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J. Biol. Chem., Vol. 280, Issue 15, 14402-14412, April 15, 2005
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From the
Department of Biochemistry, George S. Wise Faculty of Life Sciences, Tel Aviv University, Ramat Aviv, Tel Aviv 69978, Israel and the Department of Pathology, Children's Hospital of Philadelphia, Philadelphia, Pennsylvania 19104
Received for publication, January 5, 2005 , and in revised form, February 7, 2005.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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sheets stabilized by a single intradomain disulfide (1). IgG assembly is stabilized by an interchain disulfide between CL, the LC constant domain, and CH1, the first HC constant domain (2). The CH1 domain confers retention in the endoplasmic reticulum (ER) on unassembled HCs, and despite its high degree of similarity to other constant domains, only CH1 is retarded in folding and is unable to cycle from the ER chaperone BiP (3). Only upon LC expression is BiP displaced from CH1, which completes its folding, allowing assembly of HC2LC2 and secretion (4–7).
Although BiP binds transiently a wide variety of proteins (8), its associations with Ig HC and LC are among the best characterized. In vivo, BiP binds transiently to nascent LC via the unfolded VL domain and is released when this domain folds (9–12). Non-secreted LC mutants display more persistent association with BiP (13–19). BiP also binds HC, where in addition to its interaction that seems irreversible with CH1, it binds transiently other domains. In vitro studies defined specific heptameric sequences throughout LC and HC, which serve as BiP binding sites (18, 20). The affinity for binding sites in CH1 is not obviously different from that for other heptamers, so presumably, BiP binding sites in the incompletely folded CH1 remain exposed, enabling continued interaction with BiP and ER retention (3, 7, 21). It has been suggested that CH1 is unique in its propensity to expose BiP binding sites following CH2 and CH3 homodimerization, because CH1 is the only constant domain unable to homodimerize (7, 20).
Recognizing the unique folding status of CH1, we undertook to identify structural elements within this domain, which control folding, chaperone interactions, and retention of unassembled HC. Several lines of evidence focused our attention on cysteines in CH1. Contribution of exposed thiols to ER retention is well documented (22–27). Thus, in the absence of LC, CH1-conferred retention could have resulted from continued exposure of the LC-interacting cysteine. However, even without such a cysteine, a truncated 
1 HC containing only VH and CH1 was still not secreted (21). Although this construct could no longer form the CL-CH1 disulfide, its CH1 oxidative folding, reflected by intradomain disulfide bond formation and consequent secretion, still required LC (21). Hence, possible contribution of the LC-interacting cysteine to CH1-conferred retention should be in the context of the two additional CH1 cysteines, which form the intradomain disulfide.
In this work we studied possible interrelations between all three CH1 cysteines, and, as shown below, substituting each of them with serine, singly or in combinations, yielded surprising structure-function relationships with respect to folding, assembly, and secretion. Remarkably, a mutant chain lacking all three CH1 cysteines was secretion-competent. We provide a mechanistic explanation for this phenomenon by showing that, although in CH1 neither the intradomain nor the interchain native disulfide was essential for secretion, these bonds affect each other's formation. In addition to providing a molecular basis for the coordinated folding of CH1 and its assembly with LC (7), we discuss a model in which CH1 cysteines employ thiol-mediated retention, and their interplay maintains the stable BiP binding of unassembled Ig HC.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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constructs were based on pJDEVNPC2b, containing genomic DNA encoding full-length murine HC with a known constant region (Ref. 28; accession number j00461). The CH1 exon, flanked by intronic sequences, was excised with EcoRI and PvuII. A NotI-containing linker was ligated to the vector at the EcoRI and PvuII sites, generating the CH1-less pJDEVNPC2b
CH1. Exons CH1, CH2, or CH3 were PCR-amplified, using primers (Table I) and pJDEVNPC2b as a template, and subcloned into pBluescript (pBS, Stratagene) at EcoRI and XbaI sites, to generate pBS-CH1, pBS-CH2, and pBS-CH3, respectively. Site-directed mutagenesis analyses of CH1 were performed using pBS-CH1 as a template and either forward mutagenic primer (Table I) or the complementary (reverse) mutagenic primer. The mutated CH1 was cut by EcoRI and XbaI and inserted into pBS. Double and triple mutations in CH1 were introduced successively. All DNA constructs were sequenced. Wild-type and mutated CH1, as well as CH2 and CH3, were excised from the respective pBS constructs and re-introduced into pJDEVNPC2bCH1 with EcoRI and NotI. Amplification and mutagenesis of 
I was performed by PCR (see primers in Table I) on pTM1-I cDNA (29) and cloned into pCDNA3 (InvitroGen) at the EcoRI and NotI sites.
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50% confluent) were transfected using the calcium phosphate method (30), with 5 µg of the various constructs, with or without equimolar amounts of the pJDEI vector encoding genomic full-length murine I LC (14) or cDNA of wild-type or mutant . Where indicated, 8 µg of constructs encoding HA-tagged wild-type or T19G BiP (31) was co-transfected. Cells were analyzed 42–48 h post-transfection. Metabolic Labeling and Steady-state Analyses—Secretion was monitored by re-feeding cells with 1.5 ml of fresh medium that was collected after 3–5 h. For metabolic labeling, cells were starved for 1 h in methionine-deficient medium and then either pulse-labeled for 30 min with [35S]methionine (250 µCi/ml) and chased in complete medium, or labeled for 3–4 h. In all experiments, cells and media were separated by centrifugation at 4 °C, cells were washed with ice-cold phosphate-buffered saline and lysed (300 µl/2 x 106 cells) in ice-cold NET buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5 mM EDTA, 0.5% Nonidet P-40) supplemented with 2 mM phenylmethanesulfonyl fluoride (Sigma) and 100 units/ml aprotinin (Roche Applied Science). For samples analyzed by non-reducing SDS-PAGE, the phosphate-buffered saline and NET buffers included freshly added N-ethylmaleimide (20 mM, Sigma). Cell lysates were centrifuged (20,000 x g, 20 min, 4 °C), and supernatants were collected.
Immunoprecipitation, Treatment with Glycosidases, and Electrophoresis—Protein A-Sepharose (Repligen) was added to cell lysates or media to quantitatively precipitate and associated proteins. To promote BiP release, 10 mM Mg2+-ATP was added to the lysates, whereas otherwise apyrase (10 units/ml, Sigma) was included to prevent the release of BiP (3). Samples were rotated for 4 h at 4 °C, washed three times with borate wash buffer (7), and once with phosphate-buffered saline. Where indicated, immunoprecipitated proteins were treated with endoglycosidase H (endo H) or peptide: N-glycosidase F (New England Biolabs), according to the manufacturer's instructions. Proteins were resolved by SDS-PAGE under reducing or non-reducing conditions and electroblotted onto nitrocellulose. For detection of metabolically labeled proteins, blots were autoradiographed and quantified by densitometry. Subsequently, blots were probed with the following antibodies: horseradish peroxidase (HRP)-conjugated anti-mouse 2b (SouthernBiotech), HRP-conjugated anti-mouse I (SouthernBiotech), biotin-conjugated anti-BiP (SouthernBiotech) followed by HRP-conjugated avidin (Jackson), anti-HA (clone 12CA5) followed by HRP-conjugated anti-mouse IgG (Jackson). The HRP was visualized by enhanced chemiluminescence (ECL).
Limited Proteolysis—Limited proteolysis of intracellular HCs was carried out on Protein A precipitates under optimized time, temperature, and enzyme concentrations. Papain (Sigma) was added at 5 µg/ml to precipitates resuspended in 100 mM sodium acetate (pH 5.5)/50 mM cysteine/1 mM EDTA and incubated at 37 °C. Precipitates resuspended in phosphate-buffered saline were either incubated at 30 °C with 5 µg/ml of trypsin (Difco) or at 37 °C with 0.1 µg/ml elastase (ICN).
Homology Modeling and Sequence Alignment—The potential BiP binding sites were based on alignment of high BiP-binding score heptapeptides (32) with CH1 sequence of 2b (28). Structural alignment of CH1 of 2b was carried out via the Swiss-Model web site and using as template the sequence of an identical CH1 whose structure is available (Protein Data Bank ID: 1MAM
[PDB]
(33)).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2b HC (designated ), Cys-140 and Cys-195 formed the intradomain disulfide, whereas Cys-128 formed the interchain bond with LC (see Fig. 6, below). Each mutant was expressed with or without I LC (designated ) in non-lymphoid COS-7 cells, where its fate could be assayed in the absence of endogenous HC and LC.
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, based on retention of wild-type HC (WT; Fig. 1A, lanes 1 and 13; Fig. 1B; for intracellular expression of the non-secreted WT, see Fig. 3A, lane 1) and efficient secretion ofaCH1-deleted HC (CH1; Fig. 1A, lanes 2 and 14; Fig. 1B) or upon replacement of CH1 by either CH2orCH3 domains ((CH2)2 or (CH3)2, respectively; Fig. 1A, lanes 3, 15, 4, and 16; Fig. 1B). These experiments confirm and extend previous reports, that, despite their similar fold, neither CH2 nor CH3 can confer retention even when grafted in place of CH1. Importantly, all secreted HC consisted mostly of 2, indicating that CH1 absence did not hamper HC homodimerization.
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LC (Fig. 1, B and C). Because was not directly precipitated by Protein A (Ref. 12 and Fig. 1D), its co-precipitation reflected covalent assembly into the secreted species , 2, and 22 (Fig. 1C, lanes 1, 13, and 25). In addition, escorted the 2 intermediate non-covalently, as shown by co-precipitation of monomeric in addition to a 125kDa species (Fig. 1C, lanes 1 and 25). This conclusion was verified by size-exclusion chromatography, where all secreted WT species were 150-kDa tetramers, regardless of their covalent assembly status (Supplemental Fig. S1). HCs lacking CH1 (either deleted or replaced) did not interact with co-expressed (Fig. 1C, lanes 26–28) nor was their secretion improved by (Fig. 1, A–C), corroborating their LC-independent secretion. The association of BiP with intracellular WT, but not when CH1 was either deleted or replaced (Fig. 1E), confirmed that the CH1-conferred retention was due to BiP binding. BiP was displaced from WT by co-expression of , and to a lesser extent by ATP (Fig. 1E, lanes 1–3; Fig. 1F). The only partial dissociation of HC and BiP by ATP (Fig. 1F) likely reflects their rebinding as nucleotide is degraded (see Ref. 18).
Ablation of All Three CH1 Cysteines Negates CH1-conferred Retention—Substituting each of the 3 CH1 cysteines with serine, singly or in combinations, yielded surprising structure-function relationships with respect to folding, assembly, and secretion. When expressed without LC, these mutants could be divided into two categories. The majority resembled WT and were retained intracellularly, whereas a few mutants were secreted, resembling HCs that lacked CH1 altogether. The most remarkable example of a mutant that was secreted efficiently in the absence of LC was the triple mutant C128S/C140S/C195S (Fig. 1A, lanes 11 and 23; Fig. 1B). In this mutant all 3 CH1 cysteines, which normally stabilize the conserved Ig fold, were ablated. Moreover, C128S/C140S/C195S did not interact with LC, even non-covalently (Fig. 1C, lane 35), and neither its interaction with BiP (Fig. 1F) nor its secretion (lanes 11 and 23 in Fig. 1, A and C; Fig. 1B) were affected by LC.
Several lines of evidence confirmed the bona fide secretion of C128S/C140S/C195S. First, similar maturation of N-glycans, reflected by retarded mobility and resistance to endo H, was observed for WT secreted in the presence of LC (Fig. 2A, lanes 3, 4, 7, and 8) or for C128S/C140S/C195S secreted without LC (Fig. 2A, lanes 11, 12, 15, and 16). Interestingly, secreted C128S/C140S/C195S was resolved into and a unique higher assembly species designated 2* (see below), both resistant to endo H and sensitive to peptide: N-glycosidase F (Fig. 2A, lanes 11, 12, 15, and 16; arrows). Second, C128S/C140S/C195S acquired galactose residues, as detected by Ricinus communis agglutinin,2 indicating that this mutant traversed the trans-Golgi. Third, secretion of both and 2* was completely blocked by brefeldin A (Fig. 2B). Although 2* was preferentially detected in the medium, its accumulation in brefeldin A-treated cells (Fig. 2B, lane 3) indicated that it assembled within cells and was immediately secreted.
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Mutation of Cys-195 Hampers Interchain Disulfide Formation, Causes Misfolding, and Prevents Secretion—The secretion of C128S/C140S/C195S may appear as a hierarchical contribution of exposed thiols to HC retention. As shown in Fig. 1A and quantified in Fig. 1B, when all three cysteines were present, HC retention was complete (lanes 1 and 13); substituting Cys-128 (C128S) enabled only slight LC-independent secretion (lanes 5 and 17); substituting both Cys-128 and Cys-140 (C128S/C140S) led to more significant secretion (lanes 8 and 20), and ablating all 3 CH1 cysteines resulted in efficient LC-independent secretion (lanes 11 and 23). However, this apparent hierarchy did not apply to every cysteine in CH1. Disruption of the CH1 intradomain disulfide by substituting both Cys-140 and Cys-195 did not alleviate retention (Fig. 1B). Moreover, co-expression of LC allowed us to use the ability of the various mutants to assemble with , dissociate from BiP, and be secreted as a criterion to further divide the non-secreted HCs into two categories: the properly folded ones that resembled WT and were secreted, and the misfolded ones that were not secreted even in the presence of LC. In this respect, the CH1 domain of the C140S/C195S was severely misfolded, because this mutant was unable to assemble with , either covalently or non-covalently (Fig. 1C, lane 34), even though Cys-128 was intact (see Fig. 6).
We next asked whether disrupting the intradomain disulfide by singly mutating Cys-140 or Cys-195 was equivalent to the pairwise substitution in C140S/C195S. Evidently, a mutant lacking only Cys-195 resembled the double mutant C140S/C195S, because C195S was neither secreted (Fig. 1B) nor assembled with (Fig. 1C, lane 31), and what appeared as LC-facilitated secretion was negligible and hardly contained -only species not associated with LC (Fig. 1, B and C (lanes 7, 19, and 31)). Intracellular C195S seemed to be as misfolded as C140S/C195S, because even in the presence of it associated with BiP in an ATP-dependent manner (Fig. 1F) and did not bind LC (Fig. 1C, lane 31) even though Cys-128 was intact. Indeed, ablation of Cys-128 in addition to Cys-195 (C128S/C195S) resulted in a phenotype very similar to that of C195S (Fig. 1, A–C; see also Fig. 6).
When the intradomain disulfide bond was disrupted by mutating Cys-140 alone, the resulting C140S was as efficiently retained as C195S or C140S/C195S (Fig. 1, A (lanes 6 and 18) and B). However, unlike these mutants, C140S responded to the presence of LC like WT. C140S assembled reasonably efficiently into 2 and 22 (Fig. 1C, lanes 18 and 30), dissociated from BiP (Fig. 1F) and was secreted (Fig. 1, B and C (lanes 6, 18, and 30)). We conclude that a CH1 domain lacking Cys-140, one of the two cysteines that make up the native intradomain disulfide bond, is not misfolded, whereas CH1 lacking its partner Cys-195 is (see Fig. 6).
Mutation of Cys-128 Retards CH1 Oxidation and Allows Opportunistic Interchain Disulfides without Hampering Secretion—The failure to form the interchain disulfide in the absence of Cys-195, which forms the intradomain disulfide, suggested interrelations between these two bonds. We therefore performed a reciprocal experiment and disrupted the interchain disulfide by substituting Cys-128. Without LC, C128S was secreted only marginally (5% of CH1 secretion; Fig. 1B). However, C128S was not nearly as misfolded as C195S, because it resembled WT (Fig. 3A). In the presence of LC, C128S dissociated from BiP (Fig. 1F) and was secreted efficiently (Figs. 1B, 1C (lanes 5, 17, and 29), and 3A). As expected, the major secreted covalent form of C128S was 2 rather then 2 or 22 (Figs. 1C (lanes 17 and 29) and 3A). Because WT 2 was hardly secreted (Fig. 3A, lane 4), it suggested that when a LC-interacting cysteine was absent, the secreted 2 species of C128S was associated non-covalently with two LCs, as corroborated by the abundant free LC co-precipitated by Protein A (Fig. 3A, lane 16). Interestingly, a -containing species of 47kDa, which was not formed when was expressed alone (Fig. 1D), was also co-precipitated from cells and media of C128S-expressing cells (Fig. 3A, lanes 15 and 16). This species is most likely disulfide-bonded 2 homodimers that are formed only when Cys-128 is absent, as discussed elsewhere.3
A minor, yet significant, fraction of C128S was surprisingly assembled into covalent 22 and 2, as revealed by probing with anti- (Fig. 3A, lanes 7 and 8) and anti- (Fig. 3A, lanes 15 and 16) antibodies. Because LC was not disulfide-bonded to HCs that lacked CH1 (see Fig. 1C), this result indicated that, when Cys-128 was absent, CH1 cysteines that usually formed the intradomain disulfide could form opportunistic interchain disulfides with , precluding intradomain disulfide between Cys-140 and Cys-195. Indeed, consistent with altered oxidative folding (34), all species containing covalently linked C128S and (22, 2, and ) exhibited altered electrophoretic migration in nonreducing gels (Fig. 3A, lanes 4, 8, 12, and 16). Formation of opportunistic interchain disulfides is probably allowed by inefficient engagement of Cys-140 with Cys-195 when Cys-128 is absent, as shown for a small fraction of intracellular HC monomers. These assembly species, which have not yet covalently bound to either or , could be resolved into oxidized and unoxidized forms (Fig. 3A, lanes 1–8, upper and lower panels). Whereas WT monomers were fully oxidized (lane 3), C128S monomers appeared to be mostly reduced (lane 7), yet only the oxidized monomers were secreted (lane 8). That the distorted and retarded oxidative folding was due to the mutant C128S HC and not to its opportunistically bonded LC was indicated by substituting Cys-214 in , the residue that interacts with HC (Fig. 3B). This mutation abolished not only the normal disulfide with WT, but also the opportunistic interchain disulfides with C128S. Nonetheless, WT HC monomers remained oxidized and C128S monomers remained reduced (Fig. 3B, lanes 1–12, upper and lower panels), indicating that only Cys-214 could engage a HC cysteine, either the native Cys-128 or an opportunistic partner (see Fig. 6).
Taken together, this detailed analysis of single, double, and triple cysteine substitutions shows that the effect on folding and secretion of mutating one cysteine is not equivalent to mutating the other, even when the effect on disulfide bond formation is the same. Hence, when either interchain or intradomain disulfide bond in CH1 is disrupted, formation of the other disulfide bond is affected, and thus formation of these two disulfide bonds in CH1 is interdependent.
The C128S/C140S/C195S Heavy Chain Exhibits Unusual Folding and Assembly—The triple mutant C128S/C140S/C195S was secreted independently of LC and in addition exhibited a unique assembly pattern. First, the secreted molecules were mostly typical 100-kDa 2 homodimers, indicating that this Cys-less CH1 did not interfere with HC homodimerization. Yet, a significant fraction migrated as an unusual 200-kDa species (Fig. 1A, lanes 11 and 23), which contained HC exclusively, as even in cells co-expressing , they were reactive only with anti- but not with anti- antibodies (Fig. 1C, lanes 11, 23, and 35). Excising both assembly species from non-reducing gels and individually subjecting them to reducing SDS-PAGE resolved the 100-kDa species into the expected 50-kDa monomers, whereas the 200-kDa species gave rise to a 100-kDa species (Fig. 4A). The latter probably represented atypical dimers (2*) held together via SDS-resistant interactions. Two such 2* molecules appeared to undergo conventional homodimerization into 200-kDa 4* tetramers (see Fig. 6).
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85-kDa intermediate only from C128S/C140S/C195S (Fig. 4B, lane 5, arrow). An additional 70-kDa elastase product was generated from both WT and C128S/C140S/C195S, but was more pronounced in C128S/C140S/C195S (lane 5) than in WT (lane 2). Limited proteolysis with trypsin also revealed differences between intracellular C128S/C140S/C195S and WT. Although two major fragments, ranging between 80 and 90 kDa, were generated from the mutant (Fig. 4C, lanes 8–12, arrows), only a single major fragment was generated from WT (lanes 2–6). To address the conformation of the secreted C128S/C140S/C195S, it was compared with the secreted (CH3)2, because both HCs could be collected from the medium independently of LC expression. Papain digestion yielded a 50-kDa anti--reactive product in both (CH3)2 and C128S/C140S/C195S, but only C128S/C140S/C195S gave rise to a predominant 70-kDa product (Fig. 4D, lanes 8–12, arrow). The altered proteolytic sensitivity demonstrated that the fold of intracellular C128S/C140S/C195S was distinct from that of intracellular WT, and the fold of secreted C128S/C140S/C195S was distinct from that of secreted (CH3)2. An alternative interpretation to the altered proteolytic sensitivity is that C128S/C140S/C195S could interact with a unique set of auxiliary proteins. Either way, the data points to an alternate conformation of the Cys-less CH1 mutant that is correlated with unusual assembly into 4* and is evidently compatible with secretion of HCs that are not assembled with LC.
BiP Cycles from the Cys-less CH1 to Allow Secretion of C128S/C140S/C195S—As established previously and corroborated in this study, CH1-conferred retention of HC is mediated (at least in part) by BiP. Yet, none of the 3 CH1 cysteines was included in the two identified clusters of potential BiP binding sites (Fig. 5A), obtained by multiple alignment of 2b CH1 sequence (28) with likely BiP-binding heptapeptides (32). Therefore, we investigated whether and how BiP-mediated retention operated on the Cys-less CH1. For WT, the relatively constant levels of co-precipitated BiP during the chase, detected either radioactively or with an anti-BiP antibody (Fig. 5B, middle or upper panels, respectively, lanes 8–11), presumably reflected recycling of long-lived BiP on and off the nascent HC. Consistent with Vanhove et al. (3), the diminished levels of co-precipitated BiP in the presence of (Fig. 5B, lanes 8 and 9; see also Fig. 1E, lanes 1 and 3) were interpreted as a transition from an irreversible to a reversible mode of HC binding to BiP. Interestingly, BiP association with C128S/C140S/C195S in the absence of LC resembled BiP association with WT in the presence of LC (Fig. 5B, lanes 1–4 and 9–11), suggesting that, by ablating all its cysteines, CH1 binding to BiP became reversible. This was supported by overexpression of an ATPase-defective BiP mutant (T19G), known for prolonged association with its substrates, which inhibits their secretion (10). As shown in Fig. 5C, this mutant BiP decreased the secretion of C128S/C140S/C195S by more than 1.6-fold, from 56% to only 34% secreted C128S/C140S/C195S. Apparently, under these conditions, 2* formation was also hindered, resulting in secretion of a smaller fraction of 2* (Fig. 5C), suggesting that BiP displacement, a prerequisite for native Ig assembly, was also a prerequisite for the atypical assembly of Cys-less CH1 mutant into 2*.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cysteine residues can affect Ig folding, assembly, and secretion in a number of ways. Often they participate in disulfide bonds, a characteristic of Ig superfamily proteins. Usually, disulfides serve to stabilize the fold rather then to catalyze its formation and can be replaced if the loss of folding stability is at least partially compensated for by other stabilizing mutations (35, 36). In most Ig domains, a disulfide links two sheets and is buried in the core of the domain, with conserved, hydrophobic side chains packed against it. If this intradomain disulfide fails to form, exposed thiols in this hydrophobic core adversely affect folding, leading to continued exposure of BiP binding peptides and consequent retention (18). This can clearly be the explanation for the compromised folding of all mutants lacking Cys-195, which cannot be rescued by LC expression. However, the non-reciprocal nature of the mutations described here indicates that CH1 cysteines must also have other effects. Cysteine substitutions other than Cys-195, that disrupt the same intradomain disulfide (C140S; C128S when opportunistically engaged with ; Fig. 6), can be rescued by LC, at least partially, as judged by BiP displacement and consequent secretion. Moreover, leaving a surface-exposed cysteine such as Cys-128 reduced, or replacing it with serine, would not normally be expected to have a dramatic destabilizing effect. Nonetheless, C128S displays defects in oxidative folding that are manifested even when LC is present, contrary to expectation. It seems, therefore, that Cys-128 is essential for CH1 folding and not just for linking CH1 to LC. A third effect of cysteines may be via thiol-mediated retention (23, 25), where unpaired cysteines can interact with ER proteins (37–39). This mechanism may operate in WT retention, where cysteines and Cys-128 in particular are unpaired, or in some of the single mutants that form opportunistic disulfides and have promiscuously exposed thiols.
We propose that CH1 is unique because its folding requires interdependent and concurrent formation of intradomain and interchain disulfides. This is in contrast to other Ig domains with multiple cysteines, such as CL, where the intradomain disulfide forms with high fidelity, leaving unpaired the third cysteine responsible for the interchain disulfide. The interdependent oxidation of CH1 is shown in a number of ways. Cys-128 fails to form interchain disulfide with LC when intradomain disulfide cannot form due to Cys-195 substitution. Reciprocally, in HC whose LC-interacting Cys-128 is absent, intradomain cysteines oxidize inefficiently, even allowing opportunistic engagement of Cys-140 or Cys-195 with LC. Importantly, this inefficient oxidation is inherent to HC and does not occur in LC. Therefore, one possible interpretation is that as long as a fourth cysteine provided by LC is missing, the 3 CH1 cysteines cannot adopt the proper configuration to form intradomain disulfide and HC cannot dissociate from BiP. However, the finding that LC that lacks this cysteine can still promote folding and secretion of WT HC (as well as of some mutants) demonstrates that the role of CH1 cysteines in Ig folding, assembly, and secretion is not simply to engage in disulfide bridges, but rather to affect CH1 folding, including mediating possible interactions with thiol-sensing ER proteins. When LC is provided, a cascade of interdependent folding and assembly events juxtaposes the two pairs of cysteines. Once the positioning of interacting surfaces is accomplished, disulfides form, but only to stabilize the Ig fold. BiP displacement and subsequent secretion are achieved concurrently with proper CH1 folding and this can be accomplished in the presence of CL as a folding template.
A mechanistic link between BiP-mediated and thiol-mediated retention of HC is quite probable. The capacity of BiP to bind HC may well be related to HC binding through unoxidized thiols to thiol oxidoreductases, such as PDI (40, 41), ERp57 (37), or ERp44 (38, 39). Cooperation between BiP and PDI in Ig folding was shown in vitro, suggesting that BiP prevents unfolded Ig chains from collapsing and presents them to PDI (42). A secreted version of PDI (PDI-KDEL) implicated PDI in retention of unassembled procollagen C-propeptides (43), but had no significant effect on HC retention (data not shown). The possible interaction between HC and thiol oxidoreductases was explored by co-precipitation and cross-linking experiments (including SH-reactive agents), but we were unable to demonstrate physical interaction between and either PDI, ERp57, or ERp44 (data not shown). Recent proteomic analyses revealed many ER proteins with similarity to PDI and thioredoxin (44). Although some, like ERp29 (45), do not seem to function as oxidoreductases, others, like ERp46 (46), substitute for PDI. It is not presently clear whether different ER oxidoreductases perform distinct functions and whether any of them are specific to the B-lymphocyte lineage or important for Ig assembly and quality control (44). Our results suggest that if any of these ubiquitous oxidoreductases play a role in retention and do so by binding to the HC, in vivo this interaction is too weak or too transient to be captured by the techniques we used. Nevertheless, our findings that CH1 cysteines play a key role in BiP-mediated retention may point to functional in vivo cooperation of BiP and oxidoreductases. Whether BiP presents HC to PDI or vice versa, the contribution of PDI catalytic activity to this cooperative retention could be circumvented by mutating either PDI (43) or its substrate recognition, as was done in this work.
An alternative folding pathway and a different interaction with the quality control system need to be invoked to explain HC that harbors a Cys-less CH1 and is secreted efficiently. In this case, the cascade of folding and assembly events described above cannot occur, yet retention is overcome without the need for assembly with LC. Clearly, in this situation thiol-mediated retention does not operate and BiP-mediated retention is counteracted, because the Cys-less CH1 displays the common reversible mode of association with BiP, rather than the irreversible binding characteristic of WT CH1 (3). The cycling of Cys-less CH1 on and off BiP allows this mutant to acquire a secretion-competent fold, which we show to be distinct from that of WT HC. These observations suggest that, although the native structure of CH1, as defined by crystallography, is almost invariant among HC isotypes, there is more than one conformation that the cellular quality control tolerates and considers as secretion-competent.
What would explain the reversible binding of BiP to Cys-less CH1? Clearly, potential BiP binding sequences are found in all Ig domains (20), even those that do not confer retention, such as CH2 and CH3 (7) and CL (12), and none of the features of BiP binding peptides dictates whether the binding would be reversible (32). It is likely that association with BiP is not determined by the peptide sequence per se, but rather controlled by folding rate and pathway. Therefore, the weak and reversible binding of BiP to Cys-less CH1 presumably reflects rapid sequestration of BiP binding sequences within the interior of the alternatively folded domain, just as they are in many other Ig-fold proteins (12, 18). In wild-type CH1, unoxidized cysteines may slow the folding of this domain, so as to allow it to be "caught" by BiP, and only upon non-covalent association with LC is rapid folding allowed.
A correlation between BiP displacement and Ig assembly has been shown for CH2 and CH3, which undergo homo-domain dimerization (17). On the other hand, BiP displacement from CH1 was correlated with hetero-dimerization with LC (7). Taking into account our results and geometric constrains at the hinge region, it is possible that Cys-less CH1 has acquired the capacity to homodimerize via an atypical SDS-resistant interaction and, together with the usual homodimerization of two C128S/C140S/C195S via their hinge regions, a unique tetramer is formed (see Fig. 6). Moreover, the attenuated formation of these tetramers in the presence of the T19G BiP mutant suggests that BiP displacement is required even for this atypical Cys-less CH1 homodimerization. It remains to be established whether reversible BiP binding to Cys-less CH1 reflects rapid folding and secretion that may or may not result in unusual assembly, or whether the latter is the cause of BiP displacement.
Although it is surprising that ablation of conserved disulfides in the case of CH1 does not result in a denatured protein that is retained intracellularly, there are precedents for this phenomenon. Na,K-ATPase appears to fold normally and is enzymatically functional even when all 23 cysteines in its 
subunit are mutated (47). Likewise, ablating either one of the two cysteines that form an intramolecular disulfide in bacterial lipases has no effect on their enzymatic activity. Yet, this disulfide is important for stabilizing the structure of lipase, which is otherwise easily denatured and susceptible to proteolysis (48, 49). Interestingly, the criteria for correct folding, as manifested by activity, are often less stringent than those recognized by quality control mechanisms that prevent intracellular transport and secretion (48–50). Here we provide evidence that, although the conserved CH1 cysteines generate important cues for quality control, in the form of the transiently unstable structure of the normal domain, the quality control mechanism is "duped" when CH1 cysteines are substituted. Given the frequent use of Ig domain folds in the human proteome, our model for the relationship between oxidative folding and cellular quality control may be relevant to many receptors and secreted proteins.
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FOOTNOTES |
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The on-line version of this article (available at http//www.jbc.org) contains Supplemental Fig. S1.
¶ To whom correspondence should be addressed: Tel.: 972-3-640-8984; Fax: 972-3-640-6834; E-mail: shoshbn{at}tauex.tau.ac.il.
1 The abbreviations used are: Ig, immunoglobulin; ECL, enhanced chemiluminescence; endo H, endoglycosidase H; ER, endoplasmic reticulum; , mouse 2b heavy chain; HC, Ig heavy chain; HRP, horseradish peroxidase; , mouse I light chain; LC, Ig light chain; pBS, pBluescript.
2 Y. Elkabetz, Y. Argon, and S. Bar-Nun, our unpublished observation.
3 Y. Elkabetz, A. Ofir, Y. Argon, and S. Bar-Nun, manuscript submitted for publication.
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