Misfolded endoplasmic reticulum retained subunits cause degradation of wild-type subunits of arylsulfatase A heteromers Peter Poeppel1,*, Mekky Mohamed Abouzied1,2, Christof Volker1 and Volkmar Gieselmann1 ă Institut fur Biochemie und Molekularbiologie, Rheinische-Friedrich-Wilhelms Universita Bonn, Germany ăt ă Faculty of Pharmacy, University of El-Minia, Egypt Keywords arylsulfatase A; ERAD; MLD; protein oligomerization; protein quality control Correspondence V Gieselmann, Institut fur Biochemie und ă Molekularbiologie, Rheinische-FriedrichWilhelms Universitat Bonn, Nussallee 11, ă 53115 Bonn, Germany Fax: +49 228 732416 Tel: +49 228 732411 E-mail: gieselmann@ibmb.uni-bonn.de *Present address Institut fur Biochemie, Universitat zu Koln, ă ă ă Germany Arylsulfatase A is an oligomeric lysosomal enzyme In the present study, we use this enzyme as a model protein to examine how heteromerization of wild-type and misfolded endoplasmic reticulum-degraded arylsulfatase A polypeptides affects the quality control of wild-type arylsulfatase A subunits Using a conformation sensitive monoclonal antibody, we show that, within heteromers of misfolded and wild-type arylsulfatase A, the wild-type subunits are not fully folded The results obtained show that arylsulfatase A polypeptide complexes, rather than the monomers, are subject to endoplasmic reticulum quality control and that, within a heteromer, the misfolded subunit exerts a dominant negative effect on the wild-type subunit Although it has been shown that mature lysosomal arylsulfatase A forms dimers at neutral pH, the results obtained in the present study demonstrate that, in the early biosynthetic pathway, arylsulfatase A forms oligomers with more than two subunits (Received 10 February 2010, revised May 2010, accepted 18 June 2010) doi:10.1111/j.1742-4658.2010.07745.x Introduction Many proteins form homooligomers According to crystallization and in vitro gel filtration data, the lysosomal enzyme arylsulfatase A (ASA; UniProt accession number P15289) forms dimers at neutral pH and octamers at acidic pH [1,2] ASA is a 62 kDa soluble protein with three N-linked oligosaccharide side chains [3] Within the Golgi apparatus, mannose residues of at least two of these side chains are phosphorylated The resulting mannose 6-phosphate residues are important for mannose 6-phosphate receptor-mediated lysosomal delivery of the enzyme A polymorphism that is frequent in the normal population (allele frequency of approximately 15%) causes substitution of asparagine 350 carrying the third N-linked oligosaccharide of the enzyme by serine This substitution abolishes the N-glycosylation site Therefore, this allele codes for a slightly smaller ASA with only two oligosaccharide side chains [4] This ASA has been termed pseudodeficiency ASA (pdASA) Despite the loss of one N-linked oligosaccharide side chain, the biochemical properties of the pdASA polypeptide are largely identical to those of the wild-type ASA (wtASA) [4,5] Abbreviations ASA, arylsulfatase A; ER, endoplasmic reticulum; ERAD, ER associated degradation; HA, hemagglutinin tag; MLD, metachromatic leukodystrophy; moab, monoclonal antibody; pdASA, pseudodeficiency arylsulfatase A; UGGT, UDP-glucose:glycoprotein glucosyltransferase; wtASA, wild-type arylsulfatase A 3404 FEBS Journal 277 (2010) 3404–3414 ª 2010 The Authors Journal compilation ª 2010 FEBS P Poeppel et al Deficiency of ASA causes metachromatic leukodystrophy a lysosomal storage disorder, in which the degradation of the sphingolipid 3-O sulfogalactosylceramide is interrupted [6] This leads to progressive demyelination and finally lethal neurologic symptoms ASA deficiency is frequently caused by missense mutations, which cause misfolding and endoplasmic reticulumassociated degradation (ERAD) of the respective ASAs [7] Because the pd allele is so frequent, a number of mutations causing metachromatic leukodystrophy (MLD) were identified, which occur on the background of this allele One of these missense mutations causes a P377L substitution, leading to ERAD of the respective ASA [8] ERAD occurs when a protein does not pass the ER quality control [9] Various degradation pathways exist in the ER The best characterized of these pathways is proteasomal degradation of glycoproteins, which involves the modification of their N-linked oligosaccharide side chains [10] These Glc3Man9GlcNAc2 oligosaccharide side chains are processed to a Glc1Man9GlcNAc2 structure by ER glucosidases I and II This oligosaccharide side chain allows binding to calnexin or calreticulin ER resident chaperones Deglucosylation of the Glc1Man9GlcNAc2 structure by ER glucosidase II releases glycoproteins from calnexin ⁄ calreticulin [10] UDP-glucose:glycoprotein glucosyltransferase (UGGT) functions as a folding sensor and reglucosylates the Man9GlcNAc2 oligosaccharides in case the released newly-synthesized protein is not properly folded This leads to reassociation with calnexin ⁄ calreticulin and a new cycle to achieve correct folding If the protein does not succeed in achieving the correct conformation after repetitive deglucosylation ⁄ reglucosylation cycles, it will be targeted for proteasomal degradation [11,12] It is largely unclear how UGGT recognizes specifically misfolded proteins In vitro experiments using a heterodimer of normal and misfolded RNAse B [11] demonstrated that the enzyme recognizes and reglucosylates selectively the misfolded subunit of the heterodimer Although oligomerization of proteins is a frequent phenomenon, little is known with respect to how ER quality control deals with heterooligomers of wild-type and misfolded proteins The variety of defective ASAs, which are subject to ERAD [7], and the availability of structure-sensitive monoclonal antibodies identify ASA as a model protein well suited for an investigation of the consequences of heteromerization of wild-type and defective proteins in more detail ER quality control of protein heteromers Results wtASA activity is diminished upon coexpression with misfolded ASA polypeptides In a number of experiments in which we expressed misfolded, enzymatically inactive ASA to investigate the biochemical consequences of missense mutations, we noted that this reduced the endogenous ASA activity of the transfected cells To examine this phenomenon in more detail, we coexpressed active wild-type and defective enzymes and measured ASA activity in cell lysates (Fig 1) Two aspects must be considered in the set up of this experiment: The first is the fraction of cells coexpressing wild-type and defective ASA after transient transfection To determine the percentage of cells coexpressing both types of enzyme, we transiently transfected BHK cells with various amounts of plasmid expressing either green fluorescent protein or DsRed fluorescent proteins After transfection, cells expressing both proteins were counted using an immunofluorescence microscope Independent of the amount of DNA transfected, 70–73% of cells expressed both proteins (data not shown) The second fact for consideration is that sulfatases bear a unique modification of a cysteine residue in the active center [13] Cotranslationally, this residue is converted to formylglycine, which is essential for enzyme activity [13] Overexpression of sulfatases can saturate the formylglycine-generating enzyme, so that a fraction of the newly-synthesized sulfatases remains inactive Therefore, the easiest explanation for a reduction of wtASA activity upon coexpression of a defective enzyme is that the latter competitively displaces the wild-type enzyme from the formylglycine-generating enzyme [14,15] To exclude this effect, we transiently transfected increasing amounts of a plasmid expressing wtASA and measured enzyme activity in cells Figure 1A shows that the correlation between the transfected amount of wtASA expressing plasmid and ASA activity is approximately linear in the range 0–10 ng of plasmid Transfection of more than 10 ng of plasmid does not lead to a substantial further increase of ASA activity By contrast, when the same cells were investigated by immunoprecipitation, the amount of ASA cross-reacting material correlated with the amount of transfected plasmid up to 250 ng (data not shown) Thus, at higher plasmid concentrations, most of the synthesized ASA is inactive, most likely as a result of incomplete formylglycine residue formation In the range 0–10 ng of plasmid, however, the amount of ASA activity increases proportionally, showing that it is not limited by the activity of FEBS Journal 277 (2010) 3404–3414 ª 2010 The Authors Journal compilation ª 2010 FEBS 3405 ER quality control of protein heteromers P Poeppel et al activity to approximately 50% of controls (Fig 1B) The experiments were repeated in HEK293 cells, with identical results being obtained (data not shown) A Defective ASA causes partial retention of wtASA B Fig ASA activity after coexpression of wild-type and various defective ASAs (A) Increasing amounts (0.5–40 ng) of a plasmid encoding wtASA were transiently transfected into BHK cells and enzyme activity was measured 48 h after transfection In the range 0.5–10 ng of plasmid, ASA activity increases in an almost proportional manner (B) Some 4.2 ng of plasmid expressing wtASA was cotransfected with 4.2 ng of plasmids expressing various inactive, misfolded ASAs (P377L-pdASA, D335V-ASA, T275M-ASA, P136LASA, G86D-ASA, T201C-ASA and D255H-ASA) In the control, these plasmids were replaced by the empty vector (pBEH) The activity value obtained at 40 ng was taken as 100% In all cases of coexpression of defective ASA, the wtASA activity was lowered below the level of expression of wild-type enzyme only ASA activity was determined as mmg)1 protein When expressed alone, none of the defective ASA polypeptides displays enzymatic activity (data not shown) the formylglycine-generating enzyme We choose this linear range and cotransfected 4.2 ng of plasmid expressing wtASA cDNA with 4.2 ng of plasmid expressing ASA cDNAs coding for various amino acid substituted misfolded ASAs, which have been shown to be enzymatically inactive and degraded by ERAD [7] Coexpression of seven different defective, enzymatically inactive enzymes in BHK cells caused a reduction of 3406 To verify the results shown in Fig by a different experimental approach, we performed pulse chase experiments in cells coexpressing wtASA and the defective P377L-pdASA We chose the P377L-pdASA because this is a missense mutation occurring on the background of the ASA pseudodeficiency allele As explained in the Introduction, pdASA is a naturally occurring variant lacking one of the three ASA oligosaccharide side chains The properties of pdASA, however, are largely identical to wtASA [8] Because of the loss of one N-linked oligosaccharide side chain, pdASA and P377L-pdASA have a lower apparent molecular weight by SDS ⁄ PAGE and can be easily differentiated from wtASA (Fig 2A, bottom) WtASA and P377L-pdASA were expressed separately or together in BHK cells (Fig 2) Sixteen hours after transfection, cells were treated with NH4Cl This drug interferes with the post Golgi sorting of lysosomal enzymes and causes the secretion of newly-synthesized enzymes into the medium of cultured cells When cells were transfected with the wtASA cDNA, they were pulse labeled for h and chased in the presence of NH4Cl To quantify wtASA present in the medium at different chase times, we took the amount of wtASA present in the media and cells as 100% for each time point separately and plotted the percentage of ASA found in media against the chase time (Fig 2A, top) After 20 h of chase, the majority of wtASA is secreted By contrast, the defective P377L-pdASA remains in the cells (Fig 2A, middle), which is expected as a result of the retention of the defective enzyme in the ER [8] In addition, the continuous reduction of the amount of defective enzyme during the chase period demonstrates its degradation When wtASA and P377L-pdASA were coexpressed, only the wild-type enzyme appeared in the medium, but not P377L-pdASA (Fig 2A, bottom) Quantification of the percentage of wtASA in the medium of NH4Cl-treated cells expressing either the wtASA alone or together with the P377L-pdASA revealed that the coexpression of the P377L-pdASA decreases the percentage of wtASA polypeptides in the medium to approximately half of the percentage found in cells expressing wtASA only (Fig 2B) This indicates that the defective P377L-pdASA is able to cause retention of a fraction of wtASA in the cells The quantification of total precipitated ASA (i.e signals from cells plus medium for each chase time) FEBS Journal 277 (2010) 3404–3414 ª 2010 The Authors Journal compilation ª 2010 FEBS P Poeppel et al A B C ER quality control of protein heteromers Fig Secretion and stability of wtASA is decreased by the coexpression of defective P377L-pdASA (A) BHK cells were transfected with plasmids conferring expression of wtASA or P377L-pdASA Cells expressed these enzymes alone (upper two panels) or in combination (lower panel) Cells were labeled with 370 kBq of [35S]methionine for h and chased for the times indicated in the presence of 10 mM NH4Cl ASA was immunoprecipitated from cell lysat (C) and media (M) and subjected to SDS ⁄ PAGE When cells were harvested immediately after the pulse period (left lane), ASA was only immunoprecipitated from the cells and not from the media With longer chase periods, increasing amounts of wtASA appear in the media (B) 35S-labeled wtASA polypeptides of two parallel experiments shown in (A) were quantified in the cells and the media The graph shows the percentage of wtASA present in the medium (for calculations, see text) Filled circles, cells only expressing wtASA; open circles, coexpression of defective P377LpdASA The graph demonstrates that the coexpression of defective ASA reduces the secretion of wtASA Values represent the mean, minima and maxima of two parallel experiments Labeled polypeptides were quantified using a Fuji bioimager (C) Graph showing the total amount of ASA polypeptides present in the cells and media at different chase times The amount of ASA polypeptides present after h of pulse was taken as 100% Whereas wtASA (filled circles) is stable over a time period of 20 h, P377L-pdASA (open squares) is rapidly degraded The half-life of wtASA is reduced upon coexpression of defective P377L-pdASA (open circles) The half-life of P377L-pdASA is unchanged upon coexpression of wtASA (closed squares) Values are the mean, minima and maxima of two parallel experiments Labeled polypeptides were quantified by Fuji bioimager Wild-type and misfolded ASA polypeptides form heteromers allows an investigation of whether coexpression of P377L-pdASA with wtASA reduces the stability of the latter Figure 2C shows that wtASA, after an initial slight decrease, is quite stable, with 80% of the enzyme still present after 20 h By contrast, only 20% of the P377L-pdASA is left after 20 h Upon coexpression of wild-type and defective enzyme, the amount of wtASA after 20 h is reduced to less than 50% Obviously, the defective P377L-pdASA enzyme leads to a more rapid degradation of a fraction of the wild-type enzyme By contrast, coexpression of the defective enzyme with wild-type enzyme does not enhance the half-life of the P377L-pdASA Thus, the defective enzyme has a dominant negative effect on wtASA The experiments presented in Figs and suggest an interaction of misfolded ASA and wtASA Because gel filtration and crystallization studies demonstrate that ASA forms oligomers [1,2], heteromerization of wildtype and defective ASA subunits may offer an explanation for the dominant negative effect observed To detect heteromerization of ASA in metabolic labeling ⁄ pulse experiments, wtASA was tagged with a nine amino acid hemagglutinin (HA) peptide sequence at the C-terminus to allow precipitation with monoclonal antibody specific for HA (HA moab) Figure 3A shows that the HA moab immunoprecipitates HA tagged wtASA (wtASA-HA), but not untagged pdASA or P377L-pdASA Upon cotransfection, however, the pdASA P377L-pdASA coimmunoprecipitates with wtASA-HA, showing that this experimental set up allows the examination of ASA heteromerization In addition to wtASA, various defective ASAs were fused to the HA peptide sequence This yielded plasmids designated D335V-ASA-HA, T274M-ASAHA, P136L-ASA-HA, G86D-ASA-HA and D255HASA-HA All of the respective missense mutations FEBS Journal 277 (2010) 3404–3414 ª 2010 The Authors Journal compilation ª 2010 FEBS 3407 ER quality control of protein heteromers Fig Detection of ASA heteromers A nine amino acid HA tag was added to the C-terminus of wtASA (wtASA-HA) or ASAs carrying various amino acid substitutions (D335V-ASA-HA, T274M-ASAHA, P136L-ASA-HA, G86D-ASA-HA and D255H-ASA-HA) (A) BHK cells were transiently transfected with the indicated expression vectors PdASA and P377L-pdASA only have two oligosaccharide side chains, resulting in a lower molecular weight compared to wtASA-HA Cells were labeled with 370 kBq of [35S]methionine for h and subsequently harvested Cell lysates were divided into two aliquots and ASA polypeptides were immunoprecipitated with polyclonal antiserum specific for ASA (upper panel) or HA moab (lower panel) Immunoprecipitates were resolved on SDS ⁄ PAGE and labeled polypeptides were visualized using a Fuji bioimager The polyclonal ASA antiserum precipitates all polypeptides, whereas the HA moab precipitates only the HA tagged ASA polypeptides In cotransfected cells (lanes and 5), untagged pdASA and P377LpdASA are coimmunoprecipitated with the HA tagged wtASA-HA (lower panel) (B) Different HA tagged defective ASA polypeptides, as indicated in the top line, were transiently expressed with pdASA, as indicated in the line below After transfection of BHK cells with equal amounts of vectors expressing the HA-tagged and untagged pdASAs, cells were labeled with 3.7 MBq [35S]methionine for 30 Subsequently, the cells were lysed and HA tagged ASAs were immunoprecipitated with an HA moab from cell lysates Immunoprecipitates were resolved on SDS ⁄ PAGE and labeled polypeptides were visualized using a Fuji bioimager pdASA coimmunoprecipitated with the various HA tagged defective ASA polypeptides Untransfected A P Poeppel et al Transfection anti-hASA anti-HA B Transfection Untagged anti-HA of the immunoprecipitates by SDS ⁄ PAGE revealed coimmunoprecipitation of untagged nondefective pdASA in all HA immunoprecipitates of HA tagged defective ASAs Thus, pdASA forms heteromers with all of the misfolded ASAs examined To exclude the possibility that heteromer formation is not an in vivo phenomenon but occurs after cell lysis during immunoprecipitation, we labeled cells expressing either wtASA-HA or pdASA only After harvesting, we mixed the cell lysates and performed immunoprecipiation with HA moab Under these conditions, wtASA-HA did not coimmunoprecipitate pdASA, demonstrating that heteromer formation does not occur during immunoprecipitation but in the cells (data not shown) Stoichiometry of ASA oligomers were described in MLD patients, shown to be retained in the ER [16–18] and were degraded by the proteasome [7] These defective HA tagged ASAs were coexpressed with pdASA in BHK cells (Fig 3B) Cells were metabolically labeled with [35S]methionine for 30 and HA tagged misfolded ASA polypeptides were immunoprecipitated with the HA moab Resolution 3408 In the case where ASA forms dimers in the early stages of biosynthesis, coexpression of equal amounts of wtASA-HA and pdASA will yield one-third wtASA-HA homodimers, one-third wtASA-HA ⁄ pd ASA heterodimers and one-third pdASA homodimers This predicts that coimmunoprecipitation of the untagged pdASA by the wtASA-HA should yield intensity ratios of the respective bands on SDS ⁄ PAGE of approximately FEBS Journal 277 (2010) 3404–3414 ª 2010 The Authors Journal compilation ª 2010 FEBS P Poeppel et al one-third (pdASA) and two-thirds (wtASA-HA), respectively Whereas this is the case as shown in Fig 3B, in Fig 3A, the stoichiometry is not what was expected If ASA was not present as a dimer but rather as an oligomer in the early biosynthetic pathways, differences in the transfection efficiencies of the two plasmids encoding wtASA-HA and P377L-pdASA used in Fig could account for the variation in ratio of the two associated ASA polypeptides For that reason, we decided to examine the stoichiometry of the ASA heteromers in more detail by varying the ratio of the amount of plasmids in a cotransfection experiment We transfected BHK cells with varying amounts of plasmid expressing wtASA-HA and pdASA or P377LpdASA, respectively Ratio of plasmids varied from 20% : 80% to 80% : 20%, respectively After metabolic labeling, the cell lysates were split into two aliquots One aliquot was immunoprecipitated with a polyclonal ASA antiserum precipitating all expressed ASAs to control whether the ratios of wtASA and pdASA or P377L-pdASA polypeptides really reflect the ratios of the respective plasmids used for transfection Figure shows that, except for minor deviations, this is the case The second aliquot was immunoprecipitated with the HA moab to determine the amount of the coimmunoprecipitated non-HA tagged pdASA (Fig 4B) Quantification of the immunoprecipitated wtASA-HA and coimmunoprecipitated pdASA or P377L-pdASA, respectively, revealed that one wtASAHA coimmunoprecipitates at least five non-HA tagged pdASA polypeptides This suggests that newly-synthesized ASA is present at least as a hexamer Folding status of wtASA heteromerized with mutant ASA We have recently shown that wtASA folds in a sequential way, which can be followed by immunoprecipitation with various structure-sensitive monoclonal antibodies [7] The hASA specific moab A2 [19] detects an epitope of wtASA that is already formed within the first few minutes after biosynthesis [7] The T274M substituted ASA, however, is severely misfolded, so that it does not express this epitope and cannot be immunoprecipitated by moab A2 [7] This prompted us to investigate whether the T274M-ASA affects folding of the wtASA occurring in the same heteromer Cells were transfected with wtASA-HA, wtASA-Myc and T274M-ASA-HA, respectively, or cells were cotransfected with different amounts of wtASA-Myc and T274M-ASA-HA Cell lysates were divided into three aliquots and the ASAs were immunoprecipitated either with polyclonal ASA ER quality control of protein heteromers Precipition anti-hASA A Precipition anti-hHA B Fig Stoichiometry of ASA oligomerization To determine the stoichiometry of ASA in the oligomer, BHK cells were cotransfected with plamids expressing wtASA-HA and P377L-pdASA or pdASA, respectively The ratio of wtASA expressing plasmids to pdASA or P377L-pdASA expressing plasmids, respectively, varied, as indicated at the top Cells were labeled with 4.1 MBq of [35S]methionine for 30 After harvesting, cell lysates were split into two aliquots One aliquot was precipitated with polyclonal ASA antiserum (A) and the other aliquot with the HA moab (B) Quantification of 35S-labeled ASA polypeptides using a Fuji bioimager shows that one wtASA-HA can coimmunoprecipitate at least five untagged pdASA polypeptides antiserum or Myc epitope specific moab or with the hASA moab A2 The polyclonal antiserum is able to immunoprecipitate ASA even under denaturing conditions Immunoprecipitates were subjected to SDS ⁄ PAGE followed by western blotting with the HA moab When wtASA-HA was expressed and immunoprecipitated with either polyclonal antiserum or the ASA moab A2, the HA moab detected wtASA-HA in the western blot of the immunoprecipitate This confirms that wtASA-HA is correctly folded and can therefore be immunoprecipitated with moab A2 (Fig 5, lane 1) When T274M-ASA-HA was examined in the same way, no ASA polypeptides were detected with the HA moab after immunoprecipitation with the moab A2, confirming that incorrectly folded T274M-ASA-HA cannot be immunoprecipitated by the structure-sensitive ASA moab A2 (Fig 5, lane 3) When wtASA-Myc was coexpressed with the T274MASA-HA and immunoprecipitated with the moab A2, again, no HA containing enzyme could be detected in the immunoprecipitate (Fig 5, lane 4) This shows FEBS Journal 277 (2010) 3404–3414 ª 2010 The Authors Journal compilation ª 2010 FEBS 3409 ER quality control of protein heteromers P Poeppel et al Transfection Immunoprecipitation Western Polyclonal antiserum anti-HA anti-Myc anti-HA Moab A2 anti-HA Fig Association of misfolded T274M-ASA-HA with wtASA-Myc prevents folding of wtASA-Myc BHK cells were transfected with plasmids expressing either wtASA-HA (lane 1), wtASA-Myc (lane 2) or T274M-ASA-HA alone (lane 3), or combinations of wtASA-Myc and T274M-HA (lane 4), as indicated at the top ASA was immunoprecipitated from cell lysates with either a polyclonal ASA antiserum recognizing ASA polypeptides even under denaturing conditions (upper panel), or an Myc tag specific moab (middle panel), or the structure sensitive hASA moab A2 (lower panel) recognizing an epitope that is formed early in biosynthesis [7] Immunoprecipitates were subjected to western blot analysis by HA moab In the case of the immunoprecipitation with polyclonal antiserum, the HA tagged ASAs can be detected from cells expressing wtASA-HA or T274M-ASA-HA alone and from cells coexpressing 20% wtASA-Myc and 80% T274M-ASA-HA (upper panel, lanes 1, and 4) When Myc moab is used for immunoprecipitation, T274M-ASA-HA can be detected by western blot analysis from cells coexpressing 20% wtASA-Myc and 80% T274M-ASA-HA (middle panel, lane 4) After immunoprecipitation with moab A2, wtASA-HA can be immunoprecipitated from cells and detected by western blotting (lower panel, lane 1) The defective T274M-ASAHA cannot be immunoprecipitated with the structure sensitive moab A2 Also, in the case of coexpressing wtASA-Myc with T274M-ASA-HA, the defective enzyme cannot be coimmunoprecipitated with wtASA-Myc, indicating that wtASA-Myc does not express the A2 epitope and thus is not completely folded that the wild-type enzyme associated with the misfolded T274M-ASA defective enzyme does not express the A2 epitope and therefore is not completely folded Otherwise, T274M-ASA-HA should be detectable in the immunoprecipitate As a control demonstrating 3410 wtASA-Myc and T274M-ASA-HA heteromerization, lysates from coexpressing cells were immunoprecipitated with the Myc moab Western blot analysis of these immunoprecipitates shows that the T274M-ASA-HA subunits are detectable by the HA moab after cotransfection with wtASA-Myc (Fig 5, middle) This reveals that the inability to coimmunoprecipitate the T274MASA-HA with the structure-sensitive moab A2 is not the result of a lack of heteromerization of T274MASA-HA and wtASA-Myc These results suggest that, within a heteromer, the T274M substituted ASA prevents proper folding of the wtASA Improper conformation of the wtASA in the heteromer may be the result of the insufficient time available for folding because degradation as a result of association with the defective enzyme may occur too rapidly Kifunensine is an inhibitor of ER a1,2-mannosidase I Inhibition of this enzyme blocks the pathway diverting a misfolded enzyme to the proteasome, allowing more time for proper folding [20] Therefore, wtASA and T274M-ASA-HA were coexpressed in the absence or presence of kifunensine ASA was immunoprecipitated with the moab A2 and the immunoprecipitates were probed on a western blot with the HA moab (data not shown) However, even after stabilization with kifunensine, T274M-ASA-HA could not be coimmunoprecipitated with wtASA, indicating that, under these conditions, wtASA expressing the epitope of the moab A2 was not present in the T274M-ASAHA ⁄ wtASA heteromers Discussion Oligomerization of proteins is a frequent phenomenon, but the mechanism by which heterooligomers of normal and defective proteins pass ER quality control is only poorly understood We used the lysosomal enzyme ASA to examine the consequences of heteromerization of wild-type and defective ASA in more detail We demonstrate that, within such a heteromer, the misfolded ASA exerts a dominant effect on the wtASA subunit, decreasing its stability Although we have only shown this in detail for the P377L-pdASA, the reduction of enzyme activity upon coexpression of various defective ASAs (Fig 1), as well as the capability of heteromerization for all defective ASAs investigated in the present study, strongly suggests that this applies to all defective ASA polypeptides Crystallization [2] and gel filtration experiments [1] suggest that ASA forms dimers at neutral pH These studies were performed with mature lysosomal ASA, which has passed the biosynthetic compartments and reached its final lysosomal destination Our data, FEBS Journal 277 (2010) 3404–3414 ª 2010 The Authors Journal compilation ª 2010 FEBS P Poeppel et al however, clearly show that, during the early biosynthetic stages in vivo, the enzyme forms at least hexamers, possibly octamers, which have only been described in vitro at acidic pH The unexpectedly high number of ASA monomers with an oligomer cannot be explained by the aggregation of defective ASA polypeptides in the ER because the same stoichiometry is also found with nondefective pd ASA Currently, we not have an explanation for the stoichiometry of ASA, although it is possible that as yet unknown modifications occur during the early biosynthetic stages that affect oligomerization of the enzyme In vitro experiments using heterodimers of native and misfolded RNAse B have demonstrated that, even within a RNAse heterodimer, UGGT can distinguish the native subunit from the misfolded subunit and reglucosylates only the latter [11] If this process also occurs similarly in vivo, the wtASA is expected to remain unglucosylated within the ASA heteromers and only the associated defective ASA would be reglucosylated Because wtASA is also trapped in the ER, the dominant effect of the defective enzyme may then be explained by the assumption that a single misfolded subunit causes degradation of the heteromer irrespective of the conformational status of the other subunits The results obtained for the T274M-ASA, however, offer yet another explanation The ASA moab A2 does not recognize denatured ASA [19] Recent data suggest that the antibody binds an epitope that is formed early in ASA biosynthesis when the enzyme is partially folded [7] The T274M substituted ASA does not react with moab A2, nor with any other structure-sensitive ASA moab, indicating that it is severely misfolded This allowed an investigation of the folding status of the wtASA within a heteromer with defective T274MASA If the wtASA reaches a folding state within a heteromer that allows the expression of the A2 epitope, it should be possible to immunoprecipitate the wtASA with the moab A2 and to detect the T274MASA-HA subunit afterwards in the immunoprecipitates In our experiments, however, this was not the case This suggests that, within the heteromer, the wtASA does not fully proceed through its normal folding pathway Alternatively, the folding of ASA may be catalyzed by different chaperones acting successively on the enzyme In the case where a defective subunit cannot achieve a certain conformational state, this may prevent the entire oligomer from interacting with chaperones catalyzing later steps of folding In this case, the wtASA subunit remains incompletely folded and may be a substrate of UGGT Heteromerization of defective polypeptides with their normal counterparts has been demonstrated for ER quality control of protein heteromers several membrane proteins that are defective in dominant genetic diseases For example, defective frizzled, a member of the Wnt signalling receptor family, forms oligomers in the ER and can retain wild-type frizzled in the ER [21] Similar findings were reported for a kidney anion exchanger defective in renal tubular acidosis [22], for aquaporins in dominant diabetes insipidus [23] and for the GABAA receptor subunit [24] Only for the GABAA receptor subunit were the consequences of heterooligomerization examined in detail Comparable to ASA, defective GABAA receptor subunits also form oligomers with wild-type subunits, leading to the degradation of the latter by ERAD MLD is an autosomal recessive disease because degradation of wtASA induced by defective ASA has no biological consequence This is expected because individuals with only 5–10% of the average ASA activity of the normal population are healthy [25] Obviously, even low ASA activity maintains a normal catabolism According to the results obtained in the present study, we would predict that, in carriers of defective ASA alleles, a fraction of wtASA will be degraded This fraction, however, does not suffice to lower the activity to less than 10%, which would be necessary for the disease Accordingly, the present study did not aim to reveal mechanisms causing MLD Rather, the well characterized dimerization ⁄ octamerization status of ASA, the availability of various structure-sensitive antibodies and defective enzymes, as well as the known 3D structure, all qualify this protein as an ideal tool for investigating the basic aspects of the oligomerization of proteins in vivo in more detail Materials and methods Materials Cell culture media and supplements were obtained from Invitrogen GmbH (Darmstadt, Germany) DNA restriction and modifying enzymes were purchased from Fermentas (Sankt Leon-Rot, Germany) [35S]methionine (specific activity > 39 TBqỈmmol)1) was from Hartmann Analytik GmbH (Karlsruhe, Germany) Isolation of plasmids was performed using the QIA-Plasmid Midi Kit Qiagen GmbH (Hilden, Germany) in accordance with the manufacturer’s instructions The preparation and characterization of the moab A2 has been described previously [20] Hybridoma 12JA5 expressing HA antibody and 9E10 expressing Myc antibody, were cultured in RPMI medium containing 10% fetal bovine serum The HA antibody was purified from the medium by affinity chromatography using protein A sepharose; the Myc antibody was purified by protein G sepharose from GE Healthcare GmbH (Munich, Germany) FEBS Journal 277 (2010) 3404–3414 ª 2010 The Authors Journal compilation ª 2010 FEBS 3411 ER quality control of protein heteromers P Poeppel et al Generation of HA and Myc tagged hASA To generate HA-tagged hASA proteins, hASA cDNA was amplified via PCR from a pBEH expression vector that contains the hASA cDNA [26,27] using the primers: forward: 5¢-dAAAGAATTCAAGCGTAATCTGGAACA TCGTATGGGTAGGCATGGGGATCTGGGCAATG-3¢, reverse: 5¢-dTTTGAATTCCATGTCCATGGGGGCACC GCGGTC-3¢ The PCR product was cloned via EcoRI restriction sites into the expression vector pBEH To generate wtASA-Myc proteins, oligonucleotides containing the sequence of the Myc tag were generated: a BamHI restriction site was integrated upstream of the Myc sequence and a HindIII restriction site was integrated downstream of the sequence Via these restriction sites, the Myc sequence was cloned into the pBEH vector hASA cDNA was amplified via PCR from the pBEH expression vector using the primers: forward: 5¢-dAAAGGATCCGGCATGGGGATCTGG GCAATG-3¢, reverse: 5¢-dTTTGAATTCCATGTCCATGG GGGCACCGCGGTC-3¢ The hASA was cloned via EcoRI and BamHI sites into the Myc containing pBEH expression vector in a final concentration of 10 mm The drug was also present during labeling periods Quantification of the precipitated proteins was performed after SDS ⁄ PAGE with a Fuji bioimager (Fuji, Tokyo, Japan) Pixels of the corresponding polypeptide band were integrated by the software aida (raytest GmbH, Straubenhardt, Germany) After subtraction of background values, the numbers obtained were taken as arbitrary values for the amount of 35S-labeled ASA Immunoprecipitation and western blot Cell lysates of hASA expressing cells were incubated with either monoclonal hASA antibody A2 or polyclonal ASA antiserum or the monoclonal antibody against the Myc tag The antigen antibody complex was precipitated by Pansorbin A in the case of hASA antibodies or protein G sepharose in the case of aMyc moab and washed three times with NaCl ⁄ Pi After SDS ⁄ PAGE, the proteins were blotted onto a nitrocellulose membrane hASA-HA was detected by a biotinylated HA antibody and fluorophore-labeled streptavidin Detection was performed using a Li-Cor laser scanner (Li-Cor, Lincoln, NE, USA) DNA transfection and ASA activity determination Transfection of expression plasmids into BHK cells was performed with ExGen 500 (Fermentas) Twenty four hours prior to transfection, · 104 per · 105 BHK cells were seeded onto 24-well per six-well plates, respectively For transfection, 22.5 lL per 121.5 lL 150 mm NaCl was mixed with 0.5 lg per 2.7 lg of DNA, respectively; then 1.7 lL per 8.9 lL ExGen 500 was added and incubated for 10 after mixing Transfection solution was added to 225 lL per 1215 lL DMEM containing 5% fetal bovine serum and then added to the cells Fourteen hours later, the DNA ⁄ transfection reagent containing medium was removed and replaced by serum-containing medium To determine ASA activity, cells were harvested 48 h later Twenty microliters of cell lysate, containing 20–50 lg of protein, were incubated with 200 lL of substrate solution (10 mm para-nitrocatecholsulfate in 0.5 m sodium acetate, pH 5.0, 10% w ⁄ v NaCl and 0.3% Triton X-100) for 30– 60 at 37 °C The reaction was terminated by the addition of 500 lL of m NaOH Absorption was measured at 515 nm Protein content was determined with the DC assay protein determination kit from Bio-Rad (Hercules, CA, USA) in accordance with the manufacturer’s instructions Metabolic labeling and immunoprecipitation Protocols for metabolic labeling with [35S]methionine and for subsequent immunoprecipitation of ASA have been described in detail previously [28] Secretion of 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Chem 267, 13262–13266 FEBS Journal 277 (2010) 3404–3414 ª 2010 The Authors Journal compilation ª 2010 FEBS ... C-terminus of wtASA (wtASA-HA) or ASAs carrying various amino acid substitutions (D335V-ASA-HA, T274M-ASAHA, P136L-ASA-HA, G86D-ASA-HA and D255H-ASA-HA) (A) BHK cells were transiently transfected... vector that contains the hASA cDNA [26,27] using the primers: forward: 5¢-dAAAGAATTCAAGCGTAATCTGGAACA TCGTATGGGTAGGCATGGGGATCTGGGCAATG-3¢, reverse: 5¢-dTTTGAATTCCATGTCCATGGGGGCACC GCGGTC-3¢ The... (data not shown) A Defective ASA causes partial retention of wtASA B Fig ASA activity after coexpression of wild-type and various defective ASAs (A) Increasing amounts (0.5–40 ng) of a plasmid