Cytosolic Degradation of T-cell Receptor alpha  Chains by the Proteasome*

(Received for publication, May 21, 1997, and in revised form, June 17, 1997)

Helen Yu , Geoffrey Kaung , Sumire Kobayashi and Ron R. Kopito Dagger

From the Department of Biological Sciences, Stanford University, Stanford, California 94305-5020

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

The T-cell antigen receptor (TCR) is an hetero-oligomeric membrane complex composed of at least seven transmembrane polypeptide chains that has served as a model for the assembly and degradation of integral membrane proteins in the endoplasmic reticulum (ER). Unassembled TCRalpha chains fail to mature to the Golgi apparatus and are rapidly degraded by a non-lysosomal "ER degradation" pathway that has been proposed to be autonomous to the ER. In these studies we show that the degradation of core-glycosylated TCRalpha is blocked by N-acetyl-L-leucyl-L-leucyl-L-norleucinal (ALLN) and lactacystin, implicating the proteasome in ER degradation. Either acute or chronic treatment of TCRalpha -transfected cells with proteasome inhibitors cause the core-glycosylated TCRalpha chains to progressively shift to an ~28-kDa form that lacks N-linked oligosaccharides and the N-terminal signal peptide. The susceptibility of this 28-kDa species to extravesicular protease indicates that it is not protected by the ER membrane and, hence, cytoplasmic. These data suggest a model in which TCRalpha chains that are translocated across the membrane, core-glycosylated, but fail to assemble are dislocated back to the cytoplasm for degradation by cytoplasmic proteasomes. Our data also suggest that covalent modification of TCRalpha with ubiquitin is not required for its degradation.


INTRODUCTION

The T-cell antigen receptor (TCR)1 is a hetero-oligomeric complex of at least seven polypeptide chains that has served as a model for the assembly and degradation of integral membrane proteins in the ER (1). The clonotypic alpha  subunit (TCRalpha ) is a type I membrane protein containing a short (~5-amino acid) cytoplasmic domain and a 223-residue extracellular domain that has four potential sites for N-glycosylation. Mature TCRalpha on the surface of the antigen-specific T-cell hybridoma line 2B4 migrates as a broad 42-44-kDa band (2-4). However, when expressed in the absence of other TCR subunits, TCRalpha is synthesized as a 38-kDa core-glycosylated precursor that is sensitive to digestion with endoglycosidase H and is rapidly degraded with a half-time of ~50 min (5, 6). This degradation process is not affected by inhibitors of autophagy, lysosomal proteolysis, or ER-Golgi traffic. Moreover, TCRalpha chains in these cells are localized to the "ER region" by immunofluorescence and electron microscopy (5). Together, these studies have led to the conclusion that TCRalpha degradation occurs at a site "within or closely associated with the ER" (5). However, efforts to identify ER-specific proteases that participate in TCRalpha degradation have been unsuccessful.

Several recent reports have suggested a role for the proteasome in the ER degradation of some membrane or lumenal proteins (reviewed in Refs. 7 and 8). For example, misfolded cystic fibrosis transmembrane conductance regulator (CFTR) molecules that fail to exit the ER are rapidly degraded by a process that requires covalent modification with ubiquitin and is blocked by lactacystin, a specific proteasome inhibitor (9). Degradation of other ER-restricted proteins including mutant human alpha 1-antitrypsin (10), yeast carboxypeptidase Y (11), and MHC class I heavy chains (12, 13) has also recently been shown to require proteasome activity. How these proteins, which are sequestered within the ER lumen, are recognized and delivered to the cytoplasmic proteasome complex is unknown.

In this paper we have examined the role of the ubiquitin-proteasome pathway in the ER degradation of newly synthesized TCRalpha chains. Our data suggest a model in which TCRalpha chains are first translocated into the ER, cleaved by signal peptidase, and N-glycosylated with core high mannose glycans. These chains are subsequently exported back to the cytoplasmic face of the ER, where they are deglycosylated and delivered to the proteasome for degradation. Moreover, our data suggest that ubiquitination of TCRalpha is not required for this process.


EXPERIMENTAL PROCEDURES

Cell Culture and Transfection

HEK293 cells were grown and transiently transfected by calcium phosphate precipitation as described previously (14). In some experiments, N-acetyl-L-leucinal-L-leucinal-L-norleucinal (ALLN, calpain inhibitor I, Calbiochem) or lactacystin (a kind gift from S. Omura, Kitasato Institute, Tokyo) at indicated concentrations was added to the fresh media. The cDNA corresponding to 2B4 TCRalpha (15) in pCDM8 was a kind gift from J. Bonifacino (National Institutes of Health).

Site-directed Mutagenesis

The 11 lysine residues in TCRalpha were mutated to arginine by 8 sequential rounds of polymerase chain reaction-based "megaprimer" mutagenesis (16). The mutant construct (Kalpha R) was verified by sequence analysis, which revealed the presence of an additional mutation of Phe154 to Tyr. Functional comparison with a Kalpha R lacking this additional mutation showed that this conservative change does not influence either the kinetics of degradation or its sensitivity to proteasome inhibitors.

Immunoblotting, Metabolic Labeling, and Immunoprecipitation

HEK293 cells transiently transfected with TCRalpha were processed for immunoblot analysis as described previously (9). Samples were resolved in 11% SDS-polyacrylamide gels and electroblotted to nitrocellulose. Blots were probed with the appropriate antibody, and immunoreactive bands were detected by enhanced chemiluminescence. Metabolic labeling and immunoprecipitation was carried out as described (14) with the following modifications. Cells were pulse-labeled with 500 µCi/ml [35S]Met/Cys (>1,000 Ci/mmol, NEN Life Science Products). Immunoprecipitation was performed using A2B4 (17) and/or H28-710 (18) and GammaBind Plus Sepharose (Pharmacia Biotech Inc.). The samples were fractionated in 11% SDS-polyacrylamide gel and analyzed by fluorography. For steady state labeling, cells were incubated with 125 µCi/ml [35S]Met/Cys in 90% Met/Cys-free and 10% complete media for 14 h. PNGase F (New England Biolabs) digestion was carried out on TCRalpha immunoprecipitated by H28-710 and bound to GammaBind Plus Sepharose according to the manufacturer's directions.

Protease Protection Assay

HEK293 cells were treated with 50 µg/ml ALLN for 12 h prior to homogenization in buffer H (150 mM NaCl, 10 mM Hepes, pH 7.4, 1 mM EGTA) supplemented with protease inhibitors using a glass/Teflon homogenizer. Unbroken cells and nuclei were removed by centrifugation at 800 × g for 5 min. Total cell membranes from the resulting post-nuclear supernatant were then sedimented in a TLA100.2 rotor (Beckman) at 100,000 × g for 45 min. The membrane pellet was resuspended in buffer H containing 10 µM CaCl2. Samples were treated with Proteinase K (Life Technologies, Inc.) in the presence or absence of 1% Triton X-100 for 1 h at 0 °C. Protease digestion was terminated by the addition of phenylmethylsulfonyl fluoride to a final concentration of 5 mM. Samples were analyzed by SDS-PAGE and immunoblotting.

Cell-free Translation

Radiolabeled TCRalpha was synthesized in a coupled transcription and translation reaction using a T7-TNT kit (Promega) and 0.5 mCi/ml [35S]Met (1000 Ci/mmol, Amersham). Canine pancreatic microsomes were prepared according to Walter and Blobel (19). TCRalpha translated in the presence or absence of microsomes in 50-µl reactions were immunoprecipitated with mAb H28-710 and 50 µl of GammaBind Sepharose beads.


RESULTS

Unassembled TCRalpha Chains Are Stabilized by Inhibitors of the Proteasome

To determine if proteasomes play a role in the degradation of incompletely assembled TCRalpha , we examined the effect of proteasome inhibitors on the steady state levels of TCRalpha in HEK cells transfected with cDNA encoding the 2B4alpha clonotype. Immunoblot analysis (Fig. 1A) reveals that these cells contain a predominant immunoreactive species with a mobility of 38 kDa, corresponding to the size of core-glycosylated TCRalpha , as previously observed in transfected fibroblasts (5). A minor band with ~2-kDa faster mobility, probably corresponding to a partially glycosylated form of the protein (see below), was also detected occasionally. In the absence of proteasome inhibitors, the 38-kDa species was completely soluble in nonionic detergent. However, overnight treatment with the proteasome inhibitors ALLN or lactacystin increased the steady-state level of the 38-kDa band in the detergent-soluble fraction and also led to its appearance in the detergent-insoluble fraction. Strikingly, these proteasome inhibitors also induced the accumulation of a novel, detergent-insoluble 28-kDa band. Because the mobility of this band corresponds to the predicted mobility of core, unglycosylated TCRalpha , we examined the effects of PNGase on the TCRalpha species which accumulated in proteasome-inhibited cells.


Fig. 1. Proteasome inhibitors induce the accumulation of a 28-kDa signal-cleaved, deglycosylated form of TCRalpha . A, effect of proteasome inhibitors on steady-state levels of TCRalpha . HEK293 cells transiently transfected with TCRalpha were incubated overnight without treatment (lanes 1 and 2), with 50 µg/ml ALLN (lanes 3 and 4), or with 50 µM lactacystin (lanes 5 and 6). Cells were lysed in nonionic detergents and fractionated by centrifugation into soluble (S) and insoluble pellet (P) fractions and analyzed by immunoblotting with anti-TCRalpha mAb A2B4. The arrowhead and arrow indicate the positions of core-glycosylated (38-kDa) and unglycosylated (28-kDa) forms of TCRalpha , respectively. Molecular mass markers are indicated on the left in kDa. Asterisk indicates the position of a partially glycosylated form of TCRalpha . B, characterization of 28-kDa TCRalpha . Cells transiently transfected with TCRalpha (lanes 3-6) or mock-transfected control cells (lanes 1 and 2) were labeled to steady state with [35S]Met/Cys in the presence of ALLN (20 µg/ml). Cells were lysed with nonionic detergents and separated into soluble (S, lanes 1, 3, and 5) and insoluble (P, lanes 2, 4, and 6) fractions. TCRalpha was immunoprecipitated with mAb H28-710. Equivalent amounts of the immunoprecipitate were treated with PNGase F (lanes 3 and 4) or left untreated (lanes 1, 2, 5, and 6). TCRalpha forms were separated by 15% SDS-PAGE and visualized by fluorography. The positions of core-glycosylated TCRalpha (TCRalpha (+CHO)) and unglycosylated TCRalpha (TCRalpha (-CHO)), with or without signal sequences (SS), are indicated. Products of cell-free transcription/translation with (lanes 7 and 8) or without (lane 9) TCRalpha cDNA in the presence (lanes 8 and 9) or absence (lane 7) of canine pancreas microsomes (RM) are indicated.
[View Larger Version of this Image (37K GIF file)]

TCRalpha was immunoprecipitated from detergent-soluble and insoluble fractions of transfected HEK cells that had been metabolically labeled to steady state with [35S]Met/Cys in the presence of ALLN (Fig. 1B). In this 15% acrylamide gel the 28-kDa species was resolved into a doublet of closely spaced bands, both of which were resistant to PNGase treatment, and thus, not glycosylated. The upper band of this doublet comigrates with TCRalpha translated in a cell-free protein synthesis extract in the absence of microsomes, indicating that it has an intact signal sequence. Together, these results suggest that translocation of TCRalpha is not completely efficient and that HEK and possibly other cells possess a proteasome-mediated degradation pathway that normally masks inefficiency in the process of ER translocation.

The lower band of the 28-kDa doublet comigrates with the limit product of PNGase-deglycosylated 38-kDa TCRalpha , corresponding to the signal-cleaved form of TCRalpha .2 This suggests that some TCRalpha chains either fail to become glycosylated following translocation and signal peptide cleavage in the ER or that a fraction of glycosylated, signal-cleaved TCRalpha chains are deglycosylated in vivo.

To distinguish between these two possibilities, we examined the effect of proteasome inhibitors on the fate of newly synthesized TCRalpha (Fig. 2). Following a 10-min pulse with [35S]Met/Cys, TCRalpha migrated primarily as a detergent-soluble 38-kDa core-glycosylated protein that was rapidly degraded with a half-time of 65 min (Fig. 2A). In contrast, core-glycosylated TCRalpha in cells treated with the proteasome inhibitors ALLN (Fig. 2B) or lactacystin (Fig. 2C) was markedly stabilized. Both proteasome inhibitors induced the formation of bands corresponding to partially glycosylated TCRalpha intermediates and a 28-kDa species which accumulated over time in the detergent-insoluble fraction. Together, these data suggest that degradation of newly synthesized TCRalpha by the proteasome is preceded by progressive deglycosylation of the core-glycosylated protein.


Fig. 2. TCRalpha degradation is blocked by proteasome inhibitors. HEK293 cells transfected with TCRalpha were pulse-labeled for 10 min and chased for the times indicated without protease inhibitor (A) or in the presence of 20 µg/ml ALLN (B) or 50 µM lactacystin (C). Cells were separated into detergent-soluble and insoluble (pellet) fractions as indicated and immunoprecipitated with mAb H28-710. Band intensity was quantified by densitometry and plotted as a percentage of the signal at 0 min. In C, cells transfected with vector (lanes 1 and 4) or TCRalpha cDNA (lanes 2, 3, 5, and 6) were pulsed for 10 min (P) and chased for 180 min (C). The sizes of partially glycosylated TCRalpha forms are indicated by the asterisks.
[View Larger Version of this Image (47K GIF file)]

TCRalpha Chains Are Dislocated from the ER

We used cell fractionation and protease protection to test the possibility that TCRalpha degradation by proteasomes is associated with its dislocation from the ER to the cytoplasm. Cells were lysed by mechanical disruption,and the post-nuclear supernatant was centrifuged at 100,000 × g. A small amount (<5%) of TCRalpha (both the 38-kDa and the 28-kDa forms) was recovered in the supernatant, even after a second round of 100,000 × g centrifugation, suggesting that some TCRalpha had been released to the cytosolic fraction (data not shown). However, the majority of TCRalpha chains sedimented with the microsomal pellet fraction, suggesting that they are associated with ER membranes or are present as high molecular weight aggregates. To determine the orientation of these TCRalpha chains with respect to the ER membrane, the microsomal pellet fraction was subjected to digestion with proteinase K (Fig. 3). The endogenous lumenal proteins BiP and GRP94 were resistant to digestion by proteinase K in the absence, but not the presence, of detergent. By contrast, the ~10-kDa cytoplasmic tail of calnexin was readily cleaved by the protease indicating that this fraction contained ER vesicles that were sealed and of uniform membrane orientation. Core-glycosylated TCRalpha in the 100,000 × g pellet was completely protected from protease digestion, confirming that it had been correctly translocated. Strikingly, both bands of the 28-kDa unglycosylated doublet were highly susceptible to proteinase K digestion, indicating that they must be present on the exterior, i.e. cytoplasmic side of the vesicles. These data strongly suggest that reverse translocation of TCRalpha from the ER accompanies its degradation by the proteasome.


Fig. 3. Unglycosylated TCRalpha is cytoplasmic. Microsomes from ALLN (20 µg/ml)-treated vector (lanes 1-3) or TCRalpha -transfected cells (lanes 4-10) digested with the indicated concentrations of proteinase K in the absence (lanes 1 and 2 and 4-7) or presence (lanes 3 and 8-10) of 1% Triton X-100. Samples were separated by SDS-PAGE, transferred to nitrocellulose, and probed with antibodies to calnexin (top panel), BiP/GRP94 (middle panel), and TCRalpha (bottom panel).
[View Larger Version of this Image (32K GIF file)]

TCRalpha Degradation Does Not Require Ubiquitination of Lysines

Substrates destined for degradation by the 26 S proteasome are commonly "tagged" by the covalent attachment of multiubiquitin chains (20). Inhibition of proteasome function in vitro or in vivo usually induces the accumulation of a significant fraction of highly ubiquitinated proteins, including ER degradation substrates like CFTR (9). As TCRalpha is a small protein, attachment of even a single ubiquitin moiety (~7 kDa) would result in a readily detectable decrease in gel mobility. In the present study, no such mobility shift was observed in proteasome-inhibited cells (Figs. 1 and 2). However, ubiquitinated TCRalpha could have been missed if the ubiquitin linkages were labile to cleavage by cellular isopeptidases. To directly test whether TCRalpha ubiquitination is required for its degradation by the proteasome, we constructed a TCRalpha mutant (Kalpha R) in which all 11 lysine residues were substituted by arginine. Attachment of ubiquitin to substrates occurs via an isopeptide linkage between a lysine epsilon -amino group on the substrate and the C-terminal glycine of ubiquitin (21). Cells transfected with Kalpha R were pulse-labeled with [35S]Met/Cys for 10 min, and the Kalpha R protein was immunoprecipitated from both the detergent-soluble and insoluble fractions with anti-TCRalpha antibody (Fig. 4A). Like wild-type TCRalpha , Kalpha R was core-glycosylated and rapidly degraded. Remarkably, this degradation was efficiently inhibited by the proteasome inhibitor ALLN, giving rise to the appearance of partially and completely deglycosylated forms in both detergent-soluble and insoluble fractions. Kalpha R degradation was similarly inhibited by 5 µM clasto-lactacystin beta -lactone, the active form of lactacystin (22). These data suggest that either ubiquitination of TCRalpha is not required for its degradation by the proteasome or ubiquitin moieties can be attached to TCRalpha at alternate non-lysine residue(s). Future studies will be required to distinguish between these two possibilities.


Fig. 4. Degradation of mutant TCRalpha lacking lysines. HEK293 cells expressing lysine-less TCRalpha mutant (Kalpha R) were pulse labeled with [35S]Met/Cys in the absence (A) or presence (B) of ALLN (20 µg/ml), chased for the times indicated, and processed as described in the legend to Fig. 2.
[View Larger Version of this Image (36K GIF file)]


DISCUSSION

Selective proteolysis is the final step in the elaborate network of proofreading and editing processes that have evolved to protect eukaryotic cells against the potentially deleterious consequences of errors that can accrue between genes and proteins. These include alterations in primary sequence due to mutation or to transcriptional and translational errors, as well as the effects of inappropriate spatial and temporal expression. Selective degradation is also required to eliminate unassembled or misassembled subunits of hetero-oligomeric plasma membrane complexes such as the heptameric TCR (4). Eukaryotic cells contain two major proteolytic systems: proteasomes, which are present in the cytoplasm and the nucleus, and lysosomes. In contrast to the lysosome-mediated disposal of mature or partially assembled TCR oligomers, the rapid, nonlysosomal degradation of unassembled TCRalpha subunits had suggested the existence of a unique degradation system associated with the endoplasmic reticulum (23). However, several recent studies have demonstrated that some misfolded proteins in the ER can be degraded by cytoplasmic proteasomes following their reverse translocation from the ER (7, 8). The data in this paper demonstrate that unassembled TCRalpha subunits that have been biosynthetically translocated into the ER and core-glycosylated are exported or "dislocated" into the cytoplasm, where they are deglycosylated and degraded by the proteasome.

TCRalpha was synthesized in HEK cells as a 38-kDa core-glycosylated precursor that was rapidly degraded. Our data show that lactacystin and ALLN stabilize this core-glycosylated form of TCRalpha , implicating the proteasome in its degradation. Although the effect of ALLN in stabilizing TCRalpha has been previously reported (24), neither its activity against the proteasome nor its ability to induce the accumulation of dislocated and deglycosylated forms were recognized at that time. Our data show that either acute or chronic treatment of TCRalpha -transfected cells with proteasome inhibitors cause the core-glycosylated 38-kDa TCRalpha chains to progressively shift to an ~28-kDa form that also lacks both N-linked oligosaccharides and an N-terminal signal peptide. As signal peptidase has its active site at the lumenal face of the ER, these data establish that some TCRalpha chains must have been at least partially translocated across the ER membrane. The susceptibility of the 28-kDa species to extravesicular protease indicates that it is not protected by the ER membrane and, hence, is cytoplasmic.

Our data indicate that the majority of dislocated TCRalpha sediments at relatively low speed and is insoluble in nonionic detergent. This change in detergent solubility is probably the result of the formation of high molecular weight aggregates. TCRalpha contains an unconventional transmembrane domain that is interrupted by four polar or potentially charged amino acids. In the absence of oligomeric partners that could shield these side chains from the hydrophobic core of the lipid bilayer, these polar residues have a dominant destabilizing influence over the rest of the molecule (6, 25, 26). It is unlikely, therefore, that nascent TCRalpha chains are able to effectively partition from the hydrophilic environment of the translocon into the lipid bilayer. At the same time the remaining 16 hydrophobic residues that constitute the TCRalpha transmembrane domain are unlikely to be able to effectively partition into the cytosol and may facilitate aggregation of the undegraded dislocated chains. It is possible that the inability of this heterodox transmembrane to effectively partition into the lipid bilayer may facilitate its dislocation without ever fully dissociating from the translocon.

The data presented in this paper suggest that ubiquitination of TCRalpha chains is not required for their degradation by cytoplasmic proteasomes. Although the attachment of high molecular weight ubiquitin polymers has been demonstrated to increase the susceptibility of substrate for degradation by 26 S proteasome, modification by ubiquitin is neither a necessary (27, 28) nor a sufficient signal (29) for degradation. The requirement for ubiquitination of membrane and secretory proteins degraded by the proteasome is also variable. For example, inhibition of proteasome-mediated degradation of alpha 1-antitrypsin (10) or MHC class I heavy chain (13, 30) does not appear to lead to the accumulation of ubiquitinated forms, although the lack of an evident ubiquitin "ladder" is not sufficient evidence upon which to exclude a role for ubiquitin. By contrast, there is evidence supporting a requirement for substrate ubiquitination in the degradation of other membrane and secretory proteins including connexin 43 (31) and CFTR in mammalian cells (9) and Sec61p (32) and carboxypeptidase Y (11) in yeast.

In the absence of ubiquitination, what signals are used to target ER degradation substrates to the proteasome? In cytomegalovirus-infected cells, two gene products appear to possess the capacity to induce the dislocation of MHC class I heavy chains from the ER and accompany them to the proteasome. We speculate that in non-virus-infected cells such targeting could be accomplished by direct coupling of proteasomes to the dislocation apparatus. Possibly, the presence of a misfolded protein in association with the dislocation apparatus could provide a signal that would recruit the docking of proteasome. Such a signal could be transmitted via a transmembrane chaperone like calnexin, as has been suggested recently (10). For this model to be true, dislocation of substrate would be predicted to depend on proteasome activity. In our studies <30% TCRalpha was dislocated (as measured by the appearance of deglycosylated chains) after 3 h in the presence of proteasome inhibitor, even though >75% TCRalpha would have been degraded during the same interval in the absence of proteasome inhibitors. Although preliminary, these data suggest that dislocation of TCRalpha from the ER may be coupled to the activity of the proteasome.

Taken together, the data presented above support the conclusion that TCRalpha chains are dislocated from the ER for degradation by cytoplasmic proteasomes. Thus, TCRalpha joins a growing number of membrane and secretory proteins which appear to be disposed of by a process involving dislocation from the ER and subsequent degradation by cytoplasmic proteasomes. Since TCRalpha has served as a prototype that has largely defined the process of ER degradation, we propose that the cytosolic degradation pathway may be the major pathway for degradation of misfolded or unassembled proteins in the ER.


FOOTNOTES

*   This work was supported by National Institutes of Health Grant R01 DK43994. This work was done during the tenure of an established investigatorship of the American Heart Association (to R. R. K.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Dagger    To whom correspondence should be addressed. Tel.: 415-723-7581; Fax: 415-723-8475; E-mail: kopito{at}leland.stanford.edu.
1   The abbreviations used are: TCR, T-cell receptor; ALLN, N-acetyl-L-leucyl-L-leucyl-L-norleucinal; CFTR, cystic fibrosis transmembrane conductance regulator; HEK, human embryonic kidney 293 cells; PNGase F, protein:N-glycanase; ER, endoplasmic reticulum; MHC, major histocompatibility complex; PAGE, polyacrylamide gel electrophoresis; mAb, monoclonal antibody.
2   Under these experimental conditions, PNGase digestion was incomplete, resulting in a ladder of partially glycosylated TCRalpha forms. However, the smallest of these bands represents the completely deglycosylated form of the protein.

ACKNOWLEDGEMENTS

We are indebted to Juan Bonifacino for providing us with the 2B4 cDNA and the 2B4 mAb used in these studies. We also thank John Moorhead for generously providing us with the mAb anti-alpha H28-710. We are grateful to Cristina Ward for help in the early stages of this study and for critical discussion of the data and manuscript.


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