Substrate Specificity of the Oxidoreductase ERp57 Is Determined Primarily by Its Interaction with Calnexin and Calreticulin

Abstract

The formation of disulfides within proteins entering the secretory pathway is catalyzed by the protein disulfide isomerase family of endoplasmic reticulum localized oxidoreductases. One such enzyme, ERp57, is thought to catalyze the isomerization of non-native disulfide bonds formed in glycoproteins with unstructured disulfide-rich domains. Here we investigated the mechanism underlying ERp57 specificity toward glycoprotein substrates and the interdependence of ERp57 and the calnexin cycle for their correct folding. Our results clearly show that ERp57 must be physically associated with the calnexin cycle to catalyze isomerization reactions with most of its substrates. In addition, some glycoproteins only require ERp57 for correct disulfide formation if they enter the calnexin cycle. Hence, the specificity of ER oxidoreductases is not only determined by the physical association of enzyme and substrate but also by accessory factors, such as calnexin and calreticulin in the case of ERp57. These conclusions suggest that the calnexin cycle has evolved with a specialized oxidoreductase to facilitate native disulfide formation in complex glycoproteins. The formation of disulfides within proteins entering the secretory pathway is catalyzed by the protein disulfide isomerase family of endoplasmic reticulum localized oxidoreductases. One such enzyme, ERp57, is thought to catalyze the isomerization of non-native disulfide bonds formed in glycoproteins with unstructured disulfide-rich domains. Here we investigated the mechanism underlying ERp57 specificity toward glycoprotein substrates and the interdependence of ERp57 and the calnexin cycle for their correct folding. Our results clearly show that ERp57 must be physically associated with the calnexin cycle to catalyze isomerization reactions with most of its substrates. In addition, some glycoproteins only require ERp57 for correct disulfide formation if they enter the calnexin cycle. Hence, the specificity of ER oxidoreductases is not only determined by the physical association of enzyme and substrate but also by accessory factors, such as calnexin and calreticulin in the case of ERp57. These conclusions suggest that the calnexin cycle has evolved with a specialized oxidoreductase to facilitate native disulfide formation in complex glycoproteins. The ability to form disulfide bonds within proteins entering the secretory pathway is essential for cell survival and occurs within the endoplasmic reticulum (ER). 3The abbreviations used are: ER, endoplasmic reticulum; PDI, protein disulfide isomerase; ATCA, aurin tricarboxylic acid; AMS, 4-acetamido-4′maleimidylstilbene-2,2′-disufhonic acid; BMH, bismaleimidohexane; HA, influenza virus hemagglutinin; NEM, N-ethylmaleimide; SP cells, semipermeabilized cells; MHC, major histocompatibility complex. 3The abbreviations used are: ER, endoplasmic reticulum; PDI, protein disulfide isomerase; ATCA, aurin tricarboxylic acid; AMS, 4-acetamido-4′maleimidylstilbene-2,2′-disufhonic acid; BMH, bismaleimidohexane; HA, influenza virus hemagglutinin; NEM, N-ethylmaleimide; SP cells, semipermeabilized cells; MHC, major histocompatibility complex. For proteins with few disulfides, the process can be catalyzed by oxidation of cysteine residues to form the correct, native disulfide; however, for proteins with several disulfides, an isomerization reaction is also required to correct non-native disulfides formed following oxidation (1Jansens A. van Duijn E. Braakman I. Science.. 2002; 298: 2401-2403Google Scholar). Both these reactions are catalyzed by a group of ER-resident proteins that belong to the protein disulfide isomerase (PDI) family, which comprises over 17 members (2Ellgaard L. Ruddock L.W. EMBO Rep.. 2005; 6: 28-32Google Scholar). It is well established that PDI and several other family members are able to catalyze the formation and isomerization of disulfides in vitro, although the exact function of each of the family members in vivo is unknown. It is still an open question as to whether they all catalyze similar reactions and have distinct substrate specificities or whether they have distinct enzymatic functions related to the breaking and formation of disulfides. For one member of the PDI family, the function and substrate specificity is a little clearer. ERp57 has been shown previously to interact specifically with glycoproteins during their folding (3Oliver J.D. van der Wal F.J. Bulleid N.J. High S. Science.. 1997; 275: 86-88Google Scholar). The enzyme is physically associated with either calnexin or calreticulin (4Oliver J.D. Roderick H.L. Llewellyn D.H. High S. Mol. Biol. Cell.. 1999; 10: 2573-2582Google Scholar) and is therefore ideally placed to catalyze correct disulfide formation within proteins entering the calnexin/calreticulin cycle (referred to subsequently just as the calnexin cycle). In addition, the ability of ERp57 to catalyze the refolding of substrates in vitro is greatly enhanced if the substrate is bound to calnexin (5Zapun A. Darby N.J. Tessier D.C. Michalak M. Bergeron J.J. Thomas D.Y. J. Biol. Chem.. 1998; 273: 6009-6012Google Scholar). Recently, substrates for the reduction or isomerization reaction catalyzed by ERp57 have been identified by trapping mixed disulfides between enzyme and substrate (6Jessop C.E. Chakravarthi S. Garbi N. Hammerling G.J. Lovell S. Bulleid N.J. EMBO J.. 2007; 26: 28-40Google Scholar). Strikingly, there was an overrepresentation of substrate proteins with cysteine-rich domains containing little secondary structure, suggesting that the main function of ERp57 is in the isomerization of non-native disulfides. ERp57 has also been shown to function independently from the calnexin cycle. It is a component of the MHC class I loading complex where it forms a disulfide-linked complex with tapasin and is thought to either stabilize the complex or facilitate correct assembly of class I molecules (7Dick T.P. Bangia N. Peaper D.R. Cresswell P. Immunity.. 2002; 16: 87-98Google Scholar, 8Peaper D.R. Wearsch P.A. Cresswell P. EMBO J.. 2005; 24: 3613-3623Google Scholar). Recently, ERp57 has been demonstrated to isomerize interchain disulfides in the major capsid protein, VP1, of simian virus 40 (9Schelhaas M. Malmstrom J. Pelkmans L. Haugstetter J. Ellgaard L. Grunewald K. Helenius A. Cell.. 2007; 131: 516-529Google Scholar). The ability to dissociate VP1 pentamers by ERp57 does not require the substrate to interact with the calnexin cycle. Hence, it is still unclear how ERp57 recognizes its substrates, and in particular, whether this recognition is solely determined by an interaction with the calnexin cycle. The recognition of substrates by PDI is somewhat clearer in that one particular domain within the protein (the b′ domain) has been shown to be primarily responsible for substrate recognition and peptide binding (10Pirneskoski A. Klappa P. Lobell M. Williamson R.A. Byrne L. Alanen H.I. Salo K.E. Kivirikko K.I. Freedman R.B. Ruddock L.W. J. Biol. Chem.. 2004; 279: 10374-10381Google Scholar). The corresponding domain within ERp57 has been shown to be responsible for interaction with the calnexin cycle (11Russell S.J. Ruddock L.W. Salo K.E. Oliver J.D. Roebuck Q.P. Llewellyn D.H. Roderick H.L. Koivunen P. Myllyharju J. High S. J. Biol. Chem.. 2004; 279: 18861-18869Google Scholar), suggesting that for ERp57, substrate recognition must occur outside this domain or is determined solely by substrate interaction with calnexin via its oligosaccharide side chain. Hence, the aim of our study was to evaluate the necessity of the calnexin cycle both for ERp57 to recognize its substrates and for correct folding of glycoproteins. ERp57 was found to be required for the efficient folding of one substrate, influenza virus hemagglutinin (HA), but only when it entered the calnexin cycle. HA did not require ERp57 to fold if it was blocked from entering the calnexin cycle. In contrast, β1-integrin does not fold efficiently either if ERp57 was depleted or if ERp57 is blocked from entering the calnexin cycle (6Jessop C.E. Chakravarthi S. Garbi N. Hammerling G.J. Lovell S. Bulleid N.J. EMBO J.. 2007; 26: 28-40Google Scholar). Although ERp57 may be dispensable for the folding of some glycoproteins, the interaction with calnexin commits them to an ERp57-dependent fate. We also found that the majority of ERp57 substrates need to enter the calnexin cycle to be acted upon by the enzyme, demonstrating that substrate specificity is primarily dependent upon substrate entry into the calnexin cycle. Antibodies and Cell Lines—Mouse monoclonal antibody (8E3) to β1-integrin was a gift from Martin Humphries (University of Manchester, Manchester, UK). A rabbit polyclonal antibody to calnexin was as described previously (12Allen S. Bulleid N.J. Biochem. J.. 1997; 328: 113-119Google Scholar). Rabbit polyclonal anti-calreticulin was purchased from Stressgen (Ann Arbor, MI), mouse monoclonal anti-V5 antibody was from Invitrogen, PaSta.1 (mouse monoclonal to tapasin) and R.RING.4C (purified rabbit serum to TAP-1) were from Peter Cresswell (Yale University School of Medicine, New Haven, CT), and V5-agarose beads were from Sigma. ERp57-/- cells and mouse fibroblasts were a gift from Natalio Garbi and Günter Hämmerling, (German Cancer Research Center, Heidelberg, Germany). Transcription and Translation in Vitro—A cDNA clone encoding influenza virus HA strain A/Japan/305/57 (H2N2) was a gift from Mary-Jane Gething (University of Melbourne, Melbourne, Australia). A cDNA coding for tapasin was a gift from Peter Cresswell (Yale University School of Medicine). Transcription and translation in semipermeabilized (SP) cells were performed essentially as described previously (13Wilson R. Allen A.J. Oliver J. Brookman J.L. High S. Bulleid N.J. Biochem. J.. 1995; 307: 679-687Google Scholar). HA and tapasin cDNA were linearized with BamHI and transcribed using T7 polymerase. β1-integrin, Ero1α, and HLA-B35 mRNA were prepared exactly as described previously (6Jessop C.E. Chakravarthi S. Garbi N. Hammerling G.J. Lovell S. Bulleid N.J. EMBO J.. 2007; 26: 28-40Google Scholar, 14Chambers J.E. Jessop C.E. Bulleid N.J. J. Biol. Chem.. 2008; 283: 1862-1869Google Scholar, 15Cabibbo A. Pagani M. Fabbri M. Rocchi M. Farmery M.R. Bulleid N.J. Sitia R. J. Biol. Chem.. 2000; 275: 4827-4833Google Scholar). Transcripts were translated using rabbit reticulocyte lysate (Flexilysate, Promega) with SP cells added as required. Where appropriate, SP cells were preincubated with 1 mm castanospermine for 5 min or with γ-interferon (200 units/ml) for 48 h. Initiation of protein synthesis was allowed to proceed for 5 min at 30 °C before inhibition with 1 mm ATCA (Sigma) followed by incubation at 30 °C to allow elongation and post-translational modification. At specified times, 25 mm NEM was added at 4 °C to prevent disulfide exchange. β1-integrin translation products were immunoisolated prior to electrophoresis; otherwise, SP cells were isolated and resuspended in SDS-PAGE sample buffer (31.25 mm Tris-HCl, pH 6.8, 2% w/v SDS, 5% v/v glycerol, 0.01% w/v bromphenol blue). Electrophoresis and Western for SDS-PAGE were resuspended in SDS-PAGE sample and was added to where were for 5 min before and proteins were to for Western or in and and products were by using UK). For Western was blocked with in mm mm pH antibody were performed for 1 at °C with secondary or rabbit to were in and at °C for 1 h. were using enhanced substrate and UK). of ERp57 Cell ERp57 was to to were linearized with before into fibroblasts with to the cell were with (Sigma) for before were isolated and for of mRNA was translated in the of or ERp57-/- SP ATCA was added 5 and NEM was added 30 were was and were with for was added and at for 5 and was added for 1 at For was added to mm for min at 30 °C prior to NEM For was added to 1 mm for the cells in were for min with 1 mm at 4 and was by the of mm were subsequently and ERp57 was immunoisolated prior to SDS-PAGE and Western and substrate were performed exactly as described previously (6Jessop C.E. Chakravarthi S. Garbi N. Hammerling G.J. Lovell S. Bulleid N.J. EMBO J.. 2007; 26: 28-40Google Scholar). in the for ERp57 to facilitate efficient disulfide formation upon the substrate protein Garbi N. Hammerling G.J. M. J. Biol. Chem.. Scholar). For some proteins HA and β1-integrin, there is a of folding in an ERp57 cell however, for such as virus and there is such (6Jessop C.E. Chakravarthi S. Garbi N. Hammerling G.J. Lovell S. Bulleid N.J. EMBO J.. 2007; 26: 28-40Google Scholar, Garbi N. Hammerling G.J. M. J. Biol. Chem.. Scholar). a in the folding of HA in the of ERp57 be to ERp57 the only oxidoreductase that can catalyze correct disulfide formation in HA, or it be a of HA entering the calnexin by other PDI family this we followed disulfide formation of HA in a SP cell in vitro translation In such a SP cells prepared from cells in are added to a reticulocyte lysate in the of an and (13Wilson R. Allen A.J. Oliver J. Brookman J.L. High S. Bulleid N.J. Biochem. J.. 1995; 307: 679-687Google Scholar). The reticulocyte lysate does not added disulfide formation can occur and within proteins into the ER of the SP cells N.J. R. Biochem. J.. Scholar). The of the and of HA have been Braakman I. Mol. 2005; Scholar), to folding by the of translation products the of translation products was following distinct were We have previously demonstrated that these to and HA (13Wilson R. Allen A.J. Oliver J. Brookman J.L. High S. Bulleid N.J. Biochem. J.. 1995; 307: 679-687Google Scholar). The is not as we have shown previously that it can be with (13Wilson R. Allen A.J. Oliver J. Brookman J.L. High S. Bulleid N.J. Biochem. J.. 1995; 307: 679-687Google Scholar). the translation products were the HA as a distinct the demonstrating that disulfide bonds The protein also the but as a that a of Hence, in our SP cell HA was translated and and the protein was into a form with a corresponding to that of the The formed disulfide but these were in as described previously J. Scholar), in the of HA does not fold the of an of ERp57 disulfide formation within HA, we a in the of either or ERp57-/- At in the folding pathway were A and 1 and the are between and ERp57-/- cells, it was that the of ERp57 the formation of the that the native disulfides not formed efficiently and there was an of at in the ERp57-/- cells These results previously Garbi N. Hammerling G.J. M. J. Biol. Chem.. Scholar) and that our SP cell can the folding by a of ERp57. In the of ERp57, HA still interact with the calnexin but the of the oxidoreductase have to the in correct disulfide this we a in the of the can be from entering the calnexin cycle by of the oligosaccharide side chain. the of this we that HA was with to calnexin or calreticulin following translation in the of or ERp57-/- when castanospermine was in the HA was demonstrating that castanospermine blocked the interaction between HA and calnexin or calreticulin In addition, in the of HA formed native disulfide bonds with similar in both and ERp57-/- cells and and results were previously when HA was in isolated in the of castanospermine Helenius A. EMBO J.. Scholar). Hence, the of correct disulfide formation in the ERp57-/- cells was to the of the protein to the calnexin cycle where the protein from other oxidoreductases. HA enter the calnexin cycle where it ERp57 for efficient folding. It has been previously that the main for HA to with the calnexin cycle is to of protein and to prevent of the protein for Helenius A. EMBO J.. Scholar, Helenius J. Braakman I. Helenius A. S. 1995; Scholar). Our results suggest that HA can fold efficiently in the of ERp57 if it is from with the calnexin suggesting that HA has been from other oxidoreductases can catalyze correct disulfide the in to a of in disulfide formation with HA in ERp57-/- cells be a of a of oxidation or isomerization of non-native disulfides. whether there is a in we the of disulfide oxidation by with the In this SP cells were with NEM to and to NEM, and with with cysteine residues and in the by cysteine Hence, if disulfides formed during the cysteine residues be with AMS, in a HA was translated in the of or ERp57-/- cells and the translation products were with AMS, the with the protein and These results clearly that the with the ERp57-/- cells is not to oxidation and is most to be to a of isomerization of disulfides. The is that an PDI, the formation of both native and non-native disulfides and that ERp57 the non-native disulfides. The formation of non-native disulfides is to be in substrates with of disulfides. These results that the essential function of ERp57 is as an isomerase a of for ERp57 and other oxidoreductases that they are able to catalyze distinct reactions within the ERp57 with in with is that the b′ domain of PDI a binding (10Pirneskoski A. Klappa P. Lobell M. Williamson R.A. Byrne L. Alanen H.I. Salo K.E. Kivirikko K.I. Freedman R.B. Ruddock L.W. J. Biol. Chem.. 2004; 279: 10374-10381Google Scholar) and that the binding of ERp57 to calnexin is by its b′ domain (11Russell S.J. Ruddock L.W. Salo K.E. Oliver J.D. Roebuck Q.P. Llewellyn D.H. Roderick H.L. Koivunen P. Myllyharju J. High S. J. Biol. Chem.. 2004; 279: 18861-18869Google Scholar, P. J.D. S. M. Thomas D.Y. K. Scholar). prevent ERp57 with calnexin or we to the interaction between these proteins by a into the b′ domain of ERp57, which has previously been shown to binding to calnexin in vitro P. J.D. S. M. Thomas D.Y. K. Scholar). for ERp57 substrate we of the that during the reduction of a non-native a mixed disulfide between the enzyme and substrate can be if the cysteine in the oxidoreductase is to We cell that were of ERp57, both have been to and such an we were able to whether an association of ERp57 with calnexin and calreticulin the trapping of mixed disulfides between enzyme and glycoprotein that the complex formation between ERp57 and calnexin or calreticulin in we added a to cells, the cells, and for the of calreticulin or calnexin It has been demonstrated previously that calnexin and calreticulin form with ERp57 that can be by with (4Oliver J.D. Roderick H.L. Llewellyn D.H. High S. Mol. Biol. Cell.. 1999; 10: 2573-2582Google Scholar). between ERp57 and calreticulin or calnexin were isolated from cell and ERp57 A and and and to a calnexin was with the antibody from the and ERp57 cell and calreticulin were from immunoisolated from cells demonstrating the specificity of the interaction with ERp57 A and and The and the ERp57 did not with calreticulin or calnexin and Hence, we were able to cell of ERp57 that are not associated with calreticulin or calnexin and have the to form mixed disulfides with substrates cell from the cell were and with the several mixed disulfides between ERp57 and substrate proteins were the cell was a reduction in the and of mixed disulfides was The of castanospermine to the cell prior to cell did not these mixed disulfides between ERp57 and substrate that these did not require association of ERp57 with calnexin The that the of mixed disulfides did not upon castanospermine also that the in mixed disulfide formation is not to the substrates to interact with ERp57 to their in the calnexin cycle. The of the as mixed disulfides was by their when We have previously been able to substrates of ERp57 by of the mixed disulfides followed by and (6Jessop C.E. Chakravarthi S. Garbi N. Hammerling G.J. Lovell S. Bulleid N.J. EMBO J.. 2007; 26: 28-40Google Scholar). We a similar with the mixed disulfides isolated from the cell A of mixed disulfides from the cell is for a few were following from the cell and the of isolated was for for most of the however, and were It that the ability of ERp57 to form mixed disulfides with these proteins is of its association with a that is with is the interaction of ERp57 with tapasin D.R. Wearsch P.A. Cresswell P. EMBO J.. 2005; 24: 3613-3623Google Scholar). an we also translated either or tapasin mRNA into SP cells isolated from the ERp57 cell Both proteins were able to form mixed disulfides with ERp57, as by with the antibody of between the translation and ERp57 and The were still when the were in the of castanospermine 4 and The in of the and tapasin translation products the inhibition of in the of castanospermine and These results clearly that these particular proteins not need to enter the calnexin cycle to to ERp57. the for ERp57 to enter the calnexin cycle to recognize substrate we translated MHC class I in the of or SP cells and immunoisolated mixed disulfides with either tapasin or We have previously demonstrated that MHC class I forms a of when translated in the of SP cells ERp57 J.E. Jessop C.E. Bulleid N.J. J. Biol. Chem.. 2008; 283: 1862-1869Google Scholar). These have been and to a and a complex and are of the complex. In addition, a of mixed disulfides are formed between and ERp57, which can be by their with the antibody and by the that they occur outside the complex J.E. Jessop C.E. Bulleid N.J. J. Biol. Chem.. 2008; 283: 1862-1869Google Scholar, A. M. M. T.P. 2007; Scholar). was translated in the of the ERp57 cell the and the ERp57 were still the mixed disulfides between and ERp57 were The at in 4 to ERp57 translated in this cell as it is also when is added to the translation reaction These results that the interaction occurs of the ERp57 association with In contrast, the interaction between ERp57 and calnexin is for ERp57 to form mixed disulfides with chain. These results that the ability of ERp57 to and upon most substrates an interaction between the substrate glycoprotein and The main of a in ERp57, which its for is to the formation of mixed disulfides with substrates. that for ERp57 to function as an isomerase or it must be associated with the calnexin cycle. the ability of ERp57 to interact with a of proteins does not to be by the or by suggesting that the interaction with these proteins is not by the as that required for calnexin how ERp57 can form a interaction with both calreticulin and tapasin within the MHC class I complex Cresswell P. 1998; Scholar). ERp57 the of It in with results with HA suggest that the function of ERp57 is if proteins are from entering the calnexin cycle. we have shown previously that when β1-integrin is from entering the calnexin it efficiently (6Jessop C.E. Chakravarthi S. Garbi N. Hammerling G.J. Lovell S. Bulleid N.J. EMBO J.. 2007; 26: 28-40Google Scholar). These were by castanospermine in the translation which β1-integrin entering the calnexin cycle and a in folding (6Jessop C.E. Chakravarthi S. Garbi N. Hammerling G.J. Lovell S. Bulleid N.J. EMBO J.. 2007; 26: 28-40Google Scholar). whether this was to ERp57 into the calnexin we investigated whether β1-integrin fold in the ERp57 cell In this cell β1-integrin be to fold as ERp57 is and with the calnexin cycle. when the are in the of β1-integrin does not interact with the calnexin we can whether in folding can be by the of the ERp57 that is not in association with β1-integrin is a complex glycoprotein with over disulfides and β1-integrin as a distinct when the protein is (6Jessop C.E. Chakravarthi S. Garbi N. Hammerling G.J. Lovell S. Bulleid N.J. EMBO J.. 2007; 26: 28-40Google Scholar) castanospermine was during translation of β1-integrin in the of ERp57 cells, a in disulfide formation was and A of non-native disulfides was the in the of the protein into a in the of of the each that a was in the of castanospermine Hence, for this substrate, there is a to with the calnexin cycle to correct disulfide such an interaction does not allow correct folding by to other oxidoreductases ERp57. the of unstructured cysteine-rich domains and the correct folding in the of The of ERp57 in the folding of β1-integrin the for an oxidoreductase to be associated with calnexin to facilitate disulfide isomerization within complex glycoproteins. of oxidoreductases in the ER that each enzyme has the ability to a of substrates or that each enzyme a of reaction (2Ellgaard L. Ruddock L.W. EMBO Rep.. 2005; 6: 28-32Google Scholar). It is from our results with ERp57 that a of these can specificity is related to the of glycoproteins into the calnexin but this specificity is not a of a interaction of ERp57 with it is by the of a oligosaccharide side chain. In addition, the essential function of ERp57 is as an isomerase as oxidation of substrates can occur in the of the Hence, one of the for the of ER oxidoreductases is the that the substrates for disulfide formation can be into which require both and with oxidoreductases to catalyze or The calnexin cycle and ERp57 is one of such a of substrates, but it may also be the case that substrates with require oxidoreductases to and facilitate disulfide L. Mol. Biol. Cell.. 2002; Scholar) and Sitia R. EMBO J.. 2008; Scholar, R. J. K. Thomas D.Y. K. Science.. 2008; Scholar) are to with and may well facilitate disulfide reactions within such a complex. The of a oxidoreductase for glycoprotein folding within M. Helenius A. EMBO J.. 1998; Scholar) that substrates entering the calnexin cycle in these the correct disulfides. The calnexin cycle may have evolved to a function distinct from protein folding such as of or proteins L. M. Helenius A. Science.. 1999; Scholar). of the of glycoproteins to be a of the of if the glycoprotein has a complex of disulfides. Hence, an be to the association of an oxidoreductase with the calnexin cycle to allow the isomerization of non-native disulfides, folding. an oxidoreductase that is not associated with the calnexin ERp57 does not need to to the glycoprotein as it be into to its substrate by its binding to Hence, the function of PDI, which is via the b′ is in ERp57 that when the binding to calnexin is it can facilitate disulfide isomerization with its substrates. the specificity of ER oxidoreductases may in be by their interaction with other proteins a interaction between enzyme and substrate with the We Natalio Günter Hämmerling, Martin Peter and Gething for used during this We also and for the