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• W3088186192 abstract "α1-antitrypsin (AAT) regulates the activity of multiple proteases in the lungs and liver. A mutant of AAT (E342K) called ATZ forms polymers that are present at only low levels in the serum and induce intracellular protein inclusions, causing lung emphysema and liver cirrhosis. An understanding of factors that can reduce the intracellular accumulation of ATZ is of great interest. We now show that calreticulin (CRT), an endoplasmic reticulum (ER) glycoprotein chaperone, promotes the secretory trafficking of ATZ, enhancing the media:cell ratio. This effect is more pronounced for ATZ than with AAT and is only partially dependent on the glycan-binding site of CRT, which is generally relevant to substrate recruitment and folding by CRT. The CRT-related chaperone calnexin does not enhance ATZ secretory trafficking, despite the higher cellular abundance of calnexin-ATZ complexes. CRT deficiency alters the distributions of ATZ-ER chaperone complexes, increasing ATZ-BiP binding and inclusion body formation and reducing ATZ interactions with components required for ER-Golgi trafficking, coincident with reduced levels of the protein transport protein Sec31A in CRT-deficient cells. These findings indicate a novel role for CRT in promoting the secretory trafficking of a protein that forms polymers and large intracellular inclusions. Inefficient secretory trafficking of ATZ in the absence of CRT is coincident with enhanced accumulation of ER-derived ATZ inclusion bodies. Further understanding of the factors that control the secretory trafficking of ATZ and their regulation by CRT could lead to new therapies for lung and liver diseases linked to AAT deficiency. α1-antitrypsin (AAT) regulates the activity of multiple proteases in the lungs and liver. A mutant of AAT (E342K) called ATZ forms polymers that are present at only low levels in the serum and induce intracellular protein inclusions, causing lung emphysema and liver cirrhosis. An understanding of factors that can reduce the intracellular accumulation of ATZ is of great interest. We now show that calreticulin (CRT), an endoplasmic reticulum (ER) glycoprotein chaperone, promotes the secretory trafficking of ATZ, enhancing the media:cell ratio. This effect is more pronounced for ATZ than with AAT and is only partially dependent on the glycan-binding site of CRT, which is generally relevant to substrate recruitment and folding by CRT. The CRT-related chaperone calnexin does not enhance ATZ secretory trafficking, despite the higher cellular abundance of calnexin-ATZ complexes. CRT deficiency alters the distributions of ATZ-ER chaperone complexes, increasing ATZ-BiP binding and inclusion body formation and reducing ATZ interactions with components required for ER-Golgi trafficking, coincident with reduced levels of the protein transport protein Sec31A in CRT-deficient cells. These findings indicate a novel role for CRT in promoting the secretory trafficking of a protein that forms polymers and large intracellular inclusions. Inefficient secretory trafficking of ATZ in the absence of CRT is coincident with enhanced accumulation of ER-derived ATZ inclusion bodies. Further understanding of the factors that control the secretory trafficking of ATZ and their regulation by CRT could lead to new therapies for lung and liver diseases linked to AAT deficiency. The endoplasmic reticulum (ER) is responsible for ensuring that protein quality control is maintained throughout various complex steps that allow proteins to reach their native state after being synthesized as an unstructured polypeptide chain (1Ellgaard L. Helenius A. Quality control in the endoplasmic reticulum.Nat. Rev. Mol. Cell Biol. 2003; 4 (12612637): 181-19110.1038/nrm1052Crossref PubMed Scopus (1671) Google Scholar). Crucial to the function of the ER are molecular chaperones that fall into several classical chaperone families such as the heat shock protein (HSP) family, which includes HSP47 and HSP70s (to which binding immunoglobulin protein, BiP, belongs) (2Ma Y. Hendershot L.M. ER chaperone functions during normal and stress conditions.J. Chem. Neuroanat. 2004; 28 (15363491): 51-6510.1016/j.jchemneu.2003.08.007Crossref PubMed Scopus (326) Google Scholar). Another group of chaperones that is particularly important for the folding of N-linked glycosylated proteins includes the lectin chaperones calreticulin (CRT), calnexin (CNX), and calmegin (CLGN) (2Ma Y. Hendershot L.M. ER chaperone functions during normal and stress conditions.J. Chem. Neuroanat. 2004; 28 (15363491): 51-6510.1016/j.jchemneu.2003.08.007Crossref PubMed Scopus (326) Google Scholar, 3Williams D.B. Beyond lectins: the calnexin/calreticulin chaperone system of the endoplasmic reticulum.J. Cell Sci. 2006; 119 (16467570): 615-62310.1242/jcs.02856Crossref PubMed Scopus (372) Google Scholar, 4Ikawa M. Wada I. Kominami K. Watanabe D. Toshimori K. Nishimune Y. Okabe M. The putative chaperone calmegin is required for sperm fertility.Nature. 1997; 387 (9177349): 607-61110.1038/42484Crossref PubMed Scopus (243) Google Scholar). Whereas CRT is soluble, CNX and CLGN are type I transmembrane proteins. The ER lumenal domains of these chaperones are structurally related, containing a globular domain that is responsible for the binding of nascent monoglucosylated glycoproteins and a P/arm-domain responsible for the recruitment of co-chaperones, including the thiol-oxidoreductase ERp57 (5Chouquet A. Païdassi H. Ling W.L. Frachet P. Houen G. Arlaud G.J. Gaboriaud C. X-ray structure of the human calreticulin globular domain reveals a peptide-binding area and suggests a multi-molecular mechanism.PLoS ONE. 2011; 6 (21423620)e17886 10.1371/journal.pone.0017886Crossref PubMed Scopus (69) Google Scholar, 6Schrag J.D. Bergeron J.J. Li Y. Borisova S. Hahn M. Thomas D.Y. Cygler M. The structure of calnexin, an ER chaperone involved in quality control of protein folding.Mol. Cell. 2001; 8 (11583625): 633-64410.1016/s1097-2765(01)00318-5Abstract Full Text Full Text PDF PubMed Scopus (316) Google Scholar, 7Kozlov G. Pocanschi C.L. Rosenauer A. Bastos-Aristizabal S. Gorelik A. Williams D.B. Gehring K. Structural basis of carbohydrate recognition by calreticulin.J. Biol. Chem. 2010; 285 (20880849): 38612-3862010.1074/jbc.M110.168294Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar) (Fig. 1A). Unique to CRT is an acidic C-terminal region, which contains multiple low-affinity calcium-binding sites (8Baksh S. Michalak M. Expression of calreticulin in Escherichia coli and identification of its Ca2+ binding domains.J. Biol. Chem. 1991; 266 (1939178): 21458-21465Abstract Full Text PDF PubMed Google Scholar, 9Nakamura K. Zuppini A. Arnaudeau S. Lynch J. Ahsan I. Krause R. Papp S. De Smedt H. Parys J.B. Muller-Esterl W. Lew D.P. Krause K.H. Demaurex N. Opas M. Michalak M. et al.Functional specialization of calreticulin domains.J. Cell Biol. 2001; 154 (11524434): 961-97210.1083/jcb.200102073Crossref PubMed Scopus (229) Google Scholar, 10Wijeyesakere S.J. Gafni A.A. Raghavan M. Calreticulin is a thermostable protein with distinct structural responses to different divalent cation environments.J. Biol. Chem. 2011; 286 (21177861): 8771-878510.1074/jbc.M110.169193Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar) (Fig. 1A). Calcium is a key ionic component of the ER known to be important for proper protein folding and secretion. Calcium concentrations in the ER range from 100–800 μm, compared with 100 nm in the cytosol and 1–2 mm in the extracellular space (11Samtleben S. Jaepel J. Fecher C. Andreska T. Rehberg M. Blum R. Direct imaging of ER calcium with targeted-esterase induced dye loading (TED).J. Vis. Exp. 2013; 75 (23685703)e50317 10.3791/50317Google Scholar). Through its low-affinity calcium-binding sites, CRT plays a central role in ER calcium storage and cellular calcium homeostasis (12Michalak M. Corbett E.F. Mesaeli N. Nakamura K. Opas M. Calreticulin: one protein, one gene, many functions.Biochem. J. 1999; 344 (10567207): 281-292Crossref PubMed Scopus (671) Google Scholar). α1-antitrypsin (AAT) is a 52-kDa glycoprotein, a highly abundant serum serine protease inhibitor (serpin) that binds to and irreversibly inhibits members of the chymotrypsin family, serine proteases, including but not limited to neutrophil elastase, via a molecular “mouse-trap” mechanism (13Lomas D.A. Molecular mousetraps, α1-antitrypsin deficiency and the serpinopathies.Clin. Med. (Lond.). 2005; 5 (16011217): 249-25710.7861/clinmedicine.5-3-249Crossref PubMed Scopus (46) Google Scholar). AAT has several genetic variants, which fall into three main categories: normal alleles (such as M1, M2, M3, and M4), deficient alleles characterized by missense mutations (for example, the Z and S variants), and null/truncation mutants that have a stop codon insertion (the null Hong Kong (NHK) and Saar variants) (14Greene C.M. Marciniak S.J. Teckman J. Ferrarotti I. Brantly M.L. Lomas D.A. Stoller J.K. McElvaney N.G. α1-antitrypsin deficiency.Nat. Rev. Dis. Primers. 2016; 2 (27465791)16051 10.1038/nrdp.2016.51Crossref PubMed Scopus (156) Google Scholar). Expression of many of the deficient and null alleles results in misfolding and/or polymerization of their protein products (15Gooptu B. Dickens J.A. Lomas D.A. The molecular and cellular pathology of α1-antitrypsin deficiency.Trends Mol. Med. 2014; 20 (24374162): 116-12710.1016/j.molmed.2013.10.007Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). Of these, the Z mutation characterized by a Glu-to-Lys substitution at position 342 (E342K) in the mature protein is particularly debilitating (14Greene C.M. Marciniak S.J. Teckman J. Ferrarotti I. Brantly M.L. Lomas D.A. Stoller J.K. McElvaney N.G. α1-antitrypsin deficiency.Nat. Rev. Dis. Primers. 2016; 2 (27465791)16051 10.1038/nrdp.2016.51Crossref PubMed Scopus (156) Google Scholar). The Z allele encoding α1-antitrypsin Z (ATZ) is autosomal codominant with a frequency of 2–5% in Caucasians of European ancestry (14Greene C.M. Marciniak S.J. Teckman J. Ferrarotti I. Brantly M.L. Lomas D.A. Stoller J.K. McElvaney N.G. α1-antitrypsin deficiency.Nat. Rev. Dis. Primers. 2016; 2 (27465791)16051 10.1038/nrdp.2016.51Crossref PubMed Scopus (156) Google Scholar). ATZ is misfolded and forms polymers or aggregates within the cell (16Perlmutter D.H. α1-antitrypsin deficiency: a misfolded secretory protein variant with unique effects on the endoplasmic reticulum.Endoplasmic Reticulum Stress Dis. 2016; 3 (28217691): 63-7210.1515/ersc-2016-0004PubMed Google Scholar, 17Lomas D.A. New insights into the structural basis of α 1-antitrypsin deficiency.QJM. 1996; 89 (8977959): 807-81210.1093/qjmed/89.11.807Crossref PubMed Google Scholar). Polymerization also leads to reduced secretion of the protein into the serum, leading to the disease α1-antitrypsin deficiency (ATD), resulting in proteolytic damage of lung tissue and lung emphysema. The other highly studied phenotype is the accumulation of aggregated ATZ within hepatocytes resulting in cellular toxicity that, in some people, leads to liver cirrhosis and hepatocellular carcinoma (16Perlmutter D.H. α1-antitrypsin deficiency: a misfolded secretory protein variant with unique effects on the endoplasmic reticulum.Endoplasmic Reticulum Stress Dis. 2016; 3 (28217691): 63-7210.1515/ersc-2016-0004PubMed Google Scholar). Aggregated ATZ is a very well-characterized target for much of the cell's degradative machinery, including ER-associated degradation (ERAD) (18Kroeger H. Miranda E. MacLeod I. Pérez J. Crowther D.C. Marciniak S.J. Lomas D.A. Endoplasmic reticulum-associated degradation (ERAD) and autophagy cooperate to degrade polymerogenic mutant serpins.J. Biol. Chem. 2009; 284 (19549782): 22793-2280210.1074/jbc.M109.027102Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 19Wang H. Li Q. Shen Y. Sun A. Zhu X. Fang S. Shen Y. The ubiquitin ligase Hrd1 promotes degradation of the Z variant α 1-antitrypsin and increases its solubility.Mol. Cell. Biochem. 2011; 346 (20886262): 137-14510.1007/s11010-010-0600-9Crossref PubMed Scopus (21) Google Scholar), autophagy (18Kroeger H. Miranda E. MacLeod I. Pérez J. Crowther D.C. Marciniak S.J. Lomas D.A. Endoplasmic reticulum-associated degradation (ERAD) and autophagy cooperate to degrade polymerogenic mutant serpins.J. Biol. Chem. 2009; 284 (19549782): 22793-2280210.1074/jbc.M109.027102Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 20Kamimoto T. Shoji S. Hidvegi T. Mizushima N. Umebayashi K. Perlmutter D.H. Yoshimori T. Intracellular inclusions containing mutant α1-antitrypsin Z are propagated in the absence of autophagic activity.J. Biol. Chem. 2006; 281 (16365039): 4467-447610.1074/jbc.M509409200Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar, 21Feng L. Zhang J. Zhu N. Ding Q. Zhang X. Yu J. Qiang W. Zhang Z. Ma Y. Huang D. Shen Y. Fang S. Yu Y. Wang H. Shen Y. et al.Ubiquitin ligase SYVN1/HRD1 facilitates degradation of the SERPINA1 Z variant/α-1-antitrypsin Z variant via SQSTM1/p62-dependent selective autophagy.Autophagy. 2017; 13 (28121484): 686-70210.1080/15548627.2017.1280207Crossref PubMed Scopus (32) Google Scholar), and ER-to-lysosome-associated degradation (ERLAD) (22Fregno I. Fasana E. Bergmann T.J. Raimondi A. Loi M. Soldà T. Galli C. D'Antuono R. Morone D. Danieli A. Paganetti P. Anken E. Molinari M. ER-to-lysosome-associated degradation of proteasome-resistant ATZ polymers occurs via receptor-mediated vesicular transport.EMBO J. 2018; 3710.15252/embj.201899259Crossref PubMed Scopus (97) Google Scholar). 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A. 1994; 91 (8090762): 9014-901810.1073/pnas.91.19.9014Crossref PubMed Scopus (232) Google Scholar), whereas CRT's functions within these pathways are not well-studied. Elucidation of factors that influence folding, cellular retention, degradation, and secretion of ATZ in hepatocytes is important for gaining a better understanding of variable susceptibilities to liver disease in patients homozygous for the Z allele (16Perlmutter D.H. α1-antitrypsin deficiency: a misfolded secretory protein variant with unique effects on the endoplasmic reticulum.Endoplasmic Reticulum Stress Dis. 2016; 3 (28217691): 63-7210.1515/ersc-2016-0004PubMed Google Scholar). A recent study showed that UDP-glucose:glycoprotein glucosyltransferase 1 (UGGT1), the enzyme that regenerates monoglucosylated glycans on glycoproteins for CNX/CRT binding, can reduce insoluble NHK in a CRT-dependent manner (34Ferris S.P. Jaber N.S. Molinari M. Arvan P. Kaufman R.J. UDP-glucose:glycoprotein glucosyltransferase (UGGT1) promotes substrate solubility in the endoplasmic reticulum.Mol. Biol. Cell. 2013; 24 (23864712): 2597-260810.1091/mbc.e13-02-0101Crossref PubMed Scopus (30) Google Scholar). Based on this study, we hypothesized that CRT may also play a role in reducing ATZ accumulation within the cell by increasing its secretion and/or by promoting its degradation, both of which were investigated. Overall, our data provides a new and comprehensive view of CRT-specific roles in ATZ secretion and degradation. To examine cellular functions of CRT in ATZ folding and degradation, Calr−/− mouse embryonic fibroblasts (MEFs, K42 cells (9Nakamura K. Zuppini A. Arnaudeau S. Lynch J. Ahsan I. Krause R. Papp S. De Smedt H. Parys J.B. Muller-Esterl W. Lew D.P. Krause K.H. Demaurex N. Opas M. Michalak M. et al.Functional specialization of calreticulin domains.J. Cell Biol. 2001; 154 (11524434): 961-97210.1083/jcb.200102073Crossref PubMed Scopus (229) Google Scholar)) were transduced with retroviruses that encoded either WT human CRT, the Y92A mutant of human CRT that is deficient for binding monoglucosylated glycans (35Kapoor M. Ellgaard L. Gopalakrishnapai J. Schirra C. Gemma E. Oscarson S. Helenius A. Surolia A. Mutational analysis provides molecular insight into the carbohydrate-binding region of calreticulin: pivotal roles of tyrosine-109 and aspartate-135 in carbohydrate recognition.Biochemistry. 2004; 43 (14705935): 97-10610.1021/bi0355286Crossref PubMed Scopus (66) Google Scholar, 36Del Cid N. Jeffery E. Rizvi S.M. Stamper E. Peters L.R. Brown W.C. Provoda C. Raghavan M. Modes of calreticulin recruitment to the major histocompatibility complex class I assembly pathway.J. Biol. Chem. 2010; 285 (19959473): 4520-453510.1074/jbc.M109.085407Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar) or a control virus lacking calreticulin (hereafter called CRT WT, CRT Y92A, or CRT−/−, respectively). The cells were then transfected with either enhanced YFP (eYFP)-AAT or eYFP-ATZ encoding plasmids (37Dickens J.A. Ordóñez A. Chambers J.E. Beckett A.J. Patel V. Malzer E. Dominicus C.S. Bradley J. Peden A.A. Prior I.A. Lomas D.A. Marciniak S.J. The endoplasmic reticulum remains functionally connected by vesicular transport after its fragmentation in cells expressing Z-α1-antitrypsin.FASEB J. 2016; 30 (27601439): 4083-409710.1096/fj.201600430RCrossref PubMed Scopus (18) Google Scholar). AAT/ATZ levels in the media and within cells were quantified at 48 h post-transfection, utilizing eYFP fluorescence as a readout (Fig. 1B). We estimated cellular fluorescence using flow cytometry, by gating on eYFP+ cells, using untransfected cells for the background control (Fig. 1C). In the case of eYFP-AAT, there were small differences in transfection efficiency (% eYFP+ cells) between the CRT WT and CRT−/− cells and other conditions (Fig. 1D and Fig. S1A), which resulted in a parallel decrease in media fluorescence (Fig. 1E and Fig. S1B, left panels). However, in the case of eYFP-ATZ, we observed a small, nonsignificant increase in the media fluorescence of CRT WT over CRT−/− cells, which was accompanied by a reduction in cellular eYFP mean fluorescence intensity (MFI) (Fig. 1E and Fig. S1B, right panels). Fewer eYFP+ cells yielded the increase the ATZ media fluorescence in the context of CRT WT (Fig. 1D and Fig. S1A, right panels), indicating that transfection differences did not account for the observed changes. Quantitative PCR revealed no differences in the levels of eYFP-ATZ transcripts between the K42 cells (Fig. S2A). In transfections with plasmids encoding either eYFP-AAT or eYFP-ATZ, we also did not detect significant differences in cell death (% live cells) from transfection (Fig. 1D and Fig. S1A). We next calculated the ratio between the secreted and intracellular eYFP fluorescence by dividing the media fluorescence (corrected for background by subtracting the media fluorescence of the corresponding untransfected cells) by the cell fluorescence (calculated as the product of cellular eYFP MFI and the number of eYFP+ cells) (Table S1). For eYFP-AAT, the presence of CRT did not affect the ratio (Fig. 1F and Fig. S1C, left panels). In contrast, CRT expression increased the media:cell fluorescence ratio for ATZ (Fig. 1F and Fig. S1C, right panels), indicating a role for CRT in altering the distribution of ATZ between media and cells. CRT's ability to bind monoglucosylated glycoproteins is attributed to conserved residues in its globular lectin domain, of which Tyr-92 (Fig. 1A; PDB 6ENY (38Blees A. Januliene D. Hofmann T. Koller N. Schmidt C. Trowitzsch S. Moeller A. Tampé R. Structure of the human MHC-I peptide-loading complex.Nature. 2017; 551 (29107940): 525-52810.1038/nature24627Crossref PubMed Scopus (198) Google Scholar)) is crucial because its substitution causes an impairment of its ability to bind substrates such as major histocompatibility complex 1 (MHC-1) molecules (35Kapoor M. Ellgaard L. Gopalakrishnapai J. Schirra C. Gemma E. Oscarson S. Helenius A. Surolia A. Mutational analysis provides molecular insight into the carbohydrate-binding region of calreticulin: pivotal roles of tyrosine-109 and aspartate-135 in carbohydrate recognition.Biochemistry. 2004; 43 (14705935): 97-10610.1021/bi0355286Crossref PubMed Scopus (66) Google Scholar, 36Del Cid N. Jeffery E. Rizvi S.M. Stamper E. Peters L.R. Brown W.C. Provoda C. Raghavan M. Modes of calreticulin recruitment to the major histocompatibility complex class I assembly pathway.J. Biol. Chem. 2010; 285 (19959473): 4520-453510.1074/jbc.M109.085407Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). CRT WT and CRT Y92A were expressed at similar levels (Fig. S2B, top panel), which allowed further assessment of whether the ability of CRT to increase the secretory trafficking of eYFP-ATZ depended on glycan binding by CRT. In transfections with plasmids encoding eYFP-ATZ, the media fluorescence values were comparable between CRT WT and CRT Y92A, and both were slightly higher than that from CRT−/− cells (Fig. 1E, right panel). In addition, there was a significant reduction in cell MFI of CRT WT compared with both CRT−/− and CRT Y92A MEFs (Fig. 1E, right panel), along with fewer eYFP+ cells in the CRT WT condition (Fig. 1D, right panel). Interestingly, the significant increase in the eYFP-ATZ media:cell fluorescence ratio in the CRT WT condition compared with CRT−/− was also measured for the CRT Y92A mutant. However, there was also a significant difference in the media:cell fluorescence ratio between CRT WT and the CRT Y92A mutant, indicating that compared with CRT WT, the CRT Y92A mutant is less efficient at altering eYFP-ATZ distribution (Fig. 1F, right panel for averaged data; see also individual experimental trends shown in Fig. S3, A–C). The statistical analyses in Fig. 1, Fig. S1, and Fig. S3 are based on paired two-tailed t tests (Fig. S1; n = 2 conditions being compared) or repeated measures (RM) one-way analysis of variance (ANOVA) analyses (Fig. 1 and Fig. S3, A–C, n = 3 conditions being compared). Trends within experiments (Fig. S3A) are compared for the statistical analyses of Fig. 1, because the flow cytometry settings and conditions of the experiments (such as numbers of cells analyzed and transfection reagents used) were kept consistent across the conditions being compared on a given day. On the other hand, neither CRT WT nor the CRT Y92A mutant altered the media:cell fluorescence ratio for eYFP-AAT (Fig. 1F, left panel). Thus, the glycan-binding deficient CRT Y92A mutant does not affect the trafficking of eYFP-AAT but improves eYFP-ATZ secretion, although to a lesser extent than CRT WT. We further assessed whether human CRT is capable of preventing aggregation of eYFP-ATZ in an in vitro assay (Fig. 2A). Calr was knocked out using CRISPR/Cas9 gene editing in Huh7.5 cells, a human hepatocellular carcinoma-derived cell line (39Nakabayashi H. Taketa K. Miyano K. Yamane T. Sato J. Growth of human hepatoma cells lines with differentiated functions in chemically defined medium.Cancer Res. 1982; 42 (6286115): 3858-3863PubMed Google Scholar) that secretes endogenous AAT (hereafter called WT or CRT KO, respectively, for the control Huh7.5 (transduced with an empty lentiviral vector) and CRISPR/Cas9-edited cells (transduced with sgRNA that targeted CRT)). The levels of CRT were below detection limits on immunoblots, thus confirming the knockout (Fig. S2B, middle panel). The Huh7.5 CRT KO cells were transfected with plasmids encoding eYFP-ATZ, semi-permeabilized with a low concentration of digitonin (0.01%), and centrifuged to generate a supernatant fraction (which consists of cytosolic components) and a pellet fraction enriched in cell membranes, including the ER. The pellet fraction was then lysed in RIPA buffer containing 0.1% SDS and further diluted 10-fold before incubation at 37 °C in the presence of purified recombinant human CRT or BSA. Insoluble eYFP-ATZ was pelleted by centrifugation and levels of eYFP-ATZ in both the supernatant and pellet fractions were quantified by immunoblotting. Although the effect is modest, we found that the presence of increasing amounts of human CRT contributed to a reduction in the levels of insoluble eYFP-ATZ (Fig. 2, B and C). Together, these findings of Fig. 2 support the model that direct interactions between CRT and ATZ can enhance ATZ folding and solubility, which at least in part can explain the ability of CRT to enhance the secretory trafficking of ATZ. Various studies have shown roles for CNX in the binding, sequestration, and degradation of AAT and its mutants (22Fregno I. Fasana E. Bergmann T.J. Raimondi A. Loi M. Soldà T. Galli C. D'Antuono R. Morone D. Danieli A. Paganetti P. Anken E. Molinari M. 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Chem. 1995; 270 (7876241): 4697-470410.1074/jbc.270.9.4697Abstract Full Text Full Text PDF PubMed Scopus (382) Google Scholar, 26Rokeach L.A. Haselby J.A. Meilof J.F. Smeenk R.J. Unnasch T.R. Greene B.M. Hoch S.O. Characterization of the autoantigen calreticulin.J. Immunol. 1991; 147 (1919005): 3031-3039PubMed Google Scholar, 27Cabral C.M. Choudhury P. Liu Y. Sifers R.N. Processing by endoplasmic reticulum mannosidases partitions a secretion-impaired glycoprotein into distinct disposal pathways.J. Biol. Chem. 2000; 275 (10827201): 25015-2502210.1074/jbc.M910172199Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, 28Cabral C.M. Liu Y. Moremen K.W. Sifers R.N. Organizational diversity among distinct glycoprotein endoplasmic reticulum-associated degradation programs.Mol. Biol. Cell. 2002; 13 (12181335): 2639-265010.1091/mbc.e02-02-0068Crossref PubMed Scopus (0) Google Scholar, 29Liu Y. Choudhury P. Cabral" @default.
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