Grp78, Grp94, and Grp170 interact with
1-antitrypsin mutants that are retained in the endoplasmic reticulum
Bela Z. Schmidt and
David H. Perlmutter
Departments of Pediatrics, Cell Biology and Physiology, University of Pittsburgh School of Medicine, Children's Hospital of Pittsburgh, Pittsburgh, Pennsylvania
Submitted 26 May 2004
; accepted in final form 19 April 2005
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ABSTRACT
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In
1-antitrypsin (
1-AT) deficiency, a mutant form of
1-AT polymerizes in the endoplasmic reticulum (ER) of liver cells resulting in chronic hepatitis and hepatocellular carcinoma by a gain of toxic function mechanism. Although some aspects of the cellular response to mutant
1-AT Z have been partially characterized, including the involvement of several proteasomal and nonproteasomal mechanisms for disposal, other parts of the cellular response pathways, particularly the chaperones with which it interacts and the signal transduction pathways that are activated, are still not completely elucidated. The
1-AT Z molecule is known to interact with calnexin, but, according to one study, it does not interact with Grp78. To carry out a systematic search for the chaperones with which
1-AT Z interacts in the ER, we used chemical cross-linking of several different genetically engineered cell systems. Mutant
1-AT Z was cross-linked with Grp78, Grp94, calnexin, Grp170, UDP-glucose glycoprotein:glucosyltransferase, and two unknown proteins of
110130 kDa. Sequential immunoprecipitation/immunoblot analysis and coimmunoprecipitation techniques demonstrated each of these interactions without chemical cross-linking. The same chaperones were found to interact with two nonpolymerogenic
1-AT mutants that are retained in the ER, indicating that these interactions are not specific for the
1-AT Z mutant. Moreover, sucrose density gradient centrifugation studies suggest that
85% of
1-AT Z exists in heterogeneous soluble complexes with multiple chaperones and
15% in extremely large polymers/aggregates devoid of chaperones. Agents that perturb the synthesis and/or activity of ER chaperones such as tunicamycin and calcium ionophore A23187
[GenBank]
, have different effects on the solubility and degradation of
1-AT Z as well as on its residual secretion.
1-antitrypsin deficiency; molecular chaperones; endoplasmic reticulum quality control; endoplasmic reticulum retention
1-ANTITRYPSIN (
1-AT) deficiency is the most common genetic cause of liver disease in children and emphysema/destructive lung disease in adults (35). The liver disease is caused by a gain of toxic function mechanism, which somehow results from the retention of a mutant
1-AT molecule in the endoplasmic reticulum (ER) of liver cells. The lung disease involves a loss of function mechanism in which the lack of
1-AT secretion from the liver into the blood and, in turn, transfer to the lung permits uninhibited proteolytic damage to the connective tissue matrix of the lung.
A prospective nationwide screening study in Sweden that began 30 years ago has indicated that only a subgroup (
10%) of individuals with the classic homozygous PIZZ form of
1-AT deficiency develop clinically significant liver disease during childhood (42, 43). Our studies of genetically engineered skin fibroblast cell lines from PIZZ individuals with and without liver disease have indicated that there is a lag in the ER degradation of mutant
1-AT Z in the subgroup that is "susceptible" to liver disease and, therein, that differences in ER degradation/quality control are important determinants of the liver disease phenotype in this deficiency (49). Studies done by a number of laboratories (37, 46) have indicated that the ER degradation pathway for mutant
1-AT Z involves several mechanisms including ubiquitin-dependent and -independent proteasomal mechanisms as well as nonproteasomal mechanisms. Autophagy and a tyrosine phosphatase-dependent reaction have been implicated as potential nonproteasomal mechanisms (4, 45).
Examination of the structure of
1-AT and other serpins by Carrell and Lomas has shown that the mutant
1-AT Z is prone to polymerization by a loop-sheet insertion mechanism (8, 27, 28). Moreover, by showing that the secretion defect of mutant
1-AT Z is partially reversed by the introduction of additional mutations in the
1-AT Z molecule that prevent its polymerization, they have provided evidence that polymerization plays a role in the ER retention of
1-AT Z. Recently, we have shown that a naturally occurring mutation of
1-AT, called
1-AT Saar, which causes truncation of the COOH terminus of
1-AT, is retained in the ER even though it does not polymerize. In fact, when the Saar mutation is introduced into the
1-AT Z molecule, the resulting
1-AT Z + Saar mutant is retained in the ER to the same, or perhaps even to a greater, extent as
1-AT Z itself, even though this double mutant no longer has polymerogenic properties (25). These results imply that there are different mechanisms for ER retention of polymerogenic and nonpolymerogenic mutants of
1-AT or that polymerization is an effect, rather than the cause, of ER retention.
A growing body of literature has characterized the role of ER chaperones in determining the fate of glycoproteins. The ER chaperones facilitate the folding of wild-type proteins that is necessary for translocation out of the ER to the appropriate destination. They also play a role in the quality control mechanism, inhibiting secretion of incompletely folded or misfolded proteins by retaining them in the ER and/or by targeting them for degradation. Grp78 (BiP) is the most well-studied ER chaperone. It is a nonglycosylated protein of the ER lumen that belongs to the heat shock protein (Hsp)70 family (17). It binds transiently to newly synthesized proteins, more extensively to misfolded proteins, and plays a role in the gating of the translocon pore. Grp78 also plays a crucial role as a sensor of unfolded proteins in the ER and triggering the unfolded protein response (UPR), caused by the accumulation of unfolded proteins in the ER. The involvement of Grp78 in maintaining the solubility of misfolded proteins has been demonstrated in many cases (29, 39).
Grp94 is a resident ER member of the Hsp90 protein family (1, 36). Similarly to Grp78, it is also induced under many conditions, leading to the accumulation of unfolded proteins in the ER. Compared with Grp78 and calnexin, Grp94 interacts with only a restricted set of proteins and seems to bind to advanced folding intermediates and incompletely assembled oligomers. Grp170 (Orp150) is an ER resident glycoprotein with a COOH-terminal ER retention signal (15). The physiological role of Grp170 is poorly understood. It has been shown to interact with both Grp78 and Grp94 and secretory proteins, and it may be cooperatively involved in the folding of proteins.
Calnexin (p88 or IP90) is a type I transmembrane protein with ER retention signal in its COOH terminus (9, 22, 23, 38, 48). Calnexin binds monoglucosylated high-mannose oligosaccharides produced by the partial deglucosylation of the Glc3Man9GlcNAc2 core or by the reglucosylating action of UDP-glucose glycoprotein:glucosyltransferase (UGGT) on Man79GlcNAc2 oligosaccharides (34). In addition to retaining misfolded proteins, calnexin also increases the folding efficiency of glycoproteins, preventing premature oligomerization and suppressing the formation of nonnative disulfide bridges (22). UGGT (47) adds a single glucose unit to Man79GlcNAc2 oligosaccharides covalently attached to partially folded proteins (7). It is the only component of the "calnexin cycle" identified so far that has been shown to be able to specifically recognize unfolded proteins. Chaperones have also been shown to interact with each other to form chaperone networks in the ER (16, 31, 44). These networks are thought to facilitate the transient sequential interactions involved in folding of cargo.
There are relatively limited data on the interaction of mutant
1-AT Z with ER chaperones. This mutant has been shown to interact with calnexin in cell lines and in cell-free microsomal systems (5, 37, 49). There are conflicting reports about Grp78. Early reports suggested that
1-AT Z did not interact with Grp78 in mouse hepatoma cell lines (nor did the
1-AT Hong Kong mutant) or in the liver of a transgenic mouse model (18). However, recent studies in a human embryonic kidney cell line suggest an association with Grp78 but not with Grp94 (5). Interactions with calnexin and UGGT have been demonstrated with the
1-AT Hong Kong mutant that is prematurely truncated at its COOH terminus (10, 21), but because there are significant differences in the properties of
1-AT Hong Kong compared with
1-AT Z, the interactions with
1-AT Hong Kong are not necessarily reflective of what happens with
1-AT Z.
Thus, in this study, we used a chemical cross-linking approach to provide a systematic unbiased analysis of the ER chaperones associated with mutant
1-AT Z. We also examined the possibility that nonpolymerogenic mutants
1-AT Saar and
1-AT Z + Saar interact differently with ER chaperones. Because chaperone interactions have been shown to be very sensitive to detergents, we chose a cross-linking approach to increase the probability of detecting all interactions. The interactions found by cross-linking were subsequently verified without cross-linking.
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MATERIALS AND METHODS
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Materials.
Dithiobis(succimidylpropionate) (DSP) and the bicinchoninic acid (BCA) protein assay were purchased from Pierce (Rockford, IL). Tunicamycin, protein G-bearing formalin-fixed Streptococcus sp. cells, and grade VI apyrase were from Sigma (St. Louis, MO). Reagents for analytical gel electrophoresis were from Bio-Rad (Hercules, CA). Goat anti-human
1-AT was purchased from Diasorin (Stillwater, MN), and rabbit anti-human
1-AT was purchased from DAKO (Carpinteria, CA). Rabbit anti-Grp170 serum was a kind gift of Dr. John Subjeck (Roswell Park Cancer Institute, Buffalo, NY). Rabbit anti-UDP-glucose glycoprotein:glucosyltransferase serum was generously provided by Dr. Armando Parodi (Institute for Biotechnological Research, University of San Martin, Buenos Aires, Argentina). Mouse anti-calnexin was obtained from Affinity Bioreagents (Golden, CO). Goat anti-Grp78 and anti-Grp94 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit antisera raised against Grp78, Grp94, the COOH-terminal sequence of canine calnexin, and glucosidase II were purchased from Stressgen (Victoria, British Columbia, Canada). Antibody to ER mannosidase II was kindly provided by Dr. Joyce Bischoff (Children's Hospital Boston, Harvard Medical School, Boston, MA). Secondary antibodies used in immunoblotting experiments (peroxidase-labeled donkey anti-goat IgG and donkey anti-rabbit IgG) were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA). Peroxidase-labeled streptavidin was from Pierce. Radioactive molecular mass markers were from Amersham Biosciences (Piscataway, NJ), and the prestained protein standard used for immunoblotting was purchased from Invitrogen (Carlsbad, CA). Cell culture reagents were from Invitrogen and Mediatech (Herndon, VA).
Cell lines.
Fibroblast cell lines that were transduced with amphotropic recombinant retroviral particles bearing
1-ATZ cDNA and have stable constitutive expression of
1-ATZ (CJZ12B) have been described previously (49). Chinese hamster ovary (ChoK1) cell lines expressing
1-ATM,
1-AT Saar,
1-AT Z, or
1-AT Z + Saar were established as described previously (25). The HepaTO/Z cell line, with inducible expression of
1-AT Z, was established from the murine hepatoma Hepa 16. Hepa 16 was first engineered for expression of tetracycline-controlled transactivator (tTa) using the pTet-Off plasmid (BD Biosciences, Palo Alto, CA) and geneticin-resistant colonies were isolated and screened by transfection with the pTREd2eGFP reporting plasmid (BD Biosciences). The clone with the highest expression level in the absence of doxycycline and lowest background in the presence of 1 µg/ml doxycycline was selected for further use (HepaTO). The
1-AT Z sequence was inserted into the pTRE2Hyg plasmid (BD Biosciences) and was used to transfect HepaTO cells. Hygromycin-resistant clones with highest expression of
1-AT in the absence of doxycycline and lowest background in the presence of 1 µg/ml doxycycline were propagated, frozen in aliquots, and used in further experiments. These cell lines are appropriate model systems because pulse-chase studies have shown that the mutants are retained in the ER (data not shown) just as they are in the liver of deficient individuals (25, 49).
Metabolic labeling, immunoprecipitation, sequential immunoprecipitation, analytical gel electrophoresis, and immunoblotting.
Cell lines were subjected to pulse-chase radiolabeling as described previously (37). For the pulse period, the cells were incubated at 37°C in 100500 µCi/ml Tran35S-label (MP Biomedicals, Irvine, CA) in Dulbecco's modified Eagle's medium lacking methionine. In pulse-chase experiments, the cells were then rinsed vigorously and incubated in their regular culture medium with excess unlabeled methionine for the indicated time intervals as the chase period. At the end of each chase period, the extracellular medium was harvested and the cells were lysed in 0.1 M PBS, 1% Triton X-100, 0.5% sodium deoxycholate, and 10 mM EDTA supplemented with the Complete protease inhibitor cocktail (Roche, Indianapolis, IN) and 2 mM PMSF. The radiolabeled cell lysates were subjected to clarification and immunoprecipitation, and immunoprecipitates were analyzed by SDS-PAGE/fluorography exactly as described previously (46). In experiments when sequential immunoprecipitation was performed, the immunoprecipitated material was released from protein G by boiling in 50 µl gel loading buffer for 5 min. The supernatant was then transferred to new tubes, and the volume was made up to 1 ml with 0.1 M PBS, 1% SDS, 1% Triton X-100, 0.5% sodium deoxycholate, and 1% bovine serum albumin and then processed for another round of immunoprecipitation. Aliquots of the radiolabeled cell lysates were also subjected to trichloroacetic acid precipitation and scintillation counting to ensure that there was equivalent incorporation between cells under comparison. In experiments when nonlabeled cells were used, equal amounts of total cellular protein were used as determined by the BCA assay. For coimmunoprecipitations, cells were lysed either in 50 mM Tris·HCl (pH 7.4), 150 mM NaCl containing 0.5% Nonidet P-40 (NP-40), and protease inhibitors as indicated above or in Chaps cell extract buffer (Cell Signaling Technology, Beverly, MA), supplemented with 50 U/ml apyrase or 20 mM CaCl2, as indicated. Subsequent washes were done in the same buffer. For immunoblotting, proteins were transferred to a supported nitrocellulose membrane (Schleicher & Schuell BioScience, Keene, NH). The membrane was blocked in Dulbecco's modified PBS, 1% bovine serum albumin, 1% nonfat dry milk powder, and 0.05% Tween 20. The same buffer was used for incubation with the primary antibody, and the secondary antibody was applied in 0.1 M PBS and 0.05% Tween 20. All washes were done with 0.1 M PBS and 0.05% Tween 20. Blots were developed with the Supersignal chemiluminescent substrate (Pierce), and antibodies were removed from membranes with Restore (Pierce) before they were reused. Autoradiographs and immunoblots were quantitated using ImageJ, a public domain Java image-processing program (downloaded from the National Institutes of Health, Bethesda, MD). Mean (SD) and Student's t-test calculations were done with Excel 2000 (Microsoft, Redmond, WA).
Chemical cross-linking.
A fresh 20 mM stock solution of DSP, a cell membrane-permeable cross-linker cleavable by reducing agents, was prepared every time in DMSO. Cells were washed three times with Dulbecco's modified PBS, and cross-linking was done on ice using 2 mM DSP in Dulbecco's modified PBS for 30 min. Then, the cross-linking solution was aspirated, and the cells were lysed in the same buffer as described above supplemented with 20 mM glycine and 20 mM N-ethyl-maleimide. In the case of the HepaTO/Z cells, cross-linking was done at room temperature for 20 min, followed by a 5-min incubation in 20 mM glycine in Dulbecco's modified PBS before lysis in 50 mM Tris·HCl (pH 7.4), 150 mM NaCl containing 0.5% NP-40, 20 mM glycine, 20 mM N-ethyl-maleimide, and protease inhibitors.
Preparation of soluble and insoluble fractions from cell lysates.
Cell lysates were prepared in 50 mM Tris·HCl (pH 8.0), 150 mM NaCl, 2 mM KCl, 2 mM MgCl2, 0.5% Triton X-100, 0.5% sodium deoxycholate, and 2 mM N-ethyl-maleimide supplemented with protease inhibitors as above and homogenized by 12 passages through a 26-gauge needle on ice (14). Insoluble material was recovered by centrifugation at 16,000 g for 20 min. Pellets were washed once and solubilized in 50 µl of 50 mM Tris·HCl (pH 6.8), 5% SDS, and 10% glycerol with 1 min of sonication and then 10 min of boiling. The volume of the resolubilized pellet was then made up to 1 ml with 0.1 M PBS, 1% SDS, 1% Triton X-100, 0.5% sodium deoxycholate, and 1% bovine serum albumin and then processed for immunoprecipitation exactly as the other samples. This protocol has been shown to provide specific separation of the
1-AT Z mutant into soluble and insoluble fractions (25).
Equilibrium centrifugation on sucrose gradients.
Cells grown to confluence were lysed in 50 mM Tris·HCl (pH 8.0), 150 mM NaCl, 2 mM KCl, 2 mM MgCl2, 0.5% Triton X-100, 0.5% sodium deoxycholate, and 2 mM N-ethyl-maleimide supplemented with the Complete protease inhibitor cocktail (Roche, Indianapolis, IN) and 2 mM PMSF. After homogenization as described above, cell lysates were cleared of debris by a 5-min centrifugation at 3,000 g, and then the cleared lysate was loaded on top of a 560% sucrose gradient prepared in 14 x 89-mm tubes. Gradients were centrifuged in a Beckman SW-41 rotor at 28,500 rpm for 18 h at 4°C, and then 500-µl fractions were collected from the top. The refractive index of fractions was measured to calculate their sucrose concentration. Fractions were split in two, and one aliquot was immunoprecipitated, whereas proteins were precipitated with trichloroacetic acid (TCA) from the other. Immunoprecipitates or resolubilized TCA precipitates were separated on 10% SDS-PAGE and transferred to supported nitrocellulose membranes for immunoblotting as described above.
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RESULTS
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Cross-linking of
1-AT Z in a human fibroblast cell line.
First, we used chemical cross-linking to determine other proteins that interact with mutant
1-AT Z in a human fibroblast cell line engineered for stable expression of
1-AT Z, the CJZ12B cell line. Previous studies (49) have shown that this cell line is a good model of the classic form of
1-AT deficiency with retention of the mutant protein in the ER. The results (Fig. 1) show that the
52-kDa
1-AT polypeptide coprecipitates polypeptides of
80,
94, and
170 kDa. The presence of
88- and
150-kDa bands could be discerned after a longer exposure of the film (data not shown). These polypeptides were specific as shown by their absence when nonimmune serum was used instead of anti-human
1-AT. A lesser amount of the
80-kDa polypeptide was coprecipitated in the absence of cross-linking. The relatively diffuse
55-kDa polypeptide represents a biosynthetic intermediate of
1-AT Z with more extensive glycosylation, as shown by previous endoglycosidase H- and N-glycosidase F studies (30, 49).
Cross-linking of
1-AT mutants in ChoK1 cells.
To exclude the possibility that these results are peculiar to the human fibroblast cell line and also to establish a model system with higher incorporation of biosynthetic radiolabeling, we subjected a ChoK1 cell line engineered for stable expression of
1-AT Z to chemical cross-linking. We also compared the ChoK1 cell line expressing
1-AT Z with ChoK1 cell lines that expressed mutant
1-AT Saar and
1-AT Z + Saar. The
1-AT Saar mutant is truncated for the carboxyl-terminal 19 amino acids. The
1-AT Z + Saar mutant has the substitution that characterizes
1-AT Z (Glu342 to Lys342) and the 19-amino acid carboxyl-terminal truncation. Our previous studies have shown that the
1-AT Saar and
1-AT Z + Saar mutants are retained in the ER, although they do not form insoluble aggregates, in contrast to
1-AT Z (25). We reasoned that a comparison of these mutants to
1-AT Z might reveal differences in interaction with chaperones that are specific for mutant
1-AT Z. The results show that there are no substantial differences between these mutants (Fig. 2). Both
1-AT Saar and
1-AT Z + Saar migrate slightly faster than
1-AT Z. Polypeptides of
80,
88,
94,
110,
130,
150, and
170 kDa coimmunoprecipitate in each case. Wild-type
1-AT (
1-AT M) comigrates with
1-AT Z but has significantly more of the fully glycosylated
55-kDa intermediate than
1-AT Z or the other mutants but much less, if any, of the coprecipitating polypeptides. We presume this is because the interactions with wild-type
1-AT are too transient to be detected, but it is also possible that interaction of chaperones with the
1-AT mutants reflect the degradative pathways that are invoked specifically by mutant but not by wild-type
1-AT. The interaction of all
1-AT mutants with the
80-kDa polypeptide was apparent in the absence of cross-linking.
We also examined the possibility that
1-AT Z interacts with other chaperones after an extensive chase period. ChoK1 Z cells were subjected to pulse labeling for 6 h and then to a chase period of 18 h. Cross-linking of these cells showed that the same
80-,
88-, and
94-kDa polypeptides interacted with residual undegraded
1-AT Z (data not shown).
To determine the identity of the coprecipitating polypeptides, we first subjected the ChoK1 cell lines to immunoprecipitation with anti-
1-AT followed by immunoblot analysis with anti-Grp78. In Figure 3A, this approach shows that the
80-kDa polypeptide is Grp78 present in roughly equivalent amounts for the
1-AT mutants. To provide further evidence that Grp78 interacts with mutant
1-AT Z, we subjected unlabeled ChoK1 Z cells lysed in the presence or absence of apyrase to immunoprecipitation with anti-Grp78 followed by immunoblot analysis with anti-
1-AT (Fig. 3B). The results show that a small amount of
1-AT Z coprecipitates with Grp78, and it is significantly increased in the presence of apyrase. ATP binding allows Grp78 to release its substrate (24); therefore, ATP depletion with apyrase has been previously used to enhance detection of complexes of Grp78 with its substrates (26, 30). Compared with the amount of
1-AT that is detected by immunoprecipitation with anti-
1-AT followed by immunoblot analysis with anti-
1-AT, densitometric analysis indicates that 15 % (SD 3) [means (SD), n = 4] of the steady-state
1-AT Z pool is complexed with Grp78 when lysates were depleted of ATP, whereas only 8% (SD 2) was in complex with Grp78 without ATP depletion (n = 4, P < 0.05; Fig. 3B). The specificity of the interaction with Grp78 is further verified by comparing ChoK1 cells that express mutant
1-AT Z to ChoK1 cells that express wild-type
1-AT M (Fig. 3C). When first immunoprecipitated with anti-Grp78,
1-AT is detected by immunoblot in cells expressing
1-AT Z to a much greater extent than in those expressing wild-type
1-AT.

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Fig. 3. A: the 80-kDa protein cross-linked to 1-AT in ChoK1 cells is Grp78. ChoK1 M, ChoK1 Saar, ChoK1 Z, and ChoK1 Z + Saar cells (indicated on top of gel) were mock treated (2 mM DSP) or subjected to cross-linking (+2 mM DSP) as indicated. The cell lysates were immunoprecipitated with antibody to 1-AT, and the immunoprecipitated proteins were blotted to nitrocellulose membranes. The membrane was developed using goat anti-human Grp78 and peroxidase-conjugated donkey anti-goat IgG. The position of Grp78 is marked on the right with arrows. B: Grp78 coimmunoprecipitates with 1-AT Z. ChoK1 Z cells were lysed in the presence or absence of 25 U/ml apyrase, and equal aliquots of cell lysates were subjected to immunoprecipitation with goat anti- 1-AT or goat anti-Grp78, as indicated. The material resulting from the immunoprecipitation was separated on 10% SDS-PAGE under reducing conditions and transferred to a nitrocellulose membrane, and the membrane was developed with biotinylated antibody against 1-AT and streptavidin-conjugated peroxidase. The position of 1-AT is marked on the right with arrow. The bottom shows quantitative results of several experiments. Results are presented as %values of the amount of 1-AT immunoprecipitated with anti- 1-AT; 1-AT Z 8% (SD 2; n = 4) was found in complex with Grp78, and this figure rose significantly to 15% (SD 3; n = 4) when lysates were depleted of ATP with apyrase (P < 0.05). C: Grp78 coimmunoprecipitates with 1-AT Z but not with wild-type 1-AT M. ChoK1 M or ChoK1 Z cells (as indicated on top) were lysed, and the cell lysates were immunoprecipitated with antibody to 1-AT or anti-Grp78, as indicated. The immunoprecipitated proteins were blotted to nitrocellulose membranes, and the membrane was developed using biotinylated antibody against 1-AT and streptavidin-conjugated peroxidase. The position of 1-AT is marked on the right with an arrow.
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We suspected that the
88-kDa polypeptide cross-linked with the
1-AT mutants was calnexin. To definitively determine that calnexin interacted with
1-AT mutants, we used the immunoprecipitation/immunoblot strategy. ChoK1 cells expressing mutant
1-AT Z were lysed in the absence or presence of 20 mM CaCl2 and then were immunoprecipitated with antibody to
1-AT, complement factor B as a control, and calnexin, and the immunoprecipitates were analyzed by immunoblot for
1-AT.
It has been shown that depletion of ER Ca2+ stores by thapsigargin decreases the association of calnexin with its substrates (6, 10, 12); therefore, an excess amount of Ca2+ was included in the lysis buffer to maintain the association of calnexin and
1-AT Z. The results show that antibody to calnexin coprecipitates
1-AT, and that increases when the lysis is done in the presence of calcium (Fig. 4). The interaction is specific, as shown by its absence using antibody to factor B. Quantitative analysis indicates that 46 % (SD 8) of the total cellular
1-AT Z is in complex with calnexin in lysates supplemented with calcium, whereas only 26 % (SD 7) of the
1-AT Z was bound to calnexin without calcium supplementation (n = 4, P < 0.05; Fig. 4).
We also suspected that the
94-kDa polypeptide in the cross-linking experiment was Grp94. To address this possibility, we subjected all of the ChoK1 cell lines to cross-linking. Lysates were immunoprecipitated with anti-
1-AT followed by immunoblot analysis for Grp94 (Fig. 5A). The results show that Grp94 is indeed cross-linked to
1-AT in ChoK1 cell lines expressing
1-AT Z,
1-AT Saar, and
1-AT Z + Saar in roughly equivalent amounts. A significantly lesser amount of Grp94 is complexed with wild-type
1-AT M. To show that this interaction occurs in the absence of cross-linking, we first used a different type of sequential immunoprecipitation protocol. ChoK1 cells expressing
1-AT Z were metabolically labeled to steady state, and then lysates were prepared and subjected to a first round of immunoprecipitation with antibody to
1-AT or a control, complement C1 inhibitor. The immunoprecipitated material was released and subjected to a second round of immunoprecipitation with anti-Grp94 antibody (Fig. 5B). The results show a 94-kDa polypeptide only when antibody to
1-AT was used in the first round. With the use of similar immunoprecipitation/immunoblot analysis as in Figs. 3B and 4, we found that 30% (SD 6) (n = 3) of the total intracellular
1-AT Z pool was complexed with Grp94 (Fig. 5C).

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Fig. 5. A: the 94-kDa protein cross-linked to 1-AT in ChoK1 cells is Grp94. ChoK1 M, ChoK1 Saar, ChoK1 Z, and ChoK1 Z + Saar cells (indicated on top of gel) were mock treated (2 mM DSP) or subjected to cross-linking (+2 mM DSP) as indicated. The cell lysates were immunoprecipitated with antibody to 1-AT, and the immunoprecipitated proteins were blotted to nitrocellulose membranes. The membrane was developed using goat anti-human Grp94 and peroxidase-conjugated donkey anti-goat IgG. The position of Grp94 is marked on the right with arrows. B: Grp94 coimmunoprecipitates with 1-AT Z. ChoK1 Z cells were radiolabeled for 26 h with 100 µCi/ml [35S]methionine, lysed, and equal aliquots of cell lysate were subjected to immunoprecipitation with the following antibodies: goat anti-Cl inhibitor and goat anti- 1-AT, as indicated on top of the lanes (1st IP). The immunoprecipitated material was released and subjected to a second round of immunoprecipitation with goat anti-Grp94 (2nd IP). The material resulting from this second immunoprecipitation was separated on 8% SDS-PAGE under reducing conditions, and the results were analyzed using fluorography. The position of Grp94 is marked on the right with arrow. C: significant portion of 1-AT Z is in complex with Grp94. ChoK1 Z cells were immunoprecipitated with goat anti- 1-AT or goat anti-Grp94, the immunoprecipitates were blotted to nitrocellulose membranes, and the membrane was developed using biotinylated antibody against 1-AT and streptavidin-conjugated peroxidase. The position of 1-AT is marked on the right with an arrow.
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Next, we examined the possibility that the
150- and
170-kDa polypeptides cross-linked to
1-AT Z were Grp170 and UGGT. Grp170 is an
150-kDa ER protein, structurally related to Hsp70, and has chaperone activity, but its specific ligands in mammalian cells are still not definitely characterized (15). UGGT is an
170-kDa protein that adds glucose to deglucosylated unfolded proteins in the ER (7, 16). Sequential immunoprecipitation with antibody to Grp170 and then with anti-
1-AT also proved that the 52-kDa form of
1-AT Z is specifically associated with Grp170 in the absence of cross-linking (Fig. 6A). Using immunoprecipitation/immunoblot analysis, we estimated that
16 % (SD 6) (n = 3) of the total intracellular
1-AT Z pool was complexed with Grp170 (data not shown). In Fig. 6B, sequential immunoprecipitation with antiserum to UGGT or nonimmune serum and then with anti-
1-AT shows that anti-UGGT specifically recognized (coprecipitated with) the 52-kDa form of
1-AT Z and that this interaction can be detected in the absence of cross-linking. Immunoprecipitation/immunoblot indicated that
12% of the total intracellular
1-AT Z pool was complexed with UGGT (data not shown).
We do not know the identity of the
110- and
130-kDa bands that coprecipitate in cross-linking experiments in each of the cell lines. These bands were not recognized by antibodies to glucosidase II or ER mannosidase II (data not shown).
Identification of chaperones in soluble complexes with
1-AT mutants under nondenaturing conditions.
To determine whether
1-AT Z interacts with Grp78 and Grp94 under nondenaturing conditions and to determine the relative molecular mass of the putative complexes, we subjected transfected ChoK1 Z cells to sucrose density gradient centrifugation under nondenaturing conditions. A previous study of cells transfected with
1-AT Z analyzed with this technique showed that
1-AT Z existed within the ER in soluble complexes of
150-kDa size and did not seem to form larger aggregates but was degraded from this soluble pool (20). However, the narrow sucrose gradient (520%) used in that study did not allow the detection of much larger complexes than the ones reported. Although the investigators used mild cell lysis conditions, known not to dissolve loop-sheet polymers of
1-AT Z, it was not reported how much
1-AT Z could be recovered from the pellet at the bottom of the sucrose gradient. Here, we used a wider gradient (560%) to allow detection of larger complexes.
When fractions from the sucrose density gradient of lysate of ChoK1 Z cells were immunoblotted for
1-AT, the results showed that the bulk (85%) of
1-AT Z was found in fractions 211, corresponding to sizes from under 66 kDa to over 443 kDa (Fig. 7A). Approximately 4% of
1-AT Z was found in fractions 1821 and
11% was recovered from the pellet at the bottom of the gradient.

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Fig. 7. 1-AT Z and 1-AT Z + Saar form soluble complexes of the same size with chaperones. A: homogenized lysates of control (2 mM DSP) or cross-linked (+2 mM DSP) ChoK1 Z and ChoK1 Z + Saar cells were subjected to separation on a 560% sucrose gradient. Twenty-three fractions were taken starting from the top of the gradient, and the pellet (P) was also recovered from the bottom of the tube. One-half of each fraction was subjected to trichloroacetic acid (TCA) precipitation, and the resolubilized TCA precipitates were separated on 10% SDS-PAGE and immunoblotted for 1-AT. Arrows on the top mark the approximate position of size markers. B: 1-AT immunoblotting results obtained from control cells in A were quantified, and the %value of the signal from each fraction compared with the total 1-AT signal was plotted against its sucrose concentration. Sucrose gradient profile of 1-AT Z ( ) is compared with that of 1-AT Z + Saar ( ). C: same as B, except 1-AT immunoblotting results of cross-linked cells were used. Cross-linked 1-AT Z ( ) is compared with cross-linked 1-AT Z + Saar ( ). D: one-half of each sucrose gradient fraction of cross-linked ChoK1 Z or ChoK1 Z + Saar cells was immunoprecipitated with antibody to 1-AT and then Grp78 was detected from the immunoprecipitated material by immunoblotting. Sucrose gradient profile of Grp78 cross-linked to 1-AT Z ( ) is compared with Grp78 cross-linked 1-AT Z +Saar ( ). E: ChoK1 Z cells were lysed in the presence of apyrase and separated on a sucrose gradient, and one-half of each fraction was subjected to TCA precipitation, and the other half was immunoprecipitated with antibody to 1-AT; then both TCA precipitates and immunoprecipitates were immunoblotted for Grp78. Sucrose density gradient profiles of Grp78 coimmunoprecipitating with 1-AT Z ( ) is compared with that of total Grp78 ( ). Sucrose density gradient profile of Grp78 cross-linked to 1-AT Z ( ), already presented in D, is shown here for comparison. F: soluble and insoluble fractions of ChoK1 Z cell lysate was prepared as described in MATERIALS AND METHODS, and the soluble fraction was analyzed as in A, top. Total cell lysate of ChoK1 Z cells was supplemented with SDS to a final concentration of 2% and then analyzed an in A, bottom. Bars on top represent the approximate position of the size markers: bovine serum albumin, 66 kDa; -amylase, 200 kDa; and apoferritin, 443 kDa.
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When fractions from the gradient loaded with ChoK1 Z + Saar lysate were analyzed in the same way, all of the
1-AT was found in fractions 211. None was found in the pellet or in the very large complexes of fractions 1821, distinguishing it from
1-AT Z. Interestingly, quantification of the signals for
1-AT Z and
1-AT Z + Saar showed almost identical sucrose gradient profiles in the range of 530% sucrose (Fig. 7B).
Because it might better preserve interactions with chaperones, we also subjected cells to cross-linking and then analyzed the lysates on sucrose density gradients (Fig. 7A). The results showed a partial shift of
1-AT Z to higher fractions within the 212 range. The results were identical for
1-AT Z + Saar. The bulk of protein was detected in fractions 310. Quantitative results (Fig. 7C) showed the near identity of sucrose density gradient profile of
1-AT Z and that of
1-AT Z + Saar with the shift toward complexes of 200443 kDa in cross-linked cells (compare Fig. 7, C with B). This is probably due to stabilization of complexes of larger size by cross-linking.
As expected,
1-AT Z was also present in the pellet at the bottom of the tube, although this insoluble
1-AT Z represented a much smaller proportion of the total
1-AT Z signal compared with that in the noncross-linked cells (compare top and top middle of Fig. 7A). The difference between the bottom ends of gradients of lysates from cross-linked and noncross-linked ChoK1 Z cells, namely, the reduction of insoluble
1-AT Z and the disappearance of the very large soluble complexes (detected in fractions 1821 of the non-cross-linked cells), is most likely due to the sequestration of
1-AT Z by the debris in lysates of cross-linked cells during the short, low-speed centrifugation done to clear lysates before application to the sucrose gradient. In every case, cell lysates of cross-linked cells produced a markedly increased pellet (data not shown).
On the basis of the results presented in Figs. 26, we suspected that the high-molecular-weight forms of the mutants in fractions 512 contained chaperones in complex with
1-AT Z or
1-AT Z+ Saar. We examined this possibility for Grp78 by subjecting sucrose gradient fractions to immunoprecipitation with antibody to
1-AT and then immunoblotting for Grp78. The results showed that Grp78 was indeed present in large complexes with
1-AT Z as well as with
1-AT Z + Saar, ranging in size from under 200 kDa to over 443 kDa (Fig. 7D). Grp78 was not present in fractions 1821 or in the pellet, indicating that the extremely high-molecular-weight forms of
1-AT Z are not associated with chaperones. Repeating the immunoblotting for Grp94 showed that the very same fractions that contain Grp78 also contain Grp94 (data not shown). These results, showing complexes of
1-AT mutants with Grp78 and Grp94 of
200600 kDa, are very similar to what has been seen for incompletely folded immunoglobulin heavy chains present in chaperone-containing complexes of heterogeneous size ranging from
140 to over
700 kDa (31).
The failure to detect Grp78 and Grp94 in complex with the extremely high-molecular-weight forms of
1-AT Z could not be attributed to insensitivity of immunoblotting. A similar conclusion could be drawn from experiments in which the soluble and insoluble fractions of lysates of ChoK1 Z cells subjected to radiolabeling and cross-linking were analyzed by SDS-PAGE and autoradiography. The results showed that, although polypeptides cross-linked to
1-AT Z could readily be detected in the soluble fraction, none was seen in the insoluble fraction (data not shown).
To show that the complexes containing Grp78 and
1-AT Z exist in the absence of cross-linking, ChoK1 Z cells were lysed in the presence of apyrase (as in Fig. 3B) and subjected to separation on a sucrose gradient (Fig. 7E). Fractions were either TCA precipitated or immunoprecipitated for
1-AT Z, and Grp78 was detected from both the TCA precipitates and from the immunoprecipitates by immunoblotting. The results showed a shift in the sucrose gradient profile of Grp78 that coimmunoprecipitates with
1-AT Z compared with total Grp78 (Fig. 7E). Probably due to the instability of these complexes in the absence of cross-linking, a smaller portion of Grp78 was detected in complexes of the size seen with cross-linking.
We suspected that the extremely high-molecular-weight forms of
1-AT Z in fractions 1821 and in the pellet, that are not associated with chaperones Grp78 and Grp94, represent relatively insoluble polymers/aggregates. To examine this possibility, we compared by sucrose density gradient analysis whole lysate to the soluble fraction of ChoK1 Z cells (Fig. 7F). The soluble fraction was prepared by a previously described homogenization/centrifugation protocol (25). This protocol has been shown to result in a pellet that contains
17% of the total cellular
1-AT Z in ChoK1 Z cells but none of the total cellular
1-AT Z + Saar in ChoK1 Z + Saar cells (25). The results in Fig. 7F show that the extremely high-molecular-weight forms of
1-AT Z in fractions 1821 and in the pellet disappear when the soluble fraction is analyzed this way, providing evidence that the extremely high-molecular-weight material is relatively insoluble
1-AT Z polymers/aggregates (compare Fig. 7, F, top, with A, top). Supplementing total cell lysate with SDS that is known to dissolve loop-sheet polymers of
1-AT Z (2, 18) also caused the disappearance of the extremely high-molecular weight forms of
1-AT Z in fractions 1821 and in the pellet (compare Fig. 7, F, bottom, with A, top).
Taken together, these results confirm previous results indicating that
1-AT mutants are retained in the ER in complex with Grp78 and Grp94, but, in this case, it is shown using conditions that permit biochemical/immunological analysis of cellular constituents but are relatively gentle and nondenaturing. Second,
15% of total cellular
1-AT Z in ChoK1 Z cells can be found in extremely large, relatively insoluble polymers/aggregates that are not associated with chaperones. Because this is not seen for
1-AT in ChoK1 Z + Saar and ChoK1 M cells, it is unlikely to be randomly aggregated protein. Third, the results suggest that most (
85%) of the
1-AT Z in the cell is in a monomeric form in heterogeneous complexes with multiple chaperones. This is because the sucrose gradient profile of the
1-AT Z mutants is so similar to that of the
1-AT Z + Saar mutant in the range of
66 kDa to over 443 kDa. However, it is not possible to exclude the existence of oligomers in complex with a lesser number of chaperones or with lesser proportion of larges chaperones. This also means that it is not possible to conclude that chaperones only interact with monomers of
1-AT Z.
There are two other important implications of these results. First, the similar disposition of most of
1-AT Z and
1-AT Z + Saar provides further evidence for the concept that polymerization is an effect rather than the cause of
1-AT Z retention within the cells. Second, the presence within the cells of heterogeneous complexes that contain multiple chaperones and
1-AT Z monomers and oligomers makes it very difficult to apply the lessons learned from studies on the polymerization of purified
1-AT Z ex vivo to what goes on in the secretory pathway in vivo.
Grp78, calnexin, Grp94, and Grp170 interact with
1-AT Z in cells of hepatocytic origin.
To determine whether
1-AT Z interacts with the same chaperones in cells of hepatocytic origin, we carried out cross-linking experiments in the HepaTO/Z cell line (Fig. 8). The results show that UGGT, Grp170, Grp94, calnexin, and Grp78 polypeptides are detected by immunoblotting of cell lysates from HepaTO/Z cells after immunoprecipitation with antibody to
1-AT.

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Fig. 8. Grp78, calnexin, Grp94, Grp170, and UGGT can be cross-linked to 1-AT Z in cells of hepatocytic origin. HepaTO/Z cells were mock-treated (2 mM DSP) or subjected to cross-linking (+2 mM DSP) as indicated. The cell lysates were immunoprecipitated with antibody to 1-AT, and the immunoprecipitated proteins were blotted to nitrocellulose membranes. The membrane was successively immunoblotted with one of the following antibodies: rabbit anti-Grp78, mouse anti-calnexin, rabbit anti-Grp94, rabbit anti-Grp170, rabbit anti-UGGT, and biotinylated goat anti- 1-AT and then developed with suitable secondary antibody.
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Fate of mutant
1-AT Z in ChoK1 Z cells when chaperones are perturbed.
Next, we examined the fate of
1-AT Z when the ChoK1 cell line was subjected to treatment with tunicamycin. Tunicamycin inhibits N-glycosylation and induces synthesis of Grp78, Grp94, and Grp170 as part of the unfolded protein response. First, we subjected the cells to chemical cross-linking (Fig. 9A). The results show that the unglycosylated form of
1-AT Z (
1-AT Z*) cross-linked increased amounts of Grp78, unglycosylated Grp94 (Grp94*), and unglycosylated Grp170 (Grp170*).

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Fig. 9. The effects of tunicamycin (Tun) on 1-AT Z processing. A: ChoK1 cells stably transfected with 1-AT Z were subjected to radiolabeling for 6 h with 250 µCi/ml [35S]methionine in the presence of vehicle as control or tunicamycin (10 µM) as indicated on top of gel, followed by chemical cross-linking. At the end of the cross-linking period, cell lysates were prepared and subjected to immunoprecipitation with anti- 1-AT serum. The immunoprecipitated material was separated on 6% SDS-PAGE under reducing conditions, and the results were analyzed using fluorography. The position of 1-AT Z, Grp78, Grp94, and Grp170 is marked on the right with arrow. *Faster migrating unglycosylated forms of 1-AT Z, Grp94, and Grp170. B: ChoK1 Z cells were preincubated with tunicamycin (10 µM) for 6 h and then subjected to radiolabeling for 30 min with 250 µCi/ml [35S]methionine in the presence of tunicamycin. At the end of the labeling period, cells were washed and incubated in culture medium containing tunicamycin and excess amount of unlabeled methionine for the time periods indicated on top of each lane. Control cells received vehicle only. Cell lysates (IC) and extracellular media (EC) were immunoprecipitated for 1-AT and results were analyzed with 10% SDS-PAGE/fluorography. Bottom: quantitative data from at least 2 experiments. The amount of intracellular or extracellular 1-AT detected at each time point is expressed as a %value of the intracellular 1-AT detected at the end of the pulse period. C: insoluble fractions prepared from cell lysates in B were resolubilized and immunoprecipitated for 1-AT, and results were analyzed with 10% SDS-PAGE/fluorography. Bottom: the amount of insoluble 1-AT detected at each time point expressed as a %value of the intracellular 1-AT detected at the end of the pulse period. Each time point represents data from at least 2 experiments.
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Second, we subjected ChoK1 Z cells to pulse-chase analysis in the absence or presence of tunicamycin (Fig. 9B). In the presence of tunicamycin, the faster-migrating unglycosylated
1-AT Z protein disappeared more slowly from the cells and secretion was completely abrogated. Examination of the insoluble fraction of lysates from the same experiment revealed that tunicamycin treatment caused accelerated partitioning of
1-AT Z into the insoluble fraction and an increase in the amount of insoluble
1-AT Z detected (Fig. 9C). After tunicamycin treatment,
1-AT Z begins to accumulate in the insoluble fraction within 1 h compared with 2 h in control. In other experiments, it could be detected in the insoluble fraction within 30 min of chase (data not shown). The studies shown in Fig. 7 indicate that the insoluble fraction includes extremely large polymers/aggregates of
1-AT Z not associated with Grp78.
Next, we examined the effect of calcium ionophore A23187
[GenBank]
on the fate of mutant
1-AT Z. In addition to its effect on calcium, A23187
[GenBank]
is known to induce increased synthesis of Grp78 and Grp94 (13). We treated ChoK1 Z cells with calcium ionophore A23187
[GenBank]
for 6 h and then subjected them to cross-linking (Fig. 10A). The results show a marked increase in association of Grp78 with
1-AT Z and a modest increase in cross-linking of polypeptide Grp94 to
1-AT Z but no change in Grp170 and UGGT. A similar increase in binding of Grp78 and Grp94 to thyroglobulin after depletion of ER Ca2+ stores by thapsigargin treatment was reported by Di Jeso et al. (12). Pulse-chase analysis performed after same treatment showed that A23187
[GenBank]
mediates a delay in the disappearance of
1-AT Z from the intracellular compartment (Fig. 10B). The amount of
1-AT Z detected in the extracellular media was also slightly decreased compared with control, but the secreted
1-AT Z migrated more rapidly. The more rapid migration of secreted glycoproteins caused by A23187
[GenBank]
has been observed before and is attributed to inhibition of the addition of sialic acid to complex oligosaccharide side chains (11).

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Fig. 10. The effects of calcium ionophore A23187
[GenBank]
on 1-AT Z processing. A: ChoK1 cells stably transfected with 1-AT Z were subjected to radiolabeling for 6 h with 250 µCi/ml [35S]methionine in the presence of vehicle as control or calcium ionophore A23187
[GenBank]
(1 µM) as indicated on top of gel, followed by chemical cross-linking. At the end of the cross-linking period, cell lysates were prepared and subjected to immunoprecipitation with anti- 1-AT serum. The immunoprecipitated material was separated on 6% SDS-PAGE under reducing conditions, and the results were analyzed using fluorography. The position of 1-AT Z, Grp78, Grp94, Grp170, and UGGT is marked on the right with arrow. B: ChoK1 Z cells were preincubated with calcium ionophore A23187
[GenBank]
(1 µM) for 6 h and then subjected to radiolabeling for 30 min with 250 µCi/ml [35S]methionine in the presence of calcium ionophore A23187
[GenBank]
. At the end of the labeling period, cells were washed and incubated in culture medium containing calcium ionophore A23187
[GenBank]
and excess amount of unlabeled methionine for the time periods indicated on top of each lane. Control cells received vehicle only. Cell lysates and extracellular media were immunoprecipitated for 1-AT, and results were analyzed with 10% SDS-PAGE/fluorography. Bottom: shows quantitative data from at least 2 experiments. The amount of intracellular or extracellular 1-AT detected at each time point is expressed as a %value of the intracellular 1-AT detected at the end of the pulse period. C: insoluble fractions prepared from cell lysates in B were resolubilized and immunoprecipitated for 1-AT, and the results were analyzed with 10% SDS-PAGE/fluorography. Bottom: amount of insoluble 1-AT detected at each time point expressed as a %value of the intracellular 1-AT detected at the end of the pulse period. Each time point represents data from at least 2 experiments.
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Next, we examined
1-AT Z in the insoluble fraction of lysates from the same pulse-chase experiment and found that calcium ionophore A23187
[GenBank]
had a very different effect from tunicamycin. There was a significant decrease in the amount of insoluble
1-AT Z in cells treated with calcium ionophore A23187
[GenBank]
(Fig. 10C).
These results indicate that agents that perturb ER chaperones have distinct effects on the fate of mutant
1-AT Z. Tunicamycin mediates an increase in the association of
1-AT Z with Grp78, Grp94, and Grp170, an increase in the partitioning of
1-AT Z into the insoluble state, and an increase in intracellular retention due to both delayed degradation and abrogation of residual secretion. The calcium ionophore mediates a more modest increase in association of
1-AT Z with Grp78 and Grp94. In the presence of the calcium ionophore, there is a delay in intracellular degradation and less insoluble
1-AT Z.
 |
DISCUSSION
|
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The results of this study indicate that mutant
1-AT Z interacts with a complex array of chaperones in the ER. In addition to calnexin,
1-AT Z interacts with Grp78, Grp94, Grp170, and UGGT in several different cell lines. These interactions were initially detected by chemical cross-linking as a method that would detect interacting proteins in an unbiased and relatively sensitive manner. On the basis of relative electrophoretic mobility, it was possible to surmise the identity of the putative interacting proteins and then by using several types of coimmunoprecipitating techniques, in both cross-linked- and noncross-linked cells, to more definitively establish the proteins that interact with mutant
1-AT Z. We estimated the fraction of the total
1-AT Z pool that is complexed with the individual chaperones under steady-state conditions: 15% was complexed with Grp78, 46% with calnexin, 30% with Grp94, 16% with Grp170, and 12% with UGGT. The sum of these fractions therefore accounts for 19% more than the total cellular pool of
1-AT Z. This may reflect experimental error. Certainly, the many technical factors involved in sequential immunoprecipitation/immunoblot analysis could result in errors of this magnitude. However, a much more likely explanation is that this reflects that
1-AT Z is in ternary complex with multiple chaperones. There is ample evidence for this in recent studies of ER chaperones including interactions between Grp170 and Grp94 as well as Grp170 and Grp78 (31, 40). Moreover, the results of coimmunoprecipitation studies from sucrose density gradient fractions here (Fig. 7) provide even stronger evidence for the extent of heterogeneous complexes of
1-AT Z with multiple chaperones.
The interaction of
1-AT Z with Grp78 is particularly important. It was demonstrated here in human fibroblasts as well as in the ChoK1 cell lines. It was demonstrated by several different techniques including sequential immunoprecipitation/immunoblot analysis without cross-linking, and the interaction was enhanced by ATP depletion with apyrase. We have also demonstrated this interaction in the HepaTO/Z cell line (Fig. 8) as well as in genetically engineered HeLa cell lines (data not shown). Finally, the interaction of
1-AT Z with Grp78 was demonstrated under nondenaturing conditions with evidence for their presence together in large soluble complexes from under
200 kDa to over
443 kDa. The functional significance of the interaction of
1-AT Z with Grp78 in mammalian cells is unknown, but Grp78 has been shown to play a role in the degradation
1-AT Z in yeast (4).
It was somewhat surprising to us that the nonpolymerogenic mutants,
1-AT Saar and
1-AT Z + Saar, had interactions with ER chaperones that were identical to those of
1-AT Z. These data indicate that the interactions of
1-AT Z with Grp78, calnexin, Grp94, Grp170, and UGGT are not a function of its polymerogenic properties. Because they provide further evidence for the similar fate of these mutants in the ER, these data also cast further doubt on the idea that the polymerogenic properties are the cause, as opposed to the effect, of ER retention of the
1-AT Z mutant.
Recent studies have shown that the mannose binding protein ER degradation-enhancing
-mannisidase-like protein (EDEM) binds substrates of the ER degradation pathway and collaborates with calnexin in the disposal of these substrates (19, 32). In fact, Oda et al. (33) have shown that the
1-AT Hong Kong mutant binds to EDEM. We were unable to detect a polypeptide that would correspond to EDEM in association with
1-AT Z,
1-AT Saar, or
1-AT Z + Saar in the cross-linking experiments described here. However, it is not possible to completely exclude the possibility that this negative result is due to the specific conditions used. We do know that the
1-AT Hong Kong mutant is much more rapidly degraded than
1-AT Z,
1-AT Saar, and
1-AT Z + Saar, and its ER degradation pathway differs from that of
1-AT Z,
1-AT Saar, and
1-AT Z + Saar in a number of other ways.
The results of this study also show quite different effects on the fate of
1-AT Z when the synthesis or functional activity of chaperones is perturbed pharmacologically. The effect of tunicamycin led to an increase in the association of
1-AT Z with Grp78, unglycosylated Grp94, and unglycosylated Grp170, probably reflecting the increase in synthesis of these chaperones as a result of the UPR. There was also an increase in the partitioning of
1-AT Z into the insoluble fraction, but it is not possible to determine whether this is due to the effect of the UPR directly, of underglycosylation, of the increase in association with the glucose regulated proteins, or due to the increased time that
1-AT Z resides in the ER as a result of the action of tunicamycin. A23187
[GenBank]
also induces the UPR, but, under the conditions used here, there was an increase in association of mutant
1-AT Z with Grp78 and Grp94 and a decrease in the accumulation of insoluble
1-AT Z, although degradation is delayed. The fact that we detected less insoluble
1-AT Z in the presence of A23187
[GenBank]
may reflect the capacity of Grp78 to suppress aggregation of substrates (41). However, again, because the two drugs have multiple effects, only some of which are overlapping, it is possible that other effects of tunicamycin and A23187
[GenBank]
account for these differences. Nevertheless, these data do provide further evidence that the processes of polymerization, aggregation, degradation, and secretion of mutant proteins such as
1-AT Z are not necessarily interrelated.
 |
GRANTS
|
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This work was supported in part by grants from the National Institutes of Health and the Alpha-1 Foundation.
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ACKNOWLEDGMENTS
|
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Dr. Li Lin established the ChoK1 cell lines used in this study. The advice and encouragement of Drs. Jeff Brodsky and John Subjeck are greatly appreciated. The authors are grateful to Jennifer Goeckeler and Craig Scott for help with the sucrose gradient experiments.
 |
FOOTNOTES
|
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Address for reprint requests and other correspondence: D. H. Perlmutter, Departments of Pediatrics, Cell Biology, and Physiology, Univ. of Pittsburgh School of Medicine, Children's Hospital of Pittsburgh, 3705 Fifth Ave., Pittsburgh, PA 15213-2583 (e-mail: David.Perlmutter{at}chp.edu)
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.
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