7, the 3'-ribose residue was oxidized with 10 mM Na metaperiodate and the pH was readjusted to 8.
Covalent labeling and analysis of 2-5A binding polypeptides
The 3'-oxidized [32P] 2-5ApC (2-5A probe) (3,000 Ci/mmole) was incubated with PBMC extract or with recombinant RNase L for 30 min at 4°C and for a further 30 min at 4°C with 20 mM NaBH3CN. The polypeptides were fractionated by 12 % (w/v) SDS-PAGE (15) and detected by autoradiography.
Proteolysis in PBMC extracts
Control or CFS PBMC extracts (50 µg of total proteins) were covalently labeled with the 2-5A probe, as described previously. Extracts were incubated with or without 10 mM human leukocyte elastase (HLE) inhibitor III (Calbiochem), diluted in DMSO, in 10 µl buffer A (20 mM Tris-HCl pH 7.5, 10 mM MgCl2, 8 mM 2-mercaptoethanol, 90 mM KCl and 0.1 mM ATP) at 37°C. At the indicated time, they were heated to 95°C in gel loading buffer for 3 min. The proteins were then separated on SDS-PAGE and detected by autoradiography. When HLE inhibitor III was used, a control was added with DMSO alone in order to rule out an effect of DMSO on HLE activity.
Proteolysis of recombinant RNase L with PBMC extracts or HLE
The recombinant RNase L (250 ng) was covalently labeled with the 2-5A probe and was incubated for the indicated time in 10 µl buffer A at 37°C with control or CFS PBMC extracts (2.5 µg of total proteins) or with 4.10-3 units of human leukocyte elastase (HLE) (Sigma-Aldrich) in presence or in absence of 10 mM HLE inhibitor III. The proteins were then separated on SDSPAGE and detected by autoradiography.
Proteolysis of endogenous RNase L with HLE
The extract from Daudi lymphoblastoid cells (50 µg of total proteins) was covalently labeled with the 2-5A probe and was incubated for 30 min at 37°C in 10 µl buffer A with HLE . The proteins were then separated on SDS-PAGE and detected by autoradiography.
RNase L activity
The assay of RNase L nuclease activity was adapted from Carroll et al. (16) and Dong et al (16,17). Briefly, the RNase L oligonucleotide substrate C11U2C7 (Xeragon) was labeled with pCp [32P] (5 mCi/ml) with T4 RNA ligase (Amersham) in 50 mM Hepes pH 7.5, 15 mM MgCl2, 2 mM DTT, 5 µg/ml bovine serum albumin and 25 µM ATP for 15 min at 30°C.
Recombinant RNase L (400 ng) was digested, or not, by HLE (5.10-3 units) or by a low concentration of PBMC CFS extract (200 ng of total proteins) and was then incubated with or without 100 nM of 2-5A for 10 min at 4°C. Aliquots from this reaction mixture were covalently labeled with the 2-5A probe and the amounts of 83 kDa and 37 kDa RNase L were quantified after SDS-PAGE analysis and autoradiography.
For measurement of RNase L activity, aliquots of the reaction mixture were supplemented with 37.5 pmole (including 10% of C11U2C7pCp [32P]) of RNase L substrate in 10 µl of buffer A and further incubated for 30 min at 30°C. Aliquots were taken again to quantify the amounts of 83 kDa and 37 kDa RNase L after the activity incubation. The cleavage products of C11U2C7pCp [32P] were separated on a 20 % (w/v) acrylamide, 7 M urea gel in Tris acetate EDTA and analyzed by autoradiography.
N-terminal sequencing of truncated RNase L
Recombinant RNase L (10µg) were digested, or not, by HLE (0.2 units) or by minute amounts of PBMC CFS extract (5 µg of total proteins) in buffer A. After analysis by SDS-PAGE, proteins were revealed by Zinc Stain Kit for Electrophoresis (Bio Rad), or transferred by Western blot. onto a PVDF membrane and stained with Ponceau red . The major 30 kDa and 37 kDa bands were then excised from the Western blot to be analyzed on a Procise® protein sequencer (Applied Biosystem).
Recombinant RNase L (1µg) was digested, or not, by HLE (0.02 units) or by a low concentration of PBMC CFS extract (0.5 µg of total proteins) in buffer A. After separation by SDS-PAGE and transfer to a PVDF membrane, the 6xHis-tag of the recombinant RNase L was detected with Ni- NTA acid horseradish peroxidase (Ni-NTA HRP) Conjugate (QIAGEN).
6xHis-tag protein pull down
Recombinant RNaseL was incubated overnight at 4°C with Ni-NTA magnetic agarose beads (QIAGEN) in 50 mM NaH2PO4 pH 8, 300 mM NaCl, 20 mM imidazole and 50 % (v/v) glycerol. The beads were then washed twice with 50 mM NaH2PO4 pH 8, 300 mM NaCl and 20 mM imidazole to eliminate unfixed proteins. An aliquot of RNase L bound to the beads was heated to 95°C in gel loading buffer for 3 min. The other part (1µg) was digested by HLE (10-2 units) for 15 min at 30°C in phosphate buffer saline, washed twice and heated to 95°C in gel loading buffer for 3 min. 6xHis-tagged proteins were then fractionated by SDS PAGE and were detected by Zinc Stain Kit for Electrophoresis (Bio Rad).
Increased proteolytic activity in PBMC extracts of CFS patients
In our initial studies (1), we established that PBMC extracts from CFS patients were characterized by the presence of a low molecular weight (37 kDa) 2-5A binding polypeptide while a 83 kDa band was predominant in extracts from control PBMC. In these experiments, several protease inhibitors were included at the time of PBMC lysis in order to limit breakdown of native 83 kDa RNase L during the preparation and the processing of the PBMC extracts.
An essential concern was the link between this low molecular weight (37 kDa) 2-5A binding polypeptide and native RNase L. At this stage, we could not establish whether this 37 kDa 2-5A binding polypeptide was a new protein, distinct from native RNase L, or was processed from RNase L by proteolysis in intact cells.
In order to discriminate between these two possibilities, PBMC extracts were prepared in the absence of protease inhibitors from healthy individuals or from CFS patients (as described in experimental procedures) and incubated at 37°C for increasing periods of time. The 83 kDa RNase L rapidly disappeared upon incubation in PBMC CFS extract with the concomitant accumulation of a 2-5A probe labeled band migrating with an apparent molecular weight of 37 kDa (Fig. 1B). Additional minor 2-5A probe labeled polypeptides were detected as well, which probably represent unstable intermediates in the processing of RNase L. In contrast, the 83 kDa RNase L was much more stable in PBMC control extract (Fig. 1A).
In order to further demonstrate an increased proteolytic activity in CFS extracts, recombinant RNase L was covalently labeled with the 2-5A probe and incubated with a low amount of unlabeled control or CFS PBMC extract (Fig. 2). Recombinant RNase L was rapidly converted into a 37 kDa truncated RNase L when incubated with CFS extract and remained essentially stable in the presence of control extract, in keeping with an increased proteolytic activity in the former one. Fingerprint analysis, by limited proteolysis with chymotrypsin or V8 protease, revealed that the 37 kDa band arising from the digestion of the 2-5A probe labeled recombinant RNase L with CFS extracts gave rise to a proteolytic signature that was identical to that of the 37 kDa labeled material found in PBMC extracts of CFS patients (data not shown).
Purified protease-mediated degradation of recombinant RNase L as a model system
The biochemical characterization of RNase L degradation products in human PBMC extracts from CFS patients was difficult, owing to the low abundance of RNase L. We have therefore ttempt to degrade recombinant RNase L by purified proteases. In order to validate this model, degradation of recombinant RNase L by CFS extract was used as control.
Preliminary studies have established that several proteases known to be present in PBMC were able to degrade RNase L and to lead to the accumulation of a 37 kDa truncated form (data not shown). HLE appeared particularly interesting in this respect since HLE inhibitor III, a specific peptidic HLE inhibitor, partially inhibited the degradation of endogenous RNase L in PBMC CFS extract (Fig. 3). Along the same line, endogenous RNase L in a Daudi lymphoblastoid cells extract (Fig. 4A) or recombinant RNase L (Fig. 4B) were degraded to a 37 kDa truncated RNase L by purified HLE (lane 2 to 4, Fig. 4A and lane 2, Fig. 4B). It should be noted that the degradation of recombinant RNase L by PBMC CFS extract (lane 5, Fig. 4B) gave rise to an identical pattern. Moreover, the addition of HLE inhibitor III blocked the degradation of recombinant RNase L by purified HLE (lanes 3 and 4, Fig. 4B) or by PBMC CFS extracts (lanes 6 and 7, Fig. 4B).
2-5A dependent nuclease activity is maintained after proteolysis of recombinant RNase L
Evaluating the RNase L-associated biological activity in unfractionated cell extracts was difficult due to its low abundance, to the presence of inhibitors and to the presence of other nucleases. We therefore made use of the model system described above and of the short radiolabeled RNA substrate (C11U2C7 ) described by Carroll et al. (16).
Recombinant RNase L was digested by purified HLE or by a low concentration of PBMC CFS extract, as described in previous sections, before being analyzed for nucleolytic activity. As seen in Fig. 5, the C11U2C7pCp ?32P??substrate was cleaved to the expected 8-mer labeled degradation product (C7pCp ?32P?) even when the recombinant RNase L had been degraded by purified HLE or by a low concentration of CFS extract, as a source of proteases. It should be noted that the amount of CFS extract (200ng total protein) used as a source of proteases was not sufficient in its own right (no recombinant RNase L added) to degrade labeled C11U2C7 (lanes 7 and 8, Fig. 5). The nucleolytic activity observed was strictly 2-5A dependent for degraded RNase L as for intact RNase L (lanes 3 to 6, Fig. 5).
Biochemical characterization of the proteolytic degradation products of recombinant RNase L
In previous sections, the proteolysis of recombinant or endogenous RNase L was monitored by 2-5A probe labeling. This strategy was advantageous in being highly sensitive and specific for RNase L. It of course did not allow the detection of unlabeled proteolytic degradation products.
We therefore made use of a sensitive zinc-imidazole staining method to detect proteolysis products of recombinant RNase L. As shown in Fig. 6, recombinant RNase L migrated as a major 83 kDa polypeptide as expected. Upon incubation with purified HLE, RNase L was rapidly degraded into two groups of fragments migrating with molecular weights around 30 and 37 kDa, respectively. The latter had a similar electrophoretic mobility as the 2-5A labeled 37 kDa truncated form of RNase L (data not shown and Fig. 4). The same bands were detected when CFS extract was used as a source of endogenous proteases, although the pattern was complicated by the presence of PBMC extract proteins. As an example, the major 42 kDa band corresponded to (R)-actin.
The main cleavage fragments (as indicated by arrows in Fig. 6) were collected after electrophoretic blotting and processed for microsequencing of their N-terminal ends. The Nterminal sequences of the 30 kDa fragments were 500HLADFDKSI508 and 492LIDSKKAAH500 when produced by HLE or by CFS extract protease digestion, respectively. The observed electrophoretic mobilities of these 30 kDa fragments were in agreement with the calculated mass for peptides extending from the proteolytic cleavage site to the C-terminal end of RNase L.
No N-terminal sequence could be identified for the 37 kDa fragments produced by HLA or CFS extract proteolysis of recombinant RNase L, suggesting an N-terminally blocked end. This was expected if these 37 kDa fragments started at the N-terminal end of a recombinant protein produced in a baculovirus expression system. It was indeed verified that the full size recombinant RNase L had a blocked N-terminal end (data not shown). Moreover, recombinant RNase L (which is tagged with 6xHis at its N-terminal end) was digested with HLE or with CFS extract, and the 6xHis-tag in the digestion products was identified with a specific Ni-NTA HRP conjugate. As shown in Fig. 7, an 83 kDa band was labeled in intact recombinant RNaseL and a major 37 kDa band was detected after proteolysis.
These major 30 kDa and 37 kDa cleavage fragments remain associated as evidenced by pull down experiments. We took advantage of the presence of the 6-His-tag at the N-terminal end of recombinant RNase L to immobilize the enzyme on Ni-NTA magnetic agarose beads (Fig. 8, lane 1). Treatment of bound RNase L with HLE followed by extensive washings led to proteolysis of the 83 kDa material into the expected 30 kDa and 37 kDa fragments witch both remained bound to the beads (Fig. 8, lane 3). None of the 30 kDa material was released in the supernatant after proteolysis (Fig. 8, lane 2).
Increased proteolytic activity in PBMC of CFS patients
We have established here that the 37 kDa 2-5A binding polypeptide which accumulates in CFS PBMC can be generated by proteolysis of endogenous RNase L (Fig. 1) or by the incubation of recombinant RNase L with a low concentration of PBMC CFS extract (Fig. 2) in keeping with preliminary studies from our group (18,19). Increased proteolytic activity in CFS PBMC thus appears to be the major cause for the occurrence of this 37 kDa 2-5A binding polypeptide and rules out differential RNA splicing or protein splicing as discussed by Shetzline et al (20).
Proteases play an important role in numerous physiological responses and in particular in inflammation and in apoptosis. Some of the symptoms observed in CFS can be related to a dysregulation of these functions. As an example, CFS is associated with increased IL6 secretion and elevated alpha2-macroglobulin, suggesting an acute phase inflammatory response (21).
Moreover, infections by various pathogenic agents (5-8) have been described in CFS patients and can lead to inflammatory processes. Finally, an increased apoptosis has been documented in CFS PBMC (22) and RNase L is known to be involved in apoptosis in response to various stimuli (23,24). A panel of proteases known to act in cell apoptosis or in inflammatory responses have therefore been evaluated for their abilities to degrade RNase L. Caspase 3 is not activated in CFS PBMC extracts (Demettre and Bisbal, unpublished observation) and no cleavage of recombinant RNase L by caspase 3 (25) has been observed.
HLE and cathepsin G belong to the neutral serine protease family from azurophilic granules of myeloid leukocytes and are involved in host defenses against pathogens and inflammatory responses. Interestingly, the incubation of recombinant RNase L with Cathepsin G, HLE or calpain give rise to cleavage products which are similar to those found upon PBMC CFS incubation (Fig. 1 and unpublished observations). This criterion alone, however, does not allow to conclude to an implication of these proteases in the processing of RNase L. Indeed, protein cleavage by a protease at a given site often reflects protein structure only (26). An additional argument for HLE involvement in the proteolysis of RNase L in CFS PBMC extract is the observation that HLE inhibitor III, a specific peptide inhibitor of HLE (27,28), inhibits, at least in part, the accumulation of the 37 kDa truncated RNase L in these extracts (Fig. 3). Although the role of other proteases cannot be excluded at this stage, enhanced HLE activity appears to be involved in the increased proteolysis of RNase L in CFS PBMC. Whether this increased proteolytic activity results from enzyme activation, from enzyme redistribution or from other mechanisms is presently unknown.
A model to characterize RNase L proteolytic fragments
Whatever the underlying mechanism, degradation of recombinant RNase L by purified HLE represents a convenient tool to generate RNase L cleavage fragments for further biochemical characterization. Indeed, the cleavage of recombinant RNase L by HLE mimics RNase L proteolysis in CFS PBMC by the following criteria.
Firstly, the same products are generated upon cleavage of recombinant RNase L by purified HLE or by CFS extract, whether 2-5A probe labeling (Fig. 4B), zinc-imidazole staining (Fig. 6) or detection of the N-terminal His-tag (Fig. 7) are monitored.
Secondly, the 2-5A labeled 37 kDa fragments produced by the proteolysis of endogenous RNase L in a CFS PBMC extract or by the degradation of recombinant RNase L with HLE give rise to identical sets of 2-5A labeled peptides after limited proteolysis with chymotrypsin or with V8 protease (data not shown).
Thirdly, the incubation of recombinant RNase L with either HLE or CFS extracts gives rise to closely related 30 kDa fragments as indicated by the microsequencing of their N-terminal sequences (Fig. 9).
Finally, the nucleolytic activity of both sets of cleavage products is similar (Fig. 5).
Functional organization and biological activity of RNase L cleavage products
The two major polypeptides produced by the proteolysis of RNase L have molecular masses of 37 kDa and 30 kDa. The 37 kDa fragment includes the N-terminal end (since it contains the Nterminal 6xHis-tag) and the 2-5A binding site (which has been allocated to the P-loop motifs in ankyrin domains 7 and 8) (29) (Fig. 9). Its C-terminal end has not been precisely located but it most probably ends shortly after the P-loops at the end of the ankyrin domains region (Fig. 9).
The 30 kDa fragment starts in the second half of the protein kinase-homology domain (at amino acid 500 after HLE proteolysis) and extends until the C-terminal end of RNase L, thus encompassing its catalytic site (Fig. 9). Proteolysis of RNase L then removes the first half of the protein kinase homology domain. Whether the increased proteolysis of RNase L in CFS PBMC has a significant qualitative or quantitative effect on its biological activity has not been fully appreciated. Shetzline and Suhadolnik (13) have documented a slightly increased nuclease activity in affinity purified CFS PBMC extracts. Unfortunately, their experimental model could not exclude a contribution of other nucleases in the degradation of the poly (U) substrate used in their experiments.
We made use of the simplified biological model of proteolysis described above, and of a short synthetic C11U2C7 labeled substrate containing an unique and specific cleavage site for RNase L. Its 2-5A dependent cleavage by RNase L at this site gives rise to a single 8-mer cleavage fragment which can easily be resolved and quantified by gel electrophoresis. This assay confirms that the biological activity of proteolysed RNase L is not dramatically different from native RNase L in agreement with Shetzline and Suhadolnik (13). A 2-5A dependent nuclease activity remains after the proteolysis of recombinant RNase L in its 30 kDa and 37 kDa major cleavage fragments.
The 30 kDa fragment which encompasses the C-terminal catalytic site of RNase L could potentially be active, but does not include the 2-5A binding site. Interestingly in this respect, an N-terminal deleted recombinant form of RNase L lacking all ankyrin domains (which could be analogous to the 30 kDa fragment described here) exhibits a 2-5A independent nuclease activity (17). On the other hand, the 37 kDa fragment, which encompasses the 6xHis-tagged N-terminal end of RNase L and the 2-5A binding site, cannot include the catalytic site. In principle, therefore, a 2-5A dependent nuclease activity cannot be assigned to the 30 kDa or to the 37 kDa fragment alone. However, these two fragments could remain linked together by disulfide bridges or by non covalent bonds after proteolysis, as suggested by the pull down experiments (Fig. 8), and lead to the observed 2-5A dependent nuclease activity.
Alternative possibilities to bring together the 2-5A binding site and the catalytic site within a single 37 kDa polypeptide have been proposed by Shetzline and Suhadolnik (14). They imply non-conventional mechanisms as ribosomal hopping or protein splicing which have to be experimentally demonstrated.
Whatever the case, RNase L proteolysis removes the Cys-rich region (in the protein kinase homology domain) of RNase L which is believed to be involved in protein-protein interactions. It is therefore conceivable that the proteolysis of RNase L might alter its interaction with regulatory proteins or its compartmentalization. Such dysregulation could in turn lead to the degradation of cellular mRNA species which are not normal targets of native RNase L.
1. De Meirleir, K., Bisbal, C., Campine, I., De Becker, P., Salehzada, T., Demettre, E., and Lebleu, B. (2000) Am J Med 108(2), 99-105.
2. Holmes, G. P., Kaplan, J. E., Gantz, N. M., Komaroff, A. L., Schonberger, L. B., Straus, S. E., Jones, J. F., Dubois, R. E., Cunningham-Rundles, C., and Pahwa, S. (1988) Ann Intern Med 108(3), 387-9.
3. Fukuda, K., Straus, S. E., Hickie, I., Sharpe, M. C., Dobbins, J. G., and Komaroff, A.
(1994) Ann Intern Med 121(12), 953-9.
4. Komaroff, A. L., and Buchwald, D. S. (1998) Annu Rev Med 49, 1-13
5. DeFreitas, E., Hilliard, B., Cheney, P. R., Bell, D. S., Kiggundu, E., Sankey, D., Wroblewska, Z., Palladino, M., Woodward, J. P., and Koprowski, H. (1991) Proc Natl Acad Sci U S A 88(7), 2922-6.
6. Ablashi, D. V., Eastman, H. B., Owen, C. B., Roman, M. M., Friedman, J., Zabriskie, J. B., Peterson, D. L., Pearson, G. R., and Whitman, J. E. (2000) J Clin Virol 16(3), 179-91
7. Swanink, C. M., Melchers, W. J., van der Meer, J. W., Vercoulen, J. H., Bleijenberg, G., Fennis, J. F., and Galama, J. M. (1994) Clin Infect Dis 19(5), 860-4.
8. Nasralla, M., Haier, J., and Nicolson, G. L. (1999) Eur J Clin Microbiol Infect Dis 18(12), 859-65.
9. Klimas, N. G., Salvato, F. R., Morgan, R., and Fletcher, M. A. (1990) J Clin Microbiol
10. Suhadolnik, R. J., Reichenbach, N. L., Hitzges, P., Sobol, R. W., Peterson, D. L., Henry, B., Ablashi, D. V., Muller, W. E., Schroder, H. C., Carter, W. A., and Strayer, D., R. (1994) Clin Infect Dis 18 Suppl 1, S96-104.
11. Bastide, L., Demettre, E., Martinand-Mari, C., and Lebleu, B. (2002) in Chronic Fatigue Syndrome : A biological Approch (Englebienne, P., De Meirleir, K., ed), pp. 1-15, CRC press, Boca Raton.
12. Suhadolnik, R. J., Peterson, D. L., O'Brien, K., Cheney, P. R., Herst, C. V., Reichenbach, N. L., Kon, N., Horvath, S. E., Iacono, K. T., Adelson, M. E., De Meirleir, K., De Becker, P., Charubala, R., and Pfleiderer, W. (1997) J Interferon Cytokine Res 17(7), 377-85.
13. Shetzline, S. E., and Suhadolnik, R. J. (2001) J Biol Chem 276(26), 23707-11.
14. Whitaker, J. R., and Granum, P. E. (1980) Anal Biochem 109(1), 156-9.
15. Laemmli, U. K. (1970) Nature 227(259), 680-5.
16. Carroll, S. S., Chen, E., Viscount, T., Geib, J., Sardana, M. K., Gehman, J., and Kuo, L. C. (1996) J Biol Chem 271(9), 4988-92.
17. Dong, B., and Silverman, R. H. (1997) J Biol Chem 272(35), 22236-42.
18. Lebleu, B., Herst, C. V., Bastide, L., Demettre, E., De Smet, K., D'Haese, A., Roelens, S., De Meirleir, K., Le Roy, F., Silhol, M., and Englebienne, P. (2000) Eur. Cytokine Netw. 11(Special issue ICS/ISICR), 101
19. Roelens, S., Herst, C. V., D'Haese, A., De Smet, K., Frémont, M., De Meirleir, K., and Englebienne, P. (2001) Journal of Chronic Fatigue Syndrome 8(3/4), 63-82
20. Shetzline, S. E., Martinand-Mari, C., Reichenbach, N. L., Buletic, Z., Lebleu, B.,
Pfleiderer, W., Charubala, R., De Meirleir, K., De Becker, P., Peterson, D. L., Herst, C.
V., Englebienne, P., and Suhadolnik, R. J. (2002) J Interferon Cytokine Res 22(4), 443-56.
21. Cannon, J. G., Angel, J. B., Ball, R. W., Abad, L. W., Fagioli, L., and Komaroff, A. L. (1999) J Clin Immunol 19(6), 414-21
22. Vojdani, A., Ghoneum, M., Choppa, P. C., Magtoto, L., and Lapp, C. W. (1997) J Intern Med 242(6), 465-78.
23. Zhou, A., Paranjape, J., Brown, T. L., Nie, H., Naik, S., Dong, B., Chang, A., Trapp, B., Fairchild, R., Colmenares, C., and Silverman, R. H. (1997) Embo J 16(21), 6355-63.
24. Rusch, L., Zhou, A., and Silverman, R. H. (2000) J Interferon Cytokine Res 20(12), 1091- 100.
25. Roelens, S., Herst, C. V., De Smet, K., D'Haese, A., Frémont, M., De Meirleir, K., and Englebienne, P. (2001) in AAACFS Fifth International Research, Clinical and Patient Conference (Syndrome, A. A. f. C. F., ed), pp. 70, Seattle, WA, USA
26. Fontana, A., Polverino de Laureto, P., De Filippis, V., Scaramella, E., and Zambonin, M. (1997) Fold Des 2(2), R17-26
27. Powers, J. C., Gupton, B. F., Harley, A. D., Nishino, N., and Whitley, R. J. (1977)
Biochim Biophys Acta 485(1), 156-66.
28. Stein, R. L., and Trainor, D. A. (1986) Biochemistry 25(19), 5414-9.
29. Silverman, R. H. (1997) in Ribonucleases : structures and fonctions (D'Alessio, G., and Riordan, J. F., eds), pp. 515-551, Academic Press, Inc., New York
Copyright 2002 by The American Society for Biochemistry and Molecular Biology, Inc. JBC Papers in Press. Published on July 12, 2002 as Manuscript M201263200.