Association-induced folding governs surrogate light chain and pre-B cell receptor core assembly

Domain: neuroscience
Published: 2026-01-30
Editorial outcome: minor_revision

Abstract
Binding of the surrogate light chain (SLC) to the heavy chain (HC) of the pre-B cell receptor (pre-BCR) is an important quality control checkpoint during B cell development as roughly 50% of the rearranged HCs are defective. Unlike the regular light chain (LC), the SLC is a hetero-dimer of VpreB and λ5, both containing unstructured extensions, the unique regions. The molecular mechanisms that underlie the complex assembly processes which give rise to the final pre-BCR is not fully understood. Here we show, via reconstitution of the pre-BCR in vitro and in cells that λ5 plays a key role in the pre-BCR assembly. During SLC assembly, a β-strand, located between the λ5 domain and the unique region, induces structure in the largely unfolded VpreB, creating a high affinity complex. In addition, association of λ5 with the unstructured HC CH1 domain is required for its folding. This is essential for pre-BCR assembly and its release from the endoplasmic reticulum (ER). Finally, the unique region of λ5 plays a pivotal role in the antigen interaction of the SLC-HC complex. Together, our results reveal a multi-step mechanism for SLC and pre-BCR assembly, governed by association-induced folding reactions required for structural integrity and function.

Introduction
Antibodies are a cornerstone of the humoral adaptive immune system. They fulfill important functions in protecting the body against foreign molecules and pathogens. The production of antibodies critically depends on the successful progression of hematopoietic stem cells to mature B cells1. During this process, developing B lymphocytes have to pass multiple stages and checkpoints, in which the genes for heavy chains (HC) and light chains (LC) of immunoglobulins undergo recombination and scrutiny2 to produce a unique immunoglobulin sequence in each mature B cell3.

HC and LC gene rearrangements take place sequentially4. First, HCs are rearranged and expressed in the absence of LCs, in earlier developmental stages, in a membrane-bound form5. Thus, a key checkpoint during B-cell development is to monitor whether the rearrangement of the HC gene locus resulted in a functional HC protein. This is validated by the ability to productively associate with the so-called surrogate LC (SLC)6–9, often also called invariant LC. The assembly of the SLC and the HC together with the CD79a/CD79b signaling components form the pre-B cell receptor10–12 and induces an intracellular signaling cascade that promotes LC gene rearrangement and downregulation of the expression of ʎ5 and VpreB, the components of the SLC13–17. This is a critical step where approximately 50% of cells that produce a certain HC are discarded because they do not form a productive complex with the SLC18,19. Thus, the SLC has an essential function in B-cell development and antibody production.

The SLC is structurally similar to a regular LC but also shows key differences. It consists of two proteins, which are non-covalently associated: VpreB, which corresponds to the variable LC domain, and λ5, which corresponds to the constant domain of a conventional LC of the λ-subtype20–22. VpreB interacts with the VH domain of the HC, and λ5 interacts with the HC CH1 domain20,21,23. The association of the SLC with the HC involves both non-covalent interactions and a covalent intermolecular disulfide bond between λ5 and the HC CH1 domain3,24–28. VpreB has the typical V-type Ig-fold but contains only 8 out of 9 β-strands of a conventional VL domain. It lacks a J region that forms the ninth β-strand3,20,21. The missing strand is provided by λ5, which comprises in its folded domain the seven β-strands of a conventional CL domain and, in a sequence extension outside the folded domain, the additional β-strand missing in VpreB3. This additional β-strand is important for the association of λ5 and VpreB29 by a process called β-strand complementation30.

Further major differences to regular LCs are the so-called unique regions (UR) of VpreB and λ5. These are unstructured extensions with sequences different from any known protein. The UR of VpreB is 24 aa long and is located at the C-terminal end of the domain, whereas the folded domain of λ5 contains an additional β-strand and a 50 aa long UR at its N-terminus21. The URs are not required for SLC assembly29. However, the URs of VpreB and ʎ5 may replace the missing complementarity-determining regions (CDR) of VpreB21.

In the available crystal structure of the pre-BCR, the URs are only partially resolved21. It thus remains unclear what their specific contribution to SLC assembly and function is and whether they affect antigen interaction. Furthermore, despite progress in understanding structural aspects and the biological function of the SLC, little is known about the assembly process of VpreB and λ5 and, especially, the formation of the complex of the SLC with the HC, i.e., the pre-BCR. Understanding these processes is of high biological relevance because of the checkpoint function of the SLC. To address these open questions, in this study, we have reconstituted and analyzed the assembly processes leading to the SLC and the SLC-HC complexes in vitro and in mammalian cells. Our results resolve the underlying mechanisms and provide insight into the molecular quality control function of the SLC on HCs as a key step in generating a functional pre-BCR.

Results
Formation of the SLC heterodimer involves assembly-mediated folding of VpreB
While the structure of the SLC in complex with a HC fragment has been determined (Fig. 1a)21, we know little about the structure of the two individual SLC proteins, VpreB and ʎ5, and the association reactions leading to the SLC and the SLC-HC complex. To address these questions, we analyzed these processes in cells and in vitro using the wild type proteins and constructs devoid of characteristic SLC regions, the extensions called unique regions (URs) or the extra β-strand in ʎ5 (Fig. 1b).

When expressed individually in HEK293T cells, both subunits of the SLC were weakly secreted into the cell medium (Fig. 1c). Co-expression of the proteins increased their secretion (Fig. 1c), in agreement with previous studies29, concomitant with O-glycosylation of VpreB (Supplementary Fig. 1a). Furthermore, the overall levels of λ5 and VpreB increased upon co-expression, arguing for mutual stabilization in cells upon assembly (Fig. 1c). In conclusion, λ5 and VpreB on their own seem to be partially recognized as non-native by the endoplasmic reticulum (ER) quality control machinery and are thus partially retained in the ER. When they associate as the SLC, they seem to be no longer a target for the ER quality control and are thus efficiently transported out of the cell.

To gain further insights into the underlying mechanisms, we studied the structure and stability of the individual SLC proteins in vitro. The far-UV circular dichroism (CD) spectrum of λ5 (Fig. 1d) revealed that the isolated protein is characterized by β-strands and unfolded segments consistent with its structure in the SLC complex21 and the presence of an unstructured extension. In contrast to the structure of VpreB in the SLC complex, the far-UV CD spectrum of isolated VpreB (Fig. 1d) indicated that the protein is largely unfolded under the conditions tested. In agreement with this notion, nuclear magnetic resonance (NMR) spectroscopy revealed that the 15N-1H HSQC spectrum of isolated VpreB is characteristic of an unfolded protein (Fig. 1e, red spectrum). Although VpreB shares homology with the VL domain of a conventional light chain, it lacks a conserved β-strand and therefore cannot fold independently into a stable Ig-like domain. Importantly, conventional VL domains cannot replace VpreB in the pre-BCR, showing that VpreB and λ5 together provide unique structural features required for pre-BCR assembly. When unlabeled λ5 was added to isotope-labeled VpreB (Supplementary Fig. 1b, orange spectrum), the NMR spectrum of VpreB became well-resolved in agreement with association-induced folding of VpreB by λ5. Interestingly, also a peptide (λ5β) representing the additional ß-strand donated by λ5 was able to induce folding of VpreB (Fig. 1e, blue spectrum). Many of the dispersed peaks in the spectrum of this complex superimpose exactly with the resonances observed upon addition of λ5 (compare Supplementary Fig. 1b).

To analyze the conformational changes in VpreB and λ5 upon SLC formation further, hydrogen-deuterium exchange coupled to mass spectrometry (H/DX-MS) was performed (Fig. 2a, b). For VpreB, the H/D exchange and thus the conformational flexibility decreased in the SLC complex, as upon binding of the β-strand, VpreB adopts its native structure (Fig. 2a). In agreement with this, the additional β-strand in λ5 became highly protected against exchange in the SLC complex (Fig. 2b). To a lesser extent, also in λ5, a generally increased protection against H/D exchange was observed upon assembly with VpreB, which implies that upon complex formation both VpreB and λ5 undergo structural rearrangements and/or become less dynamic (Fig. 2b). Furthermore, both URs show increased protection against H/D exchange in the SLC complex compared to the individual proteins. This is unexpected since the URs were reported to be largely unstructured and protruding from the complex21. The kinetics of VpreB folding upon association with λ5 monitored via changes of the CD signal (Fig. 2c and Table 1) revealed a time constant of τ=51.0 min at 25°C, in agreement with the pronounced conformational rearrangements in VpreB and λ5 upon formation of the SLC as described above. Taken together, VpreB is an unfolded protein that can only fold in the presence of the β-strand provided by λ5. The association is accompanied by conformational changes in both proteins involving the URs.

The benefit of the intricate complementation of the VpreB fold upon association with λ5 is that the folded state of VpreB is only attained in the heterodimeric SLC complex. Furthermore, the strand insertion reaction potentially allows creating a high-affinity complex. In agreement with this idea, we determined the dissociation constant (KD) of the SLC complex to be 17.7 nM (Fig. 2d and Table 1). Association with VpreB slightly reduces the stability of λ5 (Fig. 2d and Table 1). Interestingly, for the SLC in complex with a HC fragment consisting of VH and CH1 (also referred to as Fd), the Tm is even lower (Fig. 2e and Table 1).

Quaternary structure analysis by AUC confirmed that the SLC complex, reconstituted in vitro, sediments as a heterodimer consisting of VpreB and λ5 (~3.2 S); the λ5 monomer sediments at ~1.8 S (Fig. 2f). For isolated VpreB, the sedimentation coefficient was also ~3.2 S; however, the broad profile compared to folded controls suggests that this signal arises from a heterogeneous mixture of oligomeric species in the range of dimers to tetramers. This interpretation is consistent with previous reports describing non-native dimeric or higher-order VpreB assemblies formed under certain in vitro conditions30–32. Thus, the interaction of monomeric λ5 with VpreB results in the dissociation of the VpreB dimer and the formation of the SLC heterodimer. Further association into the VpreB-ʎ5-Fd Fab-like complex gives rise to a heterotrimer with a sedimentation coefficient of ~4.5 S (Fig. 2f).

The unique regions impact folding, stability and association of the SLC
In addition to the heterodimeric nature of the SLC, a further major difference to regular LCs is the presence of sequence extensions, the URs, on both proteins. As their functions are largely unknown, we created constructs in which the URs were removed (Fig. 1b) and first analyzed their influence on the structure, association and stability of the SLC proteins. In agreement with the notion that the URs are unfolded, CD spectra of SLC ΔUR and ʎ5 ΔUR both showed an increased β-strand content, compared to the wild type, respectively, whereas only minor changes were observed for VpreB ΔUR (Fig. 3a, compare to Fig. 1d). The analysis of the thermal stability of λ5 ΔUR revealed a Tm of 58.5°C, which is slightly higher than that of λ5 (Fig. 3b and Table 1).

The λ5-UR also affected the association-coupled folding of VpreB. The time constant τ for folding was determined to be 113.5 min at 25°C (Fig. 3c and Table 1), which is about two times slower than if λ5 was used (51.0 min). This indicates that the λ5-UR, although dispensable for SLC assembly, accelerates the association-induced folding of VpreB. Furthermore, it slightly destabilizes the SLC complex as reflected in a 3°C lower Tm (Fig. 3d and Table 1) and a marginally decreased KD value (Supplementary Fig. 2a and Table 1).

Compared to the results obtained for λ5-ΔUR, the deletion of the VpreB-UR had an opposite effect on the association-induced folding of VpreB. Here the deletion resulted in faster folding compared to the wild-type (WT) complex with a rate constant τ of 40.9 min (Fig. 3e and Table 1) and a stronger binding with a KD for λ5 and VpreB ΔUR of 3.0 nM, which is sixfold tighter compared to the WT proteins (Supplementary Fig. 2b and Table 1). Also, the thermal stability of this complex is slightly increased by about 2°C compared to the WT (Fig. 3d and Table 1). Together, these results indicate that the VpreB-UR negatively impacts folding and destabilizes the SLC complex.

Interestingly, in the absence of both URs, a KD of 5.0 nM was determined for the SLC (Supplementary Fig. 2c and Table 1), which is similar to that of the complex between λ5 and VpreB ΔU (3.0 nM), supporting the idea that mainly the VpreB-UR causes the decreased affinity. The Tm of the SLC ΔUR is about 3°C higher than that of SLC (56.1 vs. 53.3°C) (Fig. 3b and Table 1). The AUC analysis revealed SLC ΔUR to be a heterodimer, while isolated VpreB ΔUR remains a homodimer and ʎ5 ΔUR a monomer (Fig. 3f).

Building on our results on the modulatory effects of the URs on SLC assembly, we asked how deleting the URs affects SLC-Fd secretion from cells. We found that whereas the URs within both, λ5 and VpreB, were not essential for inducing SLC-Fd secretion, deleting the UR of λ5 or VpreB significantly reduced their levels in cells and made the secretion of the complex less efficient (Fig. 3g). The effect was strongest, if both SLC subunits lost their URs (Fig. 3g). This was also true for a second Fd fragment tested (Supplementary Fig. 2d). These results indicate that the URs play a role in the assembly and stability of the SLC-Fd complex in cells.

In combination, our results reveal complex and differential effects of the two URs on the association and stability of the SLC. Especially the UR of ʎ5 accelerates the association of the SLC but has a negative effect on the affinity of the complex—and the URs generally seem to be important for secretion from cells.

The ʎ5 core region drives folding of CH1
During IgG assembly, the unfolded CH1 domain of the HC folds upon interaction with the CL domain of the LC33,34. It was thus reasonable to assume that the SLC, more precisely λ5, which corresponds to the CL domain, could also induce the folding of the CH1 domain. To test this hypothesis, we first analyzed the fate of an HC Fd fragment expressed in HEK293T cells in the presence or absence of the SLC proteins. When the Fd fragment was expressed alone, it was not secreted (Fig. 4a), consistent with the unfolded state of the CH1 domain which induces ER retention29,34. Co-expression of λ5 and VpreB resulted in the secretion of Fd (Fig. 4a), suggesting that the CH1 domain was folded by the SLC. Expression of the two constituent SLC proteins individually with Fd revealed that specifically λ5 rendered the Fd segment secretion-competent, while VpreB did not allow for Fd secretion (Fig. 4a). The same effects were observed for a different Fd fragment expressed together with the SLC proteins (Supplementary Fig. 3a). To extend these findings, we performed co-immunoprecipitation experiments of Fd with both the SLC and the individual SLC proteins. Our results show that both, λ5 and VpreB, when expressed in HEK293T cells, can interact individually with Fd, Thus, for VpreB, the lacking β-strand is not a prerequisite for Fd binding. (Fig. 4b and Supplementary Fig. 3b), but only ʎ5 or the SLC are able to induce Fd secretion (Fig. 4a and Supplementary Fig. 3a).

Building on these results, we set out to analyze the potential association-induced folding of the CH1 domain by λ5 in vitro. To this end, we monitored the kinetics of CH1 folding by CD spectroscopy in the presence of the SLC or λ5. When we added the SLC to the unfolded CH1 domain, we observed folding kinetics characterized by a time constant of τ=53.9 min (Fig. 4c and Table 2). In the presence of λ5, a time constant of τ=41.4 min was determined (Fig. 4d and Table 2). As a control, VpreB was added, and here, as expected from the cell-based experiments, no folding kinetics were detected (Fig. 4e). Interestingly, the kinetics for the induced folding of CH1 by λ5 is in the same range as that observed for CH1 and CL before34. The MAK33 CH1 used in our experiments represents an IgG CH1 domain. The pre-BCR is assembled with IgM heavy chains. The CH1 domain of IgM contains a conserved glycosylation site at N46. Since this glycan is crucial for interaction with the λ5-UR, as previously shown (ref. 35), we compared glycosylated and unglycosylated CH1 (2H32). Surprisingly and in contrast to the IgG CH1 domain, we observed that glycosylated CH1 could not be folded by λ5 but only by the SLC in vitro with a rate constant τ=66 min, whereas unglycosylated CH1 required τ=104 min, indicating that glycosylation accelerates CH1 folding by SLC (Supplementary Fig. 3c, d). Of note, the KD between CL and CH1 is 6.2 µM34 while for CH1 and λ5, the affinity was determined to be slightly higher with a KD of 2.1 µM (Supplementary Fig. 4a and Table 2). For the complex between CH1 and SLC, the KD was further decreased to 0.5 µM (Supplementary Fig. 4b and Table 2). This higher affinity seems to be due to an interaction of VpreB with CH1 (KD 1.3 µM) (Supplementary Fig. 4c and Table 2). This interaction does not induce the folding of CH1 as shown in vitro and in cells. For the interaction of VpreB ΔUR with CH1, the KD was determined to be 3.3 µM (Supplementary Fig. 4d and Table 2). The interaction of CH1 and VpreB was also verified by AUC, which showed that the two proteins form a complex (Supplementary Fig. 4e). Thus, both the core domain and the UR of VpreB contribute to the CH1 interaction. In the case of ʎ5, the deletion of the UR does not affect its ability to interact with and fold CH1 (Supplementary Fig. 4f–k and Table 2). In summary, while both SLC proteins interact with CH1, only the core domain of ʎ5 induces the folding of CH1.

The unique regions strongly influence the SLC-VH association
The rearranged VH domain must be a focus of the SLC-checkpoint. It has been shown previously that VpreB interacts with VH21,31,32. However, our understanding of the mechanism, especially the role of the URs, is still at an early stage. To analyze this process further, the interaction of VpreB, λ5 and the SLC with two different VH domains (murine MAK33 and human 1HEZ) (Supplementary Fig. 5a) was assessed. AUC analyses showed that VpreB and VH 1HEZ form a heterodimeric complex (Fig. 5a). In this process, VpreB and VH 1HEZ, which are both homodimers, have to dissociate. Also, our analysis shows that λ5 and VH form a defined heterodimeric complex (Fig. 5b).

Surprisingly, the affinities between the antibody VH domain and SLC proteins determined by SPR are remarkably high. For VpreB and VH 1HEZ, the KD was 22.2 nM (Fig. 5c and Table 3) and for VpreB and VH Mak33 the KD was 10.1 nM (Supplementary Fig. 5b and Table 3). Compared to the isolated VpreB, the SLC binds to the VHs with markedly lower affinity; here the KD was 129.2 nM for VH 1HEZ (Fig. 5d and Table 3) and 29.6 nM for VH MAK33 (Supplementary Fig. 5c and Table 3). λ5 also interacts with the VH domains albeit with lower affinity, reflected in KD values of 171.9 nM for VH 1HEZ (Fig. 5e and Table 3) and 43.1 nM for VH MAK33 (Supplementary Fig. 5d and Table 3). Taken together, these experiments revealed unexpected features of the interactions of the SLC proteins with VH, which raised the question to what extent the URs are involved.

When we tested the interaction of λ5-ΔUR with VH 1HEZ, we could not detect a complex (Supplementary Fig. 5e), for both VH domains tested, binding was observed only with wild-type λ5 and not with λ5-ΔUR, strongly suggesting that the λ5-UR mediates this interaction. The CDR3s of the VH domains contain two negatively charged and one positively charged residue which may be involved in mediating this interaction. However, for the VpreB-UR the situation is different. First of all, VpreB ΔUR still binds to the VH domains (Supplementary Fig. 5f, g and Table 3). But, while for wild type VpreB the KD values for the two VH domains were similar, the effects of the UR deletion were strongly influenced by the respective VH domain: The KD for VH 1HEZ was 905.5 nM but it was 31.5 nM for VH MAK33 (Supplementary Fig. 5f, g and Table 3). Thus, depending on the VH domain used, the deletion of the VpreB-UR led either to a 3-fold or a 40-fold lower affinity compared to VpreB. In summary, the VpreB-UR is involved in the interaction with VH and the nature of the VH domain seems to play an important role concerning the magnitude of the effect. Moreover, the ʎ5-UR is essential for the interaction of VH and ʎ5.

The ʎ5-UR is crucial for antigen interaction
Since the SLC-Fd complex resembles a regular antibody Fab fragment, it was interesting whether this Fab-SLC is able to bind antigens and which parts of the SLC contributes to this interaction. To test this, enzyme-linked immunosorbent assay (ELISA) were setup, for which we used the C-terminally FLAG-tagged VH domain or the Fd fragment of the MAK33 antibody which binds creatine kinase36. The Fd fragment was assembled with either the SLC or the LC, respectively, to generate either a SLC-Fd (Fab-SLC) or a LC-Fd (Fab-LC) complex. In the ELISA, the antigen was immobilized, and serial dilutions of the Fab complexes were added. We observed a concentration-dependent interaction between the antigen and both “Fabs” demonstrating that the SLC-Fab complex is capable of antigen-binding. Interestingly, the Fab-SLC showed an affinity for the antigen, which was in the same range as compared to the authentic Fab fragment (8.6 vs. 12.6 nM; Fig. 6a). As expected, the antigen affinities of complexes containing only the VH domain instead of the Fd fragment were lower than those observed for the “Fab” fragments. For VH-SLC, the KD for the antigen was 94.1 nM and for VH-VL it was 155.9 nM (Fig. 6b). Isolated VH has a KD of 282.6 nM for the antigen (Fig. 6b).

To dissect which part of the SLC determines the affinity in the VH-SLC complex, the two SLC proteins were screened together with VH. For VpreB-VH, we determined a value of 260.1 nM (Fig. 6b) which is close to the value obtained for VH alone. Thus, VpreB does not seem to contribute significantly to the antigen interaction because it is unfolded in the absence of λ5. In contrast, λ5-VH exhibited a KD of 95.0 nM for the antigen (Fig. 6b) which is identical to the affinity of the SLC-VH complex.

To further dissect the contributions of the different parts of the SLC proteins to antigen binding, we also tested variants in which the UR was deleted. For VpreB-λ5-ΔUR-VH, the KD for creatine kinase was 122.0 nM (Fig. 6c, d). The deletion of the VpreB-UR in the VpreB-ΔUR-λ5-VH complex seems to slightly increase antigen affinity compared to the VH-SLC complex (67.9 vs. 94.1 nM, Fig. 6c, d). This confirms the assumption that the λ5-UR significantly contributes to the antigen interaction. However, the VpreB λ5-ΔUR-VH exhibited pronounced affinity for the antigen. It is important to note that in the presence of the λ5-ΔUR VpreB is folded. Thus, when the λ5-UR is missing, the ability of VpreB becomes visible. Taken together, these results indicate that in the context of the antigen interaction of the SLC, λ5 is the decisive factor because it establishes the folded state of VpreB and because the λ5-UR interacts strongly with the antigen.

Methods
Molecular biology and recombinant expression of the SLC proteins and antibody domains
Constructs for λ5 and VpreB variants were synthesized by GeneArt (Regensburg, Germany) and cloned into the pE-SUMOpro vector (KanR) (Lifesensors, Malvern, USA) after the SUMO Protease 1 cleavage site via the BsaI restriction site using sequence- and ligase-independent cloning47,48. Genes for antibody domains were synthesized by Geneart (Regensburg, Germany) and cloned into the pET28b vector (KanR) via the XbaI/Xho restriction sites keeping the sequence between XbaI and NcoI at the 5´ end of the nucleotide sequence of the proteins using sequence and ligase- independent cloning47,48. All constructs were sequenced by Eurofins Genomics (Ebersberg, Germany). In all λ5 variants, the C-terminal cysteine (C212), which forms a disulfide bridge with the CH1 domain of the HC in the final assembled state, was mutated to a serine.

Plasmids were transformed into the E. coli strain BL21-CodonPlus (DE3)-RIL. Cells were grown at 37°C in selective LB medium and expression was started by the addition of 1 mM isopropyl-beta-D-thiogalactopyranoside (IPTG) at an OD 600 of 0.9. After 3 h, cells were harvested (7000×g). The harvested cells were resuspended in 50 mM Tris/HCl (pH 7.5), 100 mM NaCl, and lysed using a cell disruptor (Constant Cell Disruption Systems, Northants, UK). A centrifugation step at 12,200×g for 40 min at 4°C was followed to obtain the inclusion bodies in the pellet.

Refolding and purification of the SLC proteins, VH MAK33-FLAG, the SLC heterodimer, the Fab and the Fd-SLC complex
Inclusion bodies were solubilized in 100 mM sodium phosphate, pH 7.5, 6 M GdmCl, 10 mM imidazole at 4°C for 16 h by gentle stirring. Insoluble components were removed by centrifugation (33,000×g, 1 h, 6°C). The supernatant was applied to a Ni-chelating column (Cytiva, Freiburg, Germany) pre-equilibrated in 50 mM sodium phosphate, pH 7.5, 5 M GdmCl, and 10 mM imidazole (equilibration butter). After washing with five column volumes of equilibration buffer, elution was performed applying a gradient over 10 column up to 500 mM imidazole. For reduction, the purified proteins were incubated for 1 h at RT with 10 mM β-mercaptoethanol and gentle stirring.

Refolding was carried out via drop dilution of the unfolded protein in 100 mM Tris/HCl, pH 8.0, 150 mM NaCl, 350 mM L-arginine, and 10 mM β-mercaptoethanol to a final protein concentration of 0.1 g L−1. The diluted protein was then dialyzed against 100 mM Tris/HCl, pH 8.0, 150 mM NaCl, 350 mM L-arginine, 4 mM oxidized glutathione, 0.5 mM reduced glutathione for 36 h at 10°C and simultaneous addition of His-tagged SUMO protease to remove the N-terminal His-SUMO-tag. A dialysis step against 50 mM HEPES, pH 7.4, 150 mM KCl, 30 mM imidazole for 16 h at 4°C followed. Aggregates were removed by filtration using a PVDF syringe filter with 0.22-µm pore size.

The filtered protein was applied to a Ni-chelating column, pre-equilibrated in 50 mM HEPES, 150 mM KCl, and pH 7.4 (native buffer). The target protein is in the flowthrough while the SUMO protease remains bound to the column. The flow-through containing the target protein was concentrated using Amicon® Ultra-15 3K MWCO (Merck KGaA, Darmstadt, Germany) and the concentration was determined from the E280 values determined in a Jasco V-630 Spectrophotometer (Jasco, Grossumstadt, Germany) at 280 nm using the calculated extinction coefficient.

The SLC heterodimer was purified according to the scheme described above with the following changes: The single domains were combined in an equimolar ratio before drop dilution at a concentration of 0.05 g L−1 for ʎ5. For the purification of the Fd-SLC complex, it was similar: The Fd fragment was diluted to a concentration of 0.06 g L−1 and equimolar ratios of VpreB and λ5 C212S were added. Fd-LC (Fab complex) was purified as described previously36.

Expression and purification of Ulp1-protease (SUMO-protease)
The ubiquitin-like-specific protease 1 (ULP1) (AA 403-621) from Saccharomyces cerevisiae containing an N-terminal His6-tag (Life Sensors Inc., Malvern, USA) (pET28b, KanR) was transformed into the E. coli strain BL21-CodonPlus (DE3)-RIL. Cells were grown at 37°C in selective LB medium, and expression was started by the addition of 1 mM IPTG at an OD 600 of 0.6–0.8. After 4 h, cells were harvested by centrifugation at 7000×g and 4°C. The harvested cells were resuspended in 40 mM sodium phosphate, pH 7.4, 300 mM NaCl, 40 mM imidazole (phosphate buffer), and lysed using a cell disruptor (Constant Cell Disruption Systems, Northants, UK). A centrifugation step at 33,000×g and 6°C for 1 h followed. The supernatant was applied to a pre-equilibrated Ni-chelating column. After washing with phosphate buffer for five column volumes, elution was performed applying a gradient over 15 column volumes up to 300 mM imidazole. The pooled and concentrated protein was applied to a 16/60, 75 pg, Superdex size-exclusion chromatography column (Cytiva, Freiburg, Germany) in 50 mM Tris/HCl, pH 8.0, 500 mM NaCl, 1 mM DTT at 4°C. Finally, 50% glycerol, and 1% tergitol were added to the purified protein.

Refolding and purification of antibody domains
Inclusion bodies for antibody domains were solubilized and reduced in 50 mM Tris (pH 7.5), 8 M urea, 10 mM β-mercaptoethanol at 4°C for 16 h by gentle stirring. Insoluble components were removed by centrifugation (33,000×g, 1 h, 6°C). The supernatant was applied to a Q Sepharose fast flow column (Cytiva, Freiburg, Germany) pre-equilibrated in 50 mM Tris/HCl, pH 7.5, and 5 M urea. After washing for two column volumes, elution was performed applying a gradient over 10 column volumes up to 1 M NaCl. The proteins were diluted to a protein concentration of roughly 0.1 g L−1. Refolding of CH1 MAK33 was carried out via dialysis in 250 mM Tris/HCl, pH 8.0, for 16 h at 10°C. For CH1 MAK33, VL, and VH 1HEZ, as well as VH MAK33, dialysis was against 250 mM Tris, pH 8.0, 100 mM L-arginine, 2 mM oxidized glutathione, and 0.5 mM reduced glutathione at 10°C for 16 h. The proteins were concentrated in Amicon® Ultra-15 3K MWCO (Merck KGaA, Darmstadt, Germany). Aggregates were removed by filtration using a PVDF syringe filter with 0.22-µm pore size. The filtrated protein was applied to a Superdex, 26/60, 75 pg (Cytiva, Freiburg, Germany) size-exclusion chromatography column pre-equilibrated in phosphate-buffered saline (PBS: 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, pH 7.4). After a final concentration step, protein concentrations were determined using their calculated extinction coefficients in a Jasco V-630 Spectrophotometer (Jasco, Grossumstadt, Germany) at 280 nm.

The N-glycosylated form of CH1 was transiently expressed and secreted from Expi293 cells cultured in suspension as His6-Fd, containing a TEV tag between the His6-VH and CH1 domains, and co-expressed with a His6-tagged light chain. For protein purification, 200 mL of clarified culture supernatant containing the secreted protein was directly subjected to Ni-NTA affinity chromatography. The bound protein was then incubated overnight with TEV protease, and CH1 was eluted the next day using HisTrapA buffer. The protein was further purified by size-exclusion chromatography on a Superdex 75 pg 26/60 column (Cytiva, Freiburg, Germany) pre-equilibrated with phosphate-buffered saline (PBS: 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, pH 7.4) for polishing.

Peptide
The additional β-strand of λ5 (ʎ5β) was obtained as a peptide with the sequence: THVFGSGTQLTVLS from Biomatik USA (Wilmington, USA).

Optical spectroscopy
Circular dichroism (CD) spectra were recorded in a Chirascan plus CD spectrophotometer (Applied Photophysics, Leatherhead, UK) to assess the secondary structure. Far-UV CD spectra were recorded in a 1 mm quartz cuvette in PBS buffer (100 mM Na2HPO4 × 2H2O, 18 mM KH2PO4, 27 mM KCl, 1.37 M NaCl, and pH 7.4). and measured at a protein concentration of 0.12 g L−1 between 260 and 200 nm. The scan speed was 0.5 s per point, and spectra were accumulated 16 times, buffer-corrected, and normalized for mean residue molar ellipticity.

Thermal transitions were recorded in a 1 mm path length quartz cuvette at 205 nm with a heating rate of 60°C/h from 20 to 90°C. Thermal transitions were normalized and fitted using a Boltzmann function to obtain the transition point where half of the protein is unfolded (Tm).

Far-UV CD kinetics were recorded in a Jasco J-1500 spectrometer (Jasco, Grossumstadt, Germany) in a 1 mm quartz cuvette at 205 nm and 25°C for 4 h. The protein concentration was 5 µM per domain in PBS buffer. Thermal transitions of the formed complexes were recorded in a Jasco J-1500 spectrometer (Jasco, Grossumstadt, Germany) equipped with a Peltier element in a 1 mm quartz cuvette at 205 nm with a heating rate of 60°C/h from 20 to 90°C. Kinetics were fitted using an exponential decay function to obtain the point where 66.3% of the protein is folded (τ). For each condition, two independent kinetic measurements were performed. The fitting start point was defined as the earliest time after which the signal derivative remained within a stable range, thereby excluding instrumental artefacts.

Analytical ultracentrifugation (AUC)
AUC measurements were carried out using an Optima AUC I (Beckman, Krefeld, Germany) equipped with absorbance optics. The protein concentration was 20 µM in 50 mM HEPES/KOH, pH 7.4, and 150 mM KCl. About 300 µL sample were loaded into assembled cells with quartz windows and 12-mm-path-length charcoal-filled epon double sector centerpieces. Samples were sedimented at 160,000×g (42,000 rpm) in an eight-hole Beckman-Coulter A50-ti rotor at 20°C. Sedimentation was continuously scanned with a radial resolution of 30 µm and monitored at 280 nm. Data analysis was carried out with SEDFIT using the continuous c(S) distribution mode49,50 and the data were normalized.

Hydrogen-deuterium exchange mass spectrometry (HDX-MS)
HDX was performed on an automated robotics system (HTS PAL; LEAP Technologies, Ft Lauderdale, USA) coupled to an Acquity M-Class UPLC and a HDX manager (Waters Corp., Milford, USA) as described elsewhere (Zhang et al., 2014).

About 3 µL of a 30 µM protein sample was added to 57 µL deuterated buffer (50 mM HEPES/KOH, 150 mM KCl, and pH 7.4) and incubated at 20°C for 0.17, 1, 10, 30, and 120 min. HDX-MS experiments were performed in two independent experiments, each including three technical replicates per protein and per timepoint. The exchange was stopped by adding 50 µL of quench buffer (200 mM Na2HPO4, 200 mM KH2PO4, 4 M GdmCl, and pH 2.2) to 50 µL of labeled protein at 1°C. Digestion was performed on-line using an immobilized Waters® EnzymateTM BEH Pepsin Column (2.1×30 mm; Waters Corporation, Milford, USA) at 20˚°C. Peptides were trapped at 0˚C on a VanGuard Pre-column [Acquity UPLC BEH C18 (1.7 µm, 2.1×5 mm, Waters Corporation, Milford, USA)] for 5 min. The peptides were separated using a C18 column [Acquity UPLC BEH C18 (1.0×100 mm, Waters Corporation, Milford, USA)] at 0°C by gradient elution of 0–35% (v/v) acetonitrile (0.1% v/v formic acid) in H2O (0.1% v/v formic acid) over 6 min followed by a gradient elution of 35-40% (v/v) acetonitrile (0.1% v/v formic acid) in H2O (0.1% v/v formic acid) over 7 min, both gradients at a flow rate of 40 µL min−1. Eluting peptides were detected using a Synapt G2S mass spectrometer (Waters Corporation, Milford, USA). The mass spectrometer was operated in HDMSE mode, with dynamic range enabled (data independent analysis (DIA) coupled with IMS separation) were used to separate peptides prior to collision-induced dissociation (CID) fragmentation in the transfer cell. CID data were used for peptide identification, and uptake quantification was performed at the peptide level (as CID results in deuterium scrambling).

Data were analyzed using ProteinLynx Global Server PLGS (v3.0.3) and DynamX (v3.0.0) software (Waters Corporation, Milford, USA). Search parameters in DynamX were as follows: peptide and fragment tolerances=automatic, min fragment ion matches=1, digest reagent=non-specific, false discovery rate=100. Restrictions for peptides in DynamX were as follows: minimum intensity=5000, minimum products per amino acid=0, max sequence length=20, min sequence length=5, max ppm error=0, file threshold=0. The software Deuteros51 was used to identify peptides with statistically significant increases/decreases in deuterium uptake based on replicate-derived confidence intervals (99%) and to prepare Wood´s plots. No formal statistical hypothesis testing was performed. An HDX-MS summary table is provided in Supplementary Table 1 in accordance with community reporting standards.

Constructs for cell experiments
Constructs used in this study for cell culture experiments were obtained from GeneArt (Regensburg, Germany) in a pBUDCE4.1 vector optimized for mammalian expression for SLC constructs. Fd constructs were cloned into the pcDNA 3.1 vector. All constructs were verified by sequencing.

Cell culture and transient transfections
HEK293T cells (obtained from ECACC) were cultivated in Dulbecco’s Modified Eagle’s Medium ((DMEM), high glucose (Sigma-Aldrich, Steinheim, Germany), supplemented with 10% (v/v) fetal bovine serum (Biochrom) and 1% (v/v) antibiotic-antimycotic solution (25 µg/ml amphotericin B, 10 mg/ml streptomycin, 10,000 units of penicillin (Sigma-Aldrich, Steinheim, Germany) and GlutaMAX (Thermo Fisher Scientific, Dreieich, Germany) at 37°C in a 95% humidified atmosphere containing 5% CO2. Transient DNA transfections of HEK293T cells were carried out in p35 dishes (uncoated Nunclon multidish six-well plates) using GeneCellin (BioCell Challenge, Signes, France) according to the manufacturer´s protocol. To perform co-transfections, the total amount of transfected DNA was divided equally according to the manufacturer´s protocol.

Cell lysis
All cell lysis steps were performed on ice using ice-cold solutions. Unless otherwise stated, cell lysis was performed 24 h after transfection. Cells were washed twice with PBS and then 0.5 ml NP-40 lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.5% (w/v) NaDOC, complemented with 0.5% (v/v) NP-40 (Sigma-Aldrich, Steinheim, Germany) and 1x Roche Protease Inhibitor w/o EDTA (Roche Diagnostics, Penzberg, Germany) were added to the cells. After an incubation period of 20 min, the cells were scraped from the plates, and the resulting cell lysate was centrifuged for 15 min at 20,000×g, 4°C. The supernatant was either used for further analysis or complemented with 5x Laemmli buffer containing 10% (v/v) β-mercaptoethanol. Laemmli-containing sample lysates were subsequently heated for 10 min at 95°C.

Deglycosylation experiments
Deglycosylation assays with a mix of O-glycosidase and α2-3,6,8 Neuraminidase (NEB, Frankfurt, Germany) were performed according to the manufacturer’s instructions at 37°C for 1 h. The digested sample proteins were thereafter supplemented with 5x Laemmli buffer and 10% (v/v) β-mercaptoethanol and heated for 10 min at 95°C.

Secretion experiments
For secretion assays, cells were transfected for 24 h. The cells were then washed twice with PBS (Sigma-Aldrich, Steinheim, Germany) and subsequently supplemented with 0.5 ml fresh DMEM for cultivation for another 24 h. To analyze secreted proteins, the medium was removed from the cells and centrifuged for 5 min at 300×g and 4°C. The resulting supernatant was thereafter transferred into a new reaction tube and supplemented with 0.1 volumes of 500 mM Tris/HCl (pH 7.5), 1.5 M NaCl, complemented with 10x Roche Complete Protease Inhibitor w/o EDTA (Roche Diagnostics, Penzberg, Germany). After another centrifugation step for 15 min at 20,000×g and 4°C, 5x Laemmli buffer containing 10% β-mercaptoethanol (β-Me) was added to the samples. Samples were then heated for 10 min at 95°C. The actual cell lysis was then carried out using a PBS wash step before the addition of 0.5 ml of 1x RIPA lysis buffer (50 mM Tris/HCl, pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% DOC, and 0.1% SDS) supplemented with 1x protease inhibitor, to the cells. After 20 min, the cell lysate was centrifuged for 15 min at 20,000×g and 4°C and 0.2 volumes of 5x Laemmli buffer containing 10% β-mercaptoethanol (β-Me) were added. Samples were thereafter heated to 95°C for 10 min.

Co-immunoprecipitation
Protein–protein interactions were analyzed and detected using co-immunoprecipitations (co-IPs). Before co-IP of target proteins, cells were lysed using NP-40 lysis buffer, and 2% of the cell lysate was supplemented with 5x Laemmli buffer containing 2% (v/v) β-mercaptoethanol. The remaining lysate was then incubated with 1.5 µg of mouse monoclonal anti-HA antibody for 2 h under constant rotation at 4°C. Following this, 30 µl protein A/G agarose beads (Thermo Fischer, Dreieich, Germany) were added and samples incubated for another hour under constant rotation at 4°C. Thereafter, the beads were washed three times with 1 ml NP-40 wash buffer (50 mM Tris/HCl, pH 7.5, 400 mM NaCl, 0.5% (w/v) NaDOC, and 0.5% (v/v) NP-40), including centrifugation steps for 5 min at 4°C and 620×g (2.500 rpm) in between. Finally, the proteins were eluted by adding 2x Laemmli buffer supplemented with 10% (v/v) β-mercaptoethanol and heating at 95°C for 10 min.

SDS-PAGE and immunoblotting
For immunoblots, samples were separated on 15% SDS-PAGE gels and then wet-transferred overnight at 4°C. After transferring the proteins to methanol (Sigma-Aldrich), activated PVDF membranes (Biorad), the membranes were blocked at least 3 h at RT with Tris-buffered saline supplemented with skim milk powder and Tween-20 (M-TBST; 25 mM Tris/HCl, pH 7.5, 150 mM NaCl, 5% (w/v) skim milk powder, 0.05% (v/v) Tween-20). Primary antibodies were diluted in M-TBST and applied to the membranes overnight at 4°C. After washing of the membranes (1×5 min TBS, 2×5 min TBST, 3×5 min TBS), HRP-conjugated secondary antibodies diluted in M-TBST were applied to the blots. Following a second washing procedure, proteins were then detected using Amersham ECL prime solution (Cytiva, Freiburg, Germany) on a Fusion Pulse 6 imaging system (Vilber Lourmat, Eberhardzell, Germany).

Antibodies
For western blots, the following primary antibodies were used at the dilutions listed: rabbit polyclonal anti-FLAG (F7425, Sigma-Aldrich, Steinheim, Germany) at 1:1000; rabbit polyclonal (9023) anti-HA (902302, Biolegend, Koblenz, Germany) at 1:1000; mouse monoclonal (B-6) anti-Hsc70 (sc-7298, Santa Cruz, Heidelberg, Germany) at 1:1000 and mouse monoclonal anti-V5 (680602, Biolegend, Koblenz, Germany) at 1:1000. The following HRP-conjugated secondary antibodies were used for development of Western blots at 1:10,000: mouse IgGκ-binding protein (m-IgGκ BP-HRP) (sc-516102, Santa Cruz, Heidelberg, Germany) and mouse monoclonal anti-rabbit IgG-HRP (sc-2357, Santa Cruz, Heidelberg, Germany). For IP, the following antibodies were employed: monoclonal mouse anti-HA (16B12) (901518, BioLegend, Koblenz, Germany). Uncropped scans of representative blots are provided in the Source Data file.

Enzyme-linked immunosorbent assay (ELISA)
PierceTM Streptavidin Coated Clear 96-Well plates (Thermo Fisher Scientific, Dreieich, Germany) were incubated with 90 µL of antigen solution per well at 25°C for 45 min, shaking on a Thermomixer compact (Eppendorf, Hamburg, Germany) at 350 RPM. This serves for immobilization of the antigen, which is human biotinylated muscle-type creatine kinase. After discarding the antigen solution, each well was washed three times with 150 µL of de-ionized H2O.

Next, a serial dilution of the constructs to be analyzed in a stoichiometric ratio of 1:1 with VH MAK33-FLAG was prepared in triplicates, except for Fd-SLC and Fd-LC, for which the serial dilutions were prepared directly from the purified complexes. About 50 µL of each protein concentration of the serial dilution was added to the 96-well plate and again incubated at 25°C for 45 min, shaking at 350 RPM. Another washing step of three times with 150 µL of de-ionized H2O followed. Thereafter, to each well 100 µL of an anti-FLAG antibody conjugated to horseradish peroxidase (HRP) (Sigma-Aldrich, Steinheim, Germany) in a dilution of 1:15,000 were added, and the plate was incubated at 25°C for 45 min, shaking at 350 RPM, covered from light. After discarding the solution, each well was washed three times with 150 µL of de-ionized H2O.

Finally, 100 µL of ABTS solution was added per well, and the plate was covered with a sealing foil. Enzymatic conversion of ABTS by HRP leads to an increase in absorption at a wavelength of 405 nm. The absorption was measured in an Infinite M Nano plate reader (Tecan Group, Männedorf, Switzerland) at 405 nm and 25°C. The measurement was conducted for 1 h with data intervals of 5 min. The intensities after 1 h were taken for data analysis. The absorption was plotted against the concentration on a logarithmic scale and fitted using a Hill 1 function to obtain the affinity (KD) for the antigen.

Surface plasmon resonance (SPR) analysis
The interaction analysis between λ5 and VpreB variants, CH1 and SLC variants, as well as between the VH domain and SLC variants, was performed with a Biacore X100 instrument (GE Healthcare, Freiburg, Germany) in 50 mM HEPES/KOH, pH 7.4, 150 mM KCl, 3 mM EDTA, and 0.5% Tween-20 as running buffer at 25°C. The ligands were immobilized on a CM5 sensor chip by amine coupling chemistry at pH 4–6 (Biacore manual, Cytiva Sweden AB, Uppsala, Sweden) using the amine coupling kit (Cytiva Sweden AB, Uppsala, Sweden). Multi-cycle runs with titrations of the analytes at various concentrations were measured with an association time of 3 min, a dissociation time of 5 min and a constant flow rate of 30 µL/min. The sensor surface was regenerated between each run with a 30 s injection of 1.5 M KCl at a flow rate of 30 µL/min.

For the SPR experiments monitoring binding between λ5 and VpreB variants, VpreB was immobilized on a CM5 sensor chip, and purified λ5 was injected as analyte at concentrations ranging from 0.002 to 4 µM. For the SPR experiments between CH1 and SLC variants, CH1 was immobilized on a CM5 sensor chip, and purified SLC variants were injected as analytes at concentrations ranging from 0.002 to 50 µM, depending on the variant used. For the SPR experiments monitoring binding between the VH domain and SLC variants, VH 1HEZ or VH MAK33 was immobilized on a CM5 sensor chip via amine coupling, and purified VpreB, λ5, or the SLC were injected as analytes at concentrations ranging from 0.003 to 10 µM, depending on the variant used.

Binding curves were corrected by subtraction of buffer and reference flow cell signals (binding to control flow cells and running buffer) and fitted to a 1:1 binding model. Sensorgrams were analyzed using the Biacore X100 Evaluation Software (Cytiva Sweden AB, Uppsala, Sweden) to obtain the equilibrium dissociation constant (KD), association rate constant (ka), and dissociation rate constant (kd).

Nuclear magnetic resonance (NMR) spectroscopy
All spectra were acquired at 25°C using a Bruker AVANCE 600 (Bruker, Rheinstetten, Germany). All proteins were solubilized in 50 mM HEPES, 150 mM KCl, pH 7.4, supplemented with 10% D2O employing a 5-mm-NMR tube. For the measurement of VpreB in the presence of the β-strand derived peptide λ5β, a 150 µM solution of 15N-labeled VpreB was titrated with unlabeled λ5β to yield a two fold excess of λ5-β. Prior to the experiment, the VpreB-λ5β complex was incubated for at least 2 h at RT to ensure complete folding of VpreB.

Complexes of VpreB and ʎ5 were obtained by co-purification of 13C, 15N-labeled VpreB and unlabeled λ5β. For references, the individual proteins (15N-labeled VpreB and 13C, 15N-labeled λ5β) were measured independently.

To probe the structure of VpreB in the presence or absence of folding agents such as λ5β or full-length λ5, 15N-HSQC spectra were recorded of VpreB alone at 25°C by using selective proton flip-back techniques for fast pulsing. Processing was identical for all spectra was performed in TOPSPIN 4.0.3 (Bruker Biospin). The data was further analyzed and visualized in CCPN V3.0.4 (University of Leicester, United Kingdom). The figures were created in Affinity Designer (Serif Europe, Nottingham, UK).

Statistics and reproducibility
No statistical method was used to predetermine sample size. No data was excluded from the analyses. The experiments were not randomized, and the investigators were not blinded to allocation during experiments and outcome assessment.

Data were presented as mean values, with error bars representing the standard deviation (SD), as indicated in the figure legends. The number of independent experiments is specified in the figure legends. Unless otherwise stated, all experiments were independently repeated at least two times with similar results.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In the manuscript NCOMMS-23-62879 Koenig et al. study the folding and assembly of components of the surrogate light-chain (VpreB and λ5) of the pre-B cell receptor (pre-BCR).\nThe surrogate light-chain (SLC) forms together with a membrane-bound immunoglobulin (Ig) heavy chain of class M (mIgM) and the CD79a/CD79b signaling components the pre-BCR that promotes the further development of only those pre-B cells with a productive heavy chain (HC) gene assembly. The authors find that in the absence of a HC fragment (Fd), the CL domain of λ5 is properly folded while the V domain of VpreB is unfolded. This is a well-known fact as VpreB is lacking one β-strand, that is provided by the N-terminal extension of the λ5 protein (ref 21). Interestingly, VpreB and λ5 together are forming a SLC in the absence of a Fd HC fragment. The authors also show that similar to a conventional LC, λ5 can promote the proper folding of the unstructured CH1 domain. By generating deletion mutants, the authors study the function of the unstructured unique region (UR) of VpreB and λ5. They found that the UR of VpreB and λ5, promote and reduced the speed of SLC formation.\nThe Ig domain folding studies are well conducted and described. However, this manuscript has a major problem namely that the authors are not aware of or are ignoring major biological features of the pre-BCR. A functional pre-BCR can only be formed by the first HC class expressed namely mIgM (Uebelhart et al. (2010) Nat Immunol 11: 759-765). The reason for this is that only the CH1 domain of mIgM (but not of mIgD or other mIg classes) carries a conserved asparagine (N46) that is glycosylated and that via the negatively charged sugar moiety apparently can interact with the positive charged UR of theλ5 protein. This UR contains several evolutionary highly conserved arginine residues (not at all mentioned in the manuscript). It is thought that that via these highly conserved arginine the UR of λ5 binds to the CH1 domain of mIgM and that this releases the inhibitory role of the λ5 UR for pre-BCR assembly that the authors also find in their studies. In addition, the N46/UR of λ5 interaction can promote the active (constitutive signaling) conformation of the pre-BCR. In their manuscript the authors do not mention the class of the used Fd HC fragment and thus it is questionable whether they study the formation of the biological relevant pre-BCR. Given this major problem I think that the manuscript is more suitable for a biochemical Journal than for Nature Communications.\nSpecific comments\nThe sentence on page 3 “The assembly of the SLC with the HC forms the pre-B cell receptor“ is wrong as a functional pre-B cell receptor also contains the CD79a/CD79b signaling components\nThe sentence on page 3 “The non-covalent association with the HC is supported by an intermolecular disulfide bond between λ5 and the CH1 domain“ is misleading as the intermolecular disulfide bond between λ5 and the CH1 domain establishes a covalent association between the SLC and the HC\nOn page 4 a ref is missing “In the available crystal structure of the pre BCR, the URs are only partially resolved (REF)“\nThe sentence on page 5” This is surprising as VpreB which corresponds to the VL domain of a regular LC and isolated VL domains have been shown to be folded in the preB cell receptor“ is misleading as the author later explain that the unassembled VpreB component is lacking a β-strand and thus does not correspond to a VL domain.\nOn page 6 the authors write ”Interestingly, the sedimentation coefficient of VpreB alone was ~3.2 S, which is indicative of a non-native homodimer, as described previously“. How can an unfolded protein like VpreB lacking a β-strand form a homodimer? Have the authors done these sedimentation experiment also with the VpreB that contains the β-strand peptide?\nOn page 6 the authors write” that both, λ5 and VpreB, can interact individually with Fd“. Are these binding experiments done with the unfolded VpreB or the VpreB that contains the β-strand peptide?\nOn page 6 the authors write our result ”strongly suggesting that binding of λ5 to VH is mediated exclusively by the λ5 UR“. Is this interaction shown by all the VH domains tested and does the VH domain carry a negatively charged CDR3 region or a sugar moiety that would explain this otherwise puzzling interaction?\nThe section on “The λ5-UR is crucial for antigen interaction“ is lacking a specificity control. Is the creatine kinase binding only seen by the VH MAK33 or also VH1HEZ containing SLC complexes?", "reviewer_2": "In this manuscript, König et al. examined the assembly of the pre-B cell receptor in vitro using purified proteins and in HEK293 cells by transient transfection. The pre-B cell receptor, whose crystal structure was reported in 2007, comprises a surrogate light chain (SLC) covalently paired with an antibody heavy chain. The SLC consists of two non-covalently associated proteins, VpreB and λ5. The authors show that VpreB, which is unfolded in isolation, adopts its native structure upon interaction with  5. This process (β-strand complementation) requires a β-strand from l5 to provide the missing ninth β-strand of VpreB. In addition, folding of CH1 requires interaction with λ5. The biochemical analysis of the assembly process is technically well done and the results largely justify the authors’ conclusions. However, it is not quite accurate to claim that “little is known about the assembly process of VpreB and  5” (p. 4, lines 4-5). In fact, quite a lot was already known, as evident from the references cited. For example, β-strand complementation by λ5 was described in refs. 29 and 30. Nevertheless, the present work provides the most complete picture to date of pre-B cell receptor assembly.\nPoints to address:\np. 5, 3rd paragraph. Individual SLC proteins for biophysical analysis were produced by in vitro refolding from inclusion bodies. CD and NMR analyses indicate that isolated VpreB is unfolded. However, finding appropriate conditions for in vitro folding of proteins is often not straightforward. That is to say, failure to refold a protein in vitro does not necessarily mean that the protein is unfolded when produced naturally within a cell in an environment that includes chaperones, etc. The authors should include this caveat. Having said that, it is certainly striking that addition of λ5  peptide to isolated VpreB induces folding of VpreB, as indicated by NMR.\nFig. 1c. Figure legend should include Hsc70 and explain that it is a positive control. In medium, there are two bands for VpreB in the λ5 + VpreB lane. What is the lower MW band, which does not appear (or just barely) in the VpreB alone lane? Is it another glycoform of VpreB?\nFig. 2d. Figure legend is incomplete. How exactly were the SPR measurements done? What was immobilized? What was the analyte? Are the concentrations for the λ5β peptide? The description of SPR in Materials and Methods does not answer these specific questions.\np. 6, 3rd paragraph. In AUC, isolated VpreB appears to sediment as a homodimer. How is this possible if isolated VpreB is unfolded? In looking at the sedimentation profile of isolated VpreB in Fig. 2f, the profile is very broad compared to those of the other samples, which we know are folded. Very likely the broad VpreB profile reflects non-specific aggregation rather than discrete dimerization. The authors should revise their text accordingly.\nFig. 3g. Figure legend should explain labeling of samples. Presumably, λ5DU means λ5 with URL deleted and VpreBDU means VpreB with UR deleted. Also, for comparison of intensities, the immunoblots should include  5 and VpreB with URs intact.\nFig. 4b. Figure legend does not adequately describe immunoprecipitations in the two panels.\nFig. 5c-e. Same comments about SPR as above for Fig. 2d.\np. 9, 3rd paragraph. Results of ELISA assays should be confirmed by SPR using immobilized creatine kinase. The authors have all necessary reagents to make these measurements.\nMinor:\nOn p. 2, 1st line, (REF) should give reference number.\nIn Fig. 3 legend, panels b and e should be panels b and d."}, "author_response": "POINT TO POINT REPLY TO THE REVIEWER COMMENTS\nReviewer #1\nIn the manuscript NCOMMS-23-62879 Koenig et al. study the folding and assembly of components of\nthe surrogate light-chain (VpreB and λ5) of the pre-B cell receptor (pre-BCR).\nThe surrogate light-chain (SLC) forms together with a membrane-bound immunoglobulin (Ig) heavy\nchain of class M (mIgM) and the CD79a/CD79b signaling components the pre-BCR that promotes the\nfurther development of only those pre-B cells with a productive heavy chain (HC) gene assembly. The\nauthors find that in the absence of a HC fragment (Fd), the CL domain of λ5 is properly folded while\nthe V domain of VpreB is unfolded. This is a well-known fact as VpreB is lacking one β-strand, that is\nprovided by the N-terminal extension of the λ5 protein (ref 21). Interestingly, VpreB and λ5 together\nare forming a SLC in the absence of a Fd HC fragment. The authors also show that similar to a\nconventional LC, λ5 can promote the proper folding of the unstructured CH1 domain. By generating\ndeletion mutants, the authors study the function of the unstructured unique region (UR) of VpreB and\nλ5. They found that the UR of VpreB and λ5, promote and reduced the speed of SLC formation.\nThe Ig domain folding studies are well conducted and described. However, this manuscript has a\nmajor problem namely that the authors are not aware of or are ignoring major biological features of the\npre-BCR. A functional pre-BCR can only be formed by the first HC class expressed namely mIgM\n(Uebelhart et al. (2010) Nat Immunol 11: 759-765). The reason for this is that only the CH1 domain of\nmIgM (but not of mIgD or other mIg classes) carries a conserved asparagine (N46) that is\nglycosylated and that via the negatively charged sugar moiety apparently can interact with the positive charged UR\nof theλ5 protein. This UR contains several evolutionary highly conserved arginine residues (not at all\nmentioned in the manuscript). It is thought that that via these highly conserved arginine the UR of λ5\nbinds to the CH1 domain of mIgM and that this releases the inhibitory role of the λ5 UR for pre-BCR\nassembly that the authors also find in their studies. In addition, the N46/UR of λ5 interaction can\npromote the active (constitutive signaling) conformation of the pre-BCR. In their manuscript the\nauthors do not mention the class of the used Fd HC fragment and thus it is questionable whether they\nstudy the formation of the biological relevant pre-BCR. Given this major problem I think that the\nmanuscript is more suitable for a biochemical Journal than for Nature Communications.\nWe thank the reviewer for pointing out the importance of the glycosylated mIgM C 1 domain for pre-\nH\nBCR formation. Indeed, this feature had escaped our attention. In the revised manuscript we now\nperformed the first comparative studies of glycosylated and unglycosylated C 1 domains, extending\nH\nthe scope of our study. In detail, in response to the reviewer’s comment, we established the\nexpression and purification of this glycosylated C 1 domain over the past months and performed\nH\nfolding studies with the SLC proteins and the assembled SLC complex.\nAs expected by the reviewer, using the glycosylated C 1 made a difference concerning the interaction\nH\nwith the SLC. Folding of the glycosylated C 1 domain by the SLC was faster.\nH\nInterestingly, while λ5 was sufficient to fold the unglycosylated C 1 domain, for the glycosylated\nH\nversion, the entire SLC complex was required. We incorporated these exciting results into the revised\nversion and changed our model accordingly (p. 8): “The MAK33 C 1 used in our experiments\nH\nrepresents an IgG C 1 domain. The pre-BCR is assembled with IgM heavy chains. The C 1 domain of\nH H\nIgM contains a conserved glycosylation site at N46. Since this glycan is crucial for interaction with the\nλ5 UR, as previously shown (Uebelhart et al.35), we compared glycosylated and unglycosylated C 1\nH\n(2H32). Surprinsingly and in contrast to the IgG C 1 domain, we observed that glycosylated C 1 could\nH H\nnot be folded by λ5 but only by the SLC in vitro with a rate constant τ = 66 min, whereas\nunglycosylated CH1 required τ = 104 min, indicating that glycosylation accelerates C 1 folding by SLC\nH\n(Supplementary Fig. 3c-d).”\nAnd in discussion (p. 12):’ An interesting difference was observed for the glycosylated IgM CH1-2H32\ndomain. It folds faster than the unglycosylated form in the presence of the SLC, suggesting that\nglycosylation enhances the efficiency of CH1–SLC interactions. This finding is consistent with the in\nvivo requirement of the conserved CH1 N-glycosylation for efficient μHC–SLC interaction and pre-\nBCR surface expression35. Notably, we did not observe folding of CH1-2H32, with or without\nglycosylation, in the presence of λ5 alone in vitro.’\nThe sentence on page 3 “The assembly of the SLC with the HC forms the pre-B cell receptor“ is wrong\nas a functional pre-B cell receptor also contains the CD79a/CD79b signaling components\nWe agree that our original wording was not precise, and we have revised the sentence accordingly.\nRevised text (p.3):\n“The assembly of the SLC and the HC together with the CD79a/CD79b signaling components forms\nthe pre-B cell receptor 10-12“\nThe sentence on page 3 “The non-covalent association with the HC is supported by an intermolecular\ndisulfide bond between λ5 and the CH1 domain“ is misleading as the intermolecular disulfide bond\nbetween λ5 and the CH1 domain establishes a covalent association between the SLC and the HC\nWe have revised the sentence to clarify the distinction between non-covalent interactions and the\ndisulfide bond. The revised text (p. 3) now reads:\n“The association of the SLC with the HC involves both non-covalent interactions and a covalent\nintermolecular disulfide bond between λ5 and the C 1 domain 3, 24-28”\nH\nOn page 4 a ref is missing “In the available crystal structure of the pre BCR, the URs are only partially\nresolved (REF)“\nResponse: The reference has been added, apologies for that.\nThe sentence on page 5” This is surprising as VpreB which corresponds to the VL domain of a regular\nLC and isolated VL domains have been shown to be folded in the preB cell receptor“ is misleading as\nthe author later explain that the unassembled VpreB component is lacking a β-strand and thus does\nnot correspond to a VL domain.\nWe thank the reviewer for highlighting this inconsistency. We agree that while VpreB is homologous to\na V domain, it is not equivalent, as it lacks a conserved β-strand and therefore does not correspond to\nL\na complete V domain. We have revised the sentence to clarify this distinction. The revised text (p. 5)\nL\nnow reads:\n“Although VpreB shares homology with the V domain of a conventional light chain, it lacks a\nL\nconserved β-strand and therefore cannot fold independently into a stable Ig-like domain. Importantly,\nconventional V domains cannot replace VpreB in the pre-BCR, showing that VpreB and λ5 together\nL\nprovide unique structural features required for pre-BCR assembly.”\nOn page 6 the authors write ”Interestingly, the sedimentation coefficient of VpreB alone was ~3.2 S,\nwhich is indicative of a non-native homodimer, as described previously“. How can an unfolded protein\nlike VpreB lacking a β-strand form a homodimer? Have the authors done these sedimentation\nexperiment also with the VpreB that contains the β-strand peptide?\nWe thank the reviewer for this comment. One would expect that an unfolded protein should not have\nany defined structure and therefore would be unable to oligomerize. Findings reported in the literature,\nhowever, show that assembly is compatible with disorder\n(https://www.nature.com/articles/nature25762). Of note, we observed a broad peak at approximately\n3.2 S, which roughly corresponds to the dimer–tetramer range of VpreB. Because the peak is very\nbroad rather than sharp, it likely reflects a heterogeneous mixture of oligomeric states of VpreB. An\naggregate would be much larger and therefore would not be visible in this sedimentation region.\nRegarding the suggested experiment with the β-strand peptide, we did not perform AUC\nmeasurements with VpreB in the presence of this peptide due to limited availability. However, our\nNMR data (Fig. 1e) clearly demonstrate that addition of the λ5β peptide induces folding of VpreB,\nsupporting the interpretation that λ5 contributes the missing β-strand required for proper folding.\nWe revised the manuscript to address this point more clearly (p. 6):\n“For isolated VpreB, the sedimentation coefficient was also ~3.2 S; however, the broad profile\ncompared to folded controls suggests that this signal arises from a heterogeneous mixture of\noligomeric species in the range of dimers to tetramers. This interpretation is consistent with previous\nreports describing non-native dimeric or higher-order VpreB assemblies formed under certain in vitro\nconditions 30-32.”\nOn page 6 the authors write” that both, λ5 and VpreB, can interact individually with Fd“. Are these\nbinding experiments done with the unfolded VpreB or the VpreB that contains the β-strand peptide?\nThe binding experiments shown in Fig. 4a were performed in HEK293T cells with the wild-type VpreB\nconstruct, which does not contain the additional β-strand peptide. We have revised the sentence on\npage 6 to clarify this point(p. 8):\n“…that both λ5 and VpreB, when expressed in HEK293T cells, can interact individually with Fd. Thus,\nfor VpreB, the lacking β-strand is not a prerequisite for Fd binding.”\nOn page 6 the authors write our result” strongly suggesting that binding of λ5 to VH is mediated\nexclusively by the λ5 UR“. Is this interaction shown by all the VH domains tested and does the VH\ndomain carry a negatively charged CDR3 region or a sugar moiety that would explain this otherwise\npuzzling interaction?\nWe thank the reviewer for this important question. We tested two VH domains in combination with λ5\nand λ5-ΔUR. In both cases, complex formation was observed with wild-type λ5 but not with λ5-ΔUR,\nindicating that the λ5 UR is required for VH binding. The CDR3 of the VH domains tested contains two\nnegatively charged and one positively charged residue, but no N-linked glycosylation motif. Thus, the\nobserved λ5–VH interaction cannot be attributed to CDR3 glycosylation. We have revised the\nmanuscript text to clarify these points(p. 9):.\n“…for both V domains tested, binding was observed only with wild-type λ5 and not with λ5-ΔUR,\nH\nstrongly suggesting that the λ5 UR mediates this interaction. The CDR3s of the V domains contain\nH\ntwo negatively charged and one positively charged residue which may be involved in mediating this\ninteraction”\nThe section on “The λ5-UR is crucial for antigen interaction“ is lacking a specificity control. Is the\ncreatine kinase binding only seen by the VH MAK33 or also VH1HEZ containing SLC complexes?\nWe appreciate this comment. In the present study, antigen binding to human creatine kinase (muscle\ntype) was assessed only with V MAK33 (Fab-SLC/Fab-LC and V MAK33±SLC components) using a\nH H\nstreptavidin ELISA format with biotinylated creatine kinase immobilized on the plate (Methods, ELISA),\nand we no longer have the antigen reagent available to extend the measurements at this time. We\nhave revised the text to state explicitly that the creatine kinase binding data pertain to V MAK33 and\nH\nthat testing with V 1HEZ remains to be determined in future work.\nReviewer #2 (Remarks to the Author):\nIn this manuscript, König et al. examined the assembly of the pre-B cell receptor in vitro using purified\nproteins and in HEK293 cells by transient transfection. The pre-B cell receptor, whose crystal structure\nwas reported in 2007, comprises a surrogate light chain (SLC) covalently paired with an antibody\nheavy chain. The SLC consists of two non-covalently associated proteins, VpreB and λ5. The authors\nshow that VpreB, which is unfolded in isolation, adopts its native structure upon interaction with 5.\nThis process (β-strand complementation) requires a β-strand from l5 to provide the missing ninth β-\nstrand of VpreB. In addition, folding of CH1 requires interaction with λ5. The biochemical analysis of\nthe assembly process is technically well done and the results largely justify the authors’ conclusions.\nHowever, it is not quite accurate to claim that “little is known about the assembly process of VpreB and\n5” (p. 4, lines 4-5). In fact, quite a lot was already known, as evident from the references cited. For\nexample, β-strand complementation by λ5 was described in refs. 29 and 30. Nevertheless, the present\nwork provides the most complete picture to date of pre-B cell receptor assembly.\nWe thank the reviewer for the positive comments on our study. We changed the sentence about what\nis known on SLC association.\nPoints to address:\np. 5, 3rd paragraph. Individual SLC proteins for biophysical analysis were produced by in vitro\n3\nrefolding from inclusion bodies. CD and NMR analyses indicate that isolated VpreB is unfolded.\nHowever, finding appropriate conditions for in vitro folding of proteins is often not straightforward. That\nis to say, failure to refold a protein in vitro does not necessarily mean that the protein is unfolded when\nproduced naturally within a cell in an environment that includes chaperones, etc. The authors should\ninclude this caveat. Having said that, it is certainly striking that addition of λ5 peptide to isolated\nVpreB induces folding of VpreB, as indicated by NMR.\nAs suggested by the reviewer, we have revised the paragraph on page 5 to clarify the properties of\nVpreB and to state more clearly its dependence on λ5 or the λ5β peptide for folding (p.5):\n“The far-UV CD spectrum of λ5 (Fig. 1d) revealed that the isolated protein is characterized by β-\nstrands and unfolded segments consistent with its structure in the SLC complex 21 and the presence of\nan unstructured extension. In contrast to the structure of VpreB in the SLC complex, the far-UV CD\nspectrum of isolated VpreB (Fig. 1d) indicated that the protein is largely unfolded under the conditions\ntested. In agreement with this notion, NMR spectroscopy revealed that the 15N-1H HSQC spectrum of\nisolated VpreB is characteristic of an unfolded protein (Fig. 1e, red spectrum).”\nAnd in the discussion, we added (p. 11):\n“Although the in vitro experiments demonstrate that VpreB is unfolded due to the lacking ß6 strand, its\nfolding state in the cell may be further affected by molecular chaperones until the interaction with λ5\noccurs.”\nFig. 1c. Figure legend should include Hsc70 and explain that it is a positive control. In medium, there\nare two bands for VpreB in the λ5 + VpreB lane. What is the lower MW band, which does not appear\n(or just barely) in the VpreB alone lane? Is it another glycoform of VpreB?\nWe thank the reviewer for this observation. We have revised the legend of Fig. 1c to include Hsc70\nand to clarify its role as a control for lysed cells. Regarding the two VpreB bands observed in the λ5 +\nVpreB lane of the medium fraction, the lower molecular weight band most likely corresponds to a non-\nglycosylated form of VpreB, as also shown in Supplementary Fig. 1a in deglycosylation experiments.\nThe revised legend now reads (p. 25):\n“Expression and secretion of V5-tagged λ5, FLAG-tagged VpreB, and both proteins together in\nHEK293T cells. Hsc70 was included as a positive control for lysed cells. In the λ5 + VpreB lane of the\nmedium fraction, two bands are observed for VpreB. The lower molecular weight band likely\nrepresents a non-glycosylated VpreB (see Supplementary Fig. 1a)”\nFig. 2d. Figure legend is incomplete. How exactly were the SPR measurements done? What was\nimmobilized? What was the analyte? Are the concentrations for the λ5β peptide? The description of\nSPR in Materials and Methods does not answer these specific questions.\nWe thank the reviewer for pointing this out. We have added a description of the experiment in the\nMethods section.\nRevised Method (p. 20 - 21):\nFor the SPR experiments monitoring binding between λ5 and VpreB variants, VpreB was immobilized\non a CM5 sensor chip, and purified λ5 was injected as analyte at concentrations ranging from 0.002 -\n4 µM. For the SPR experiments between CH1 and SLC variants, CH1 was immobilized on a CM5\nsensor chip, and purified SLC variants were injected as analytes at concentrations ranging from 0.002\nto 50 µM, depending on the variant used. For the SPR experiments monitoring binding between the\nVH domain and SLC variants, VH 1HEZ or VH MAK33 was immobilized on a CM5 sensor chip via\namine coupling, and purified VpreB, λ5, or the SLC were injected as analytes at concentrations\nranging from 0.003 – 10 µM, depending on the variant used.\nBinding curves were corrected by subtraction of buffer and reference flow cell signals (binding to\ncontrol flow cells and running buffer) and fitted to a 1:1 binding model. Sensorgrams were analyzed\nusing the Biacore X100 Evaluation Software (Cytiva Sweden AB, Uppsala, Sweden) to obtain the\nequilibrium dissociation constant (KD), association rate constant (ka), and dissociation rate constant\n(kd).\n4\np. 6, 3rd paragraph. In AUC, isolated VpreB appears to sediment as a homodimer. How is this\npossible if isolated VpreB is unfolded? In looking at the sedimentation profile of isolated VpreB in Fig.\n2f, the profile is very broad compared to those of the other samples, which we know are folded. Very\nlikely the broad VpreB profile reflects non-specific aggregation rather than discrete dimerization. The\nauthors should revise their text accordingly.\nThis point has also been addressed in our response to Reviewer 1 (see response to Reviewer 1,\ncomment 5). In short: indeed, this is puzzling, however, the assembly is compatible with\ndisorder(https://www.nature.com/articles/nature25762). We have revised the manuscript to address\nthis more clearly (p. 6):\n“For isolated VpreB, the sedimentation coefficient was also ~3.2 S; however, the broad profile\ncompared to folded controls suggests that this signal arises from a heterogeneous mixture of\noligomeric species in the range of dimers to tetramers. This interpretation is consistent with previous\nreports describing non-native dimeric or higher-order VpreB assemblies formed under certain in vitro\nconditions30-32.”\nFig. 3g. Figure legend should explain labeling of samples. Presumably, λ5DU means λ5 with URL\ndeleted and VpreBDU means VpreB with UR deleted. Also, for comparison of intensities, the\nimmunoblots should include 5 and VpreB with URs intact.\nWe thank the reviewer for this suggestion. We have clarified the meaning of the abbreviations in the\nlegend and added a reference to the wild-type controls, which are shown in Supplementary Fig. 3a (p.\n26):\n“Expression and secretion of murine Fd-MAK33 with ΔUR variants of λ5 and VpreB in HEK293T cells.\nλ5DU denotes λ5 with its UR deleted, and VpreBDU denotes VpreB with its UR deleted. For\ncomparison, the expression and secretion of murine Fd-MAK33 alone and in combination with wild-\ntype λ5, VpreB, and the complete SLC are shown in Supplementary Fig. 3a.”\nFig. 4b. Figure legend does not adequately describe immunoprecipitations in the two panels.\nWe agree and have expanded the legend to describe the input and immunoprecipitation panels in\nmore detail (Fig. 4b, p.26):\n“Immunoprecipitation of murine Fd-MAK33 with λ5, VpreB and the SLC in HEK293T lysates. Left:\nInput samples showing expression of Fd-MAK33-HA, λ5-V5, and VpreB-FLAG, detected by the\nindicated antibodies. Hsc70 served as a loading and secretion control. Right: Immunoprecipitation with\nan anti-HA antibody to isolate Fd-MAK33-HA, followed by immunoblotting for HA, V5, and FLAG to\nassess co-immunoprecipitation of λ5 and VpreB.”\nFig. 5c-e. Same comments about SPR as above for Fig. 2d.\nResponse:\nWe thank the reviewer for pointing this out. This issue has been addressed in our response to the\nrelated comment above. In short, the method description for SPR was extended.\np. 9, 3rd paragraph. Results of ELISA assays should be confirmed by SPR using immobilized creatine\nkinase. The authors have all necessary reagents to make these measurements.\nWe thank the reviewer for this valuable suggestion. We attempted to perform SPR measurements\nusing immobilized creatine kinase to confirm the ELISA results. However, despite multiple optimization\nattempts, including varying immobilization strategies, buffer conditions, and protein concentrations, we\nwere unable to obtain stable and reproducible binding signals. We suspect that this may be due to a\nloss of creatine kinase activity upon immobilization. Therefore, we were not able to include SPR data\non CK binding in the present manuscript.\nMinor:\nOn p. 2, 1st line, (REF) should give reference number.\nIn Fig. 3 legend, panels b and e should be panels b and d.\nThanks for detecting this! Both minor points have been addressed. The reference number has been\nadded and the panel labelling has been changed\n6"}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have now familiarised them with and cite the existing literature concerning the pre-BCR structure and function.\nThey also have now addressed most of my comments to improve their MS", "reviewer_2": "The authors have responded satisfactorily to the previous critiques."}, "author_response": "We thank reviewer for their positive and constructive assessment of the revised manuscript. We are\ngrateful for the acknowledgement that the relevant literature concerning pre-BCR structure and function\nhas now been appropriately incorporated and that the previous comments have been addressed.\nWe thank reviewer for their positive evaluation and for confirming that the responses to the previous\ncritiques were satisfactory."}]

Decision letter