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CUTTING EDGE |
Departments of
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Molecular Microbiology and Immunology and
Cellular and Developmental Biology, Oregon Health Sciences University, Portland, OR 97201
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResults and DiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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Ig variable regions exhibit extensive sequence diversity due to different combinations of germline genes and accumulation of somatic mutations. Igs are a natural model for exploring the influence of amino acid variation on protein assembly, secretion, and degradation. Quality control processes regulating Ig expression are complex and depend on isotype, assembly, and oxidation state of the Ig, as well as the maturation stage of the B cell (11, 12, 13). In a previous analysis of the negative effects of somatic mutation on Ig function, we found that 10% (16/160) of IgG2b transfectants with VH mutations were secretion impaired (14, 15). We examined the intracellular fate of four T15 Ab mutants and observed that the T15L chain, which is not secreted unless assembled with H chains, had two intracellular fates; most were degraded rapidly with a half-life of 1.3 h, whereas 5–20% of the L chain had a long half life paralleling the secretion-incompetent H chain (16). To begin to understand the differential mechanisms governing quality control of the T15L chain, we investigated the degradation of T15L chain expressed in SP2/0 myeloma cells in the absence of H chain.
We present evidence supporting the removal of nonsecreted Ig L chains via dislocation to cytoplasmic proteasomes. We show that the T15L chain localizes to the ER before degradation. Degradation of this secretion-impaired L chain is significantly decreased by proteasome-specific inhibitors, but not by inhibitors of vesicular transport or lysosome function. In addition, we also demonstrate that experimentally induced ER retention of a secretion-competent L chain leads to degradation via a pathway that is sensitive to proteasome-specific inhibitors, thus demonstrating proteasome involvement in L chain degradation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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SP2/0,SP2/0-T15L transfectant expressing the wild-type T15L
chain and the D16H chain loss variant (D16H-)
have been described (14, 17). PCM11 is an IgM
hybridoma
that is T15 Id positive (18) and expresses germline VH1
and V22 genes (G. Wiens, unpublished data), those used by T15. A
PCM11 H chain loss variant was isolated as described (17).
A T15L Thr74 to Asn replacement mutation was
created by site-directed mutagenesis (Bio-Rad, Richmond, CA), and a
stable SP2/0 transfectant was generated by electroporation
(19) and maintained in G418. All cells were grown as
described (14). Ig L chain content of lysates and
supernatants was determined by ELISA as described (20).
The percentage of intra- and extracellular Ig was calculated by
comparing the amount detected in the supernatant or lysate with the
total detected in the lysate plus supernatant.
Inhibitors
Carboxybenzyl-leucyl-leucyl-leucine vinyl sulfone (Z-L3VS; a gift from M. Bogyo and H. Ploegh, Harvard Medical School, Boston, MA) was stored at -80°C in DMSO (16 mM). Lactacystin (Calbiochem, La Jolla, CA or Kamiya Biomedical, Seattle, WA) was stored at 4°C in water (2.65 mM). Brefeldin A (BFA, Sigma-Aldrich) was dissolved in methanol (5 mg/ml) and stored at -20°C. Chloroquine (100 mM stock; Sigma-Aldrich, St. Louis, MO) was prepared in appropriate medium just before use.
Immunoreagents
Immunoreagents were used at the indicated dilution in PBS/10%
FCS: polyclonal rabbit anti-recombinant human calreticulin (1:100;
Affinity Bioreagents, Golden, CO); rabbit anti-mannosidase II
(purchased from Kelly W. Moremen, University of Georgia, Athens, GA;
1:1000); biotinylated monoclonal rat anti-murine chain
(PharMingen, San Diego, CA; 1:100); Texas red-X-conjugated goat
anti-rabbit IgG H and L chain (Molecular Probes, Eugene, OR;
1:200); and FITC-streptavidin (Zymed Laboratories, San Francisco, CA;
1:200). Murine IgG2b and IgA
(Zymed) were used in control
reactions at 10 µg per 50 µL of diluted primary Ab solution.
Immunohistochemistry and confocal microscopy
SP2/0 or SP2/0-T15L cells, grown on coverslips, were washed with PBS, treated with 3% paraformaldehyde (4 ml/well) for 15 min, washed, and further incubated in PBS containing 10% FCS/0.5% Triton X-100 for 30 min. Coverslips were then inverted onto 50 µL of PBS/10% FCS containing the primary Ab and incubated for 2 h. Primary Ab was removed by washing with PBS, and the same procedure was used with the secondary immunoreagent(s). Washed coverslips were placed onto slides containing 15 µL of 1% n-propyl gallate (Sigma-Aldrich) in 1:1 glycerol/PBS, permanently mounted, and stored at 4°C in the dark. Confocal microscopy was performed with a Leica (Netzlar, Germany) confocal laser scanning microscopy and imaging system.
Biosynthetic labeling of cells and immunoprecipitation
SP2/0-T15L cells were grown in 24-well Primaria plates
(Falcon/Becton Dickinson, Mountain View, CA) to 60–80% confluence.
Cell monolayers were washed twice with DMEM lacking cysteine and
methionine (Sigma-Aldrich) at 37°C, then incubated for 1 h at
37°C in deficient medium (1 ml per well). Cells were pulse labeled
with 70 µCi of 35S express labeling mix (NEN
Life Sciences, Boston, MA) for 15 min, washed, and chased in IMDM, 20%
FCS, for the times indicated (see Figs. 2
and 3). In experiments using
proteasome inhibitors, Z-L3VS (16 µM) or
lactacystin (25 µM) or an equivalent amount of diluent only (DMSO or
H2O, respectively) was added during the
preincubation period, as well as in the pulse and chase incubations.
BFA or an equivalent amount of methanol was present during the pulse
and chase periods. Chloroquine was present for the pulse and chase
periods. For experiments employing D16H- cells,
trypsinized cells were plated at a density of 1 x
105/ml 24 h before the experiment. After
washing, cells were labeled with 50 µCi of express labeling mix per
well and chased for the times indicated (see Fig. 3). Supernatants were
collected, and cells were lysed as described (21). L
chains were immunoprecipitated with affinity purified polyclonal rabbit
anti- (Cortex Biochemical, San Leandro, CA or ICN, Costa Mesa,
CA) followed by protein A-Sepharose as described (16).
Endoglycosidase H treatment of immunoprecipitated L chains was
performed as directed (New England Biolabs, Beverly, MA).
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Protein A-Sepharose immunoprecipitates were resuspended in
reducing SDS-PAGE sample buffer, and proteins were separated on either
10 or 12% acrylamide gels as described (16). Proteins
were transferred to polyvinylidene difluoride membranes (Bio-Rad), and
nonspecific sites were blocked with 0.05%-Tween-PBSA-1% BSA (fraction
V; Calbiochem). -chain was detected by probing blots with a 1:500
dilution of goat anti-mouse alkaline phosphatase conjugate
(Southern Biotechnology Associates, Birmingham, AL). After extensive
washing, immunoreactive bands were detected using an Immun-Lite
Chemiluminescent Substrate kit with the Immun-lite Enhancer (Bio-Rad)
as directed.
Quantitation of immunoprecipitation
All labeling experiments were quantified using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA) and IP lab gel software (Version 1.5; Analytics, Vienna, VA).
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Results and Discussion |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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Ig L chain degradation is a well-studied example of ER-associated
protein degradation, but the proteases involved and the cellular site
of degradation have not been conclusively identified. Several previous
studies demonstrated that nonsecreted L chain degradation takes place
in a pre-Golgi compartment, suggested to be the ER (22, 23). We have shown that the T15L (V22) chain is not secreted
and is rapidly degraded if not paired with a secretion-competent H
chain (16). To determine the subcellular location of T15L
chains before degradation, we used immunofluorescent confocal
microscopy. Immunofluorescent staining shows a significant overlap
(Fig. 1A, panel 3)
with anti- (panel 1) and the ER marker
calreticulin (panel 2). Conversely, the L chain
(panel 4) and the medial Golgi marker mannosidase II
(panel 5) signals did not colocalize
(panel 6). Anti- L chain Ab did not stain
untransfected SP2/0 cells, whereas the anti-calreticulin and
anti-mannosidase II Abs produced staining patterns consistent with
those observed with SP2/0-T15L cells (not shown). Staining by
anti- L chain Ab was completely blocked upon pretreatment of
SP2/0-T15L cells with intact murine IgG2b, but a similar
pretreatment with IgA had no effect (not shown). These results
suggest that most or all T15L chain is located in the ER before
degradation. Similar findings have been reported for CH12, another
nonsecreted Ig L chain (22).
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B). The mutant L chain displayed slower
electrophoretic mobility than wild-type L chain when the cells were
cultured without tunicamycin, indicating that the consensus
N-glycosylation site in the mutant had been utilized (Fig. 1C). In the presence of tunicamycin, the T74N mutant L chain
migrated comparably to the wild-type T15L, which is not glycosylated.
We found that T15L T74N remained endoglycosidase H sensitive,
consistent with confinement to an early portion of the biosynthetic
pathway (Fig. 1D). These results are in accord with the
confocal results, indicating that T15L is located in the ER but not the
Golgi and with the coimmunoprecipitation of T15 L with the ER chaperone
H chain-binding protein (BiP) (not shown). The rate of T15L degradation is decreased in the presence of specific, irreversible proteasome inhibitors
Previous studies on disposal of nonsecreted Ig L chains have
suggested the ER as the site of degradation (22, 23). An
investigation of the CH12 chain showed that degradation required ATP
and was sensitive to several serine protease inhibitors
(22). Based on these findings, it was suggested that
serine protease(s) within the ER is responsible for proteolysis of the
CH12 chain. In light of recent findings suggesting involvement of
proteasome-mediated degradation in putative ER degradation, we
investigated the possible role of this pathway in the clearance of
unassembled Ig L chains. We began by examining the effects of treating
SP2/0-T15L cells with two specific, irreversible inhibitors of
proteasome activity, Z-L3VS and lactacystin. A
marked stabilization of the T15L chain was observed in cells treated
with Z-L3VS relative to untreated cells over the
course of a 9-h chase (Fig. 2,
upper panel). The t1/2 for
degradation of T15L was 3.5 ± 0.8 h and 1.3 ± 0.5
h (n = 4, p < 0.05) in the presence
and absence of the inhibitor, respectively. As an independent
confirmation of this observation, a time course experiment was
conducted using a structurally unrelated proteasome inhibitor,
lactacystin (Fig. 2, lower panel). The
t1/2 for degradation of T15L was
4.0 h and 0.6 h, in the presence and absence of lactacystin,
respectively. Although the secretion of impaired mutant coagulation
factor IX is at wild-type levels in the presence of high concentrations
of proteasome inhibitors ALLM and ALLN (24), this was not
true of T15L; there was no detectable L chain in tissue culture
supernatants in any of the experiments (data not shown). Thus,
increasing the intracellular load of L chains does not result in
secretion, and cell lysis is minimal throughout the chase period.
To determine whether the degradation was peculiar to the SP2/0 T15L transfectant cell line, we also examined the effect of these proteasome inhibitors on the kinetics of T15L chain degradation in an H chain loss variant of the PCM11 hybridoma, which expresses an endogenous unmutated T15L chain. Similar to the SP2/0 T15L transfectant, in the absence of proteasome inhibitors, the t1/2 for the endogenous T15L chain was 1.5 h. Treatment with either lactacystin (25 µM) or Z-L3VS (16 µM) extended the t1/2 to 4.0 h (data not shown).
T15L degradation is not affected by blockade of ER to Golgi vesicular transport nor by inhibition of lysosomal function
Degradation by proteasomes does not appear to be dependent on
trafficking from the ER to the Golgi, nor is this process affected by
inhibitors of lysosomal proteases (25, 26). Results from
an earlier study indicated that the degradation of the nonsecreted
CH12 chain was not affected by agents that inhibit trafficking or
lysosomal function (22). To confirm that degradation of
the T15L chain is not dependent on trafficking from the ER to the Golgi
or on lysosomal function, we investigated L chain degradation in the
presence of BFA and chloroquine, respectively. We found that a
concentration of BFA (5 µg/ml) sufficient to completely prevent
secretion of Ig in SP2/0-T15L/T15H wild-type cells (data not shown) did
not affect degradation of T15L
(t1/2 = 1.16 ± 0.38
untreated vs 1.13 ± 0.33 BFA-treated, n = 6).
Chloroquine (25 µM) also did not affect the rapid L chain degradation
compared with untreated cells
(t1/2 = 1.37 ± 0.51
untreated vs 2.15 ± 1.03 chloroquine-treated, n =
6). This concentration of chloroquine significantly inhibited
degradation of total cellular protein (data not shown).
A secretion-competent L chain is also degraded by cytoplasmic proteasomes
To determine whether other IgL chains can be degraded by the
proteasome pathway, we investigated the D16 H chain loss variant cell
line, which efficiently secretes the D16L chain (V1-C)
(14). Pulse-chase experiments were performed using the ER
to Golgi transport inhibitor BFA, the proteasome inhibitor
Z-L3VS, and both inhibitors. In the absence of
inhibitor, secretion of D16L was complete by 4.5 h chase (Fig. 3A). Incubation with
Z-L3VS (16 µM) alone did not affect secretion
or degradation (Fig. 3B), suggesting that
Z-L3VS does not adversely affect normal secretory
pathway operations. Incubation with BFA (5 µg/ml) alone resulted in
complete inhibition of D16L chain secretion (Fig. 3C). A
marked decrease in the level of retained D16L chain over the chase
period was observed, with only 15.0 ± 1.2% of the labeled L
chain persisting by 4.5 h chase (p <
0.001 vs vehicle only). As expected, secretion was also completely
prevented in the presence of both BFA and Z-L3VS.
However, the rate of degradation of retained D16L chains decreased
dramatically compared with the rate in cells treated with BFA only
(Fig. 3D, 59.6 ± 17.9% remaining at 4.5 h,
p < 0.05 vs BFA alone). These results suggest that
ER-retained D16L chains are subject to the same or similar quality
control standards as T15L and become substrates for the proteasome.
Although the rates of T15L and D16L disposal were comparable, much
remains to be discerned about structural parameters that govern the
onset of quality control. Haas and colleagues observed almost complete
persistence of a secretion competent L chain over a 6 h chase
when transport was prevented by monensin treatment (23).
These workers subsequently reported that a nonsecreted L chain had
an increased association with BiP as well as longer intracellular half
life compared with a nonsecreted L chain. In line with the notion
that BiP association may be a key aspect of retained L chain longevity,
they demonstrate that intracellular
t1/2 correlates with the strength of
BiP binding to the variable region (27).
To our knowledge these studies provide the first evidence that ER-retained Ig L chains are degraded by the proteasomal pathway. Although the data presented here support the involvement of cytosolic proteasomes in clearance of L chains, we do not know whether the proteasome directly degrades the L chain or indirectly affects L chain stability, for example, by degrading a protease inhibitor. In a physiological setting, the ability to dispose of L chains that fail ER quality control via cytosolic proteasomes may represent an important checkpoint during B lymphocyte development. At present, it is unclear whether proteasomes are involved in the disposal of nonsecreted H chains or whether degradation of nonsecreted Ig chains by the proteasomal pathway could lead to class I presentation of Ig peptides and recognition by autoimmune T cells. The secretion-defective mutant T15 anti-phosphocholine Abs, which only partially assemble and then are retained in the ER for long periods, will be a useful model system to address these questions (16).
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Acknowledgments |
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Footnotes |
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2 T.O., G.D.W., and E.A.W. contributed equally to this work.
3 Current address: Department of Chemistry, Olin Science Center, 900 State Street, Willamette University, Salem, OR 97301.
4 Address correspondence and reprint requests to Dr. Marvin B. Rittenberg, Department of Molecular Microbiology and Immunology, Oregon Health Sciences University, 3181 Southwest Sam Jackson Park Road, Portland, OR 97201. E-mail address:
5 Abbreviations used in this paper: ER, endoplasmic reticulum; Z-L3VS, carboxybenzyl-leucyl-leucyl-leucine vinyl sulfone; BFA, brefeldin A; BiP, H chain-binding protein.
Received for publication April 9, 1999. Accepted for publication April 26, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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1-antitrypsin Z, in the endoplasmic reticulum requires proteasome activity. J. Biol. Chem. 271:22791. chains by the proteasome. J. Biol. Chem. 272:20800.This article has been cited by other articles:
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  S. M. Kelly, J. K. VanSlyke, and L. S. Musil Regulation of Ubiquitin-Proteasome System mediated Degradation by Cytosolic Stress Mol. Biol. Cell, November 1, 2007; 18(11): 4279 - 4291. [Abstract] [Full Text] [PDF]
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 S. Meister, U. Schubert, K. Neubert, K. Herrmann, R. Burger, M. Gramatzki, S. Hahn, S. Schreiber, S. Wilhelm, M. Herrmann, et al. Extensive Immunoglobulin Production Sensitizes Myeloma Cells for Proteasome Inhibition Cancer Res., February 15, 2007; 67(4): 1783 - 1792. [Abstract] [Full Text] [PDF]
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Y. Elkabetz, A. Kerem, L. Tencer, D. Winitz, R. R. Kopito, and S. Bar-Nun Immunoglobulin Light Chains Dictate Vesicular Transport-dependent and -independent Routes for IgM Degradation by the Ubiquitin-Proteasome Pathway J. Biol. Chem., May 23, 2003; 278(21): 18922 - 18929. [Abstract] [Full Text] [PDF]
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M. S. Gelman, E. S. Kannegaard, and R. R. Kopito A Principal Role for the Proteasome in Endoplasmic Reticulum-associated Degradation of Misfolded Intracellular Cystic Fibrosis Transmembrane Conductance Regulator J. Biol. Chem., March 29, 2002; 277(14): 11709 - 11714. [Abstract] [Full Text] [PDF]
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P. Mukherjee, A. Dani, S. Bhatia, N. Singh, A. Y. Rudensky, A. George, V. Bal, S. Mayor, and S. Rath Efficient Presentation of Both Cytosolic and Endogenous Transmembrane Protein Antigens on MHC Class II Is Dependent on Cytoplasmic Proteolysis J. Immunol., September 1, 2001; 167(5): 2632 - 2641. [Abstract] [Full Text] [PDF]
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S. C. Ho, S. Chaudhuri, A. Bachhawat, K. McDonald, and S. Pillai Accelerated Proteasomal Degradation of Membrane Ig Heavy Chains J. Immunol., May 1, 2000; 164(9): 4713 - 4719. [Abstract] [Full Text] [PDF]
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C. Fagioli and R. Sitia Glycoprotein Quality Control in the Endoplasmic Reticulum. MANNOSE TRIMMING BY ENDOPLASMIC RETICULUM MANNOSIDASE I TIMES THE PROTEASOMAL DEGRADATION OF UNASSEMBLED IMMUNOGLOBULIN SUBUNITS J. Biol. Chem., April 13, 2001; 276(16): 12885 - 12892. [Abstract] [Full Text] [PDF]
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G. D. Wiens, T. O'Hare, and M. B. Rittenberg Recovering Antibody Secretion Using a Hapten Ligand as a Chemical Chaperone J. Biol. Chem., October 26, 2001; 276(44): 40933 - 40939. [Abstract] [Full Text] [PDF]
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C. Fagioli, A. Mezghrani, and R. Sitia Reduction of Interchain Disulfide Bonds Precedes the Dislocation of Ig-{micro} Chains from the Endoplasmic Reticulum to the Cytosol for Proteasomal Degradation J. Biol. Chem., October 26, 2001; 276(44): 40962 - 40967. [Abstract] [Full Text] [PDF]
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