Abstract
Binding of the surrogate light chain (SLC) to the heavy chain (HC) of the pre-B cell receptor (preBCR) 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 affinit…
Abstract
Binding of the surrogate light chain (SLC) to the heavy chain (HC) of the pre-B cell receptor (preBCR) 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.
Data availability
All data were contained within the main article and Supplementary Information. The source data behind all figures and Supplementary Figs. are included in the source data. Further information can be obtained from the authors upon reasonable request. All data were analyzed using commercially available software as described in the Methods section. Custom scripts, if any, were used only for figure generation and not for data processing or statistical analysis.2H32 [https://www.rcsb.org/structure/2H32]. 1HEZ. MAK33 [https://www.rcsb.org/structure/1FH5]. Source data are provided with this paper.
References
Melchers, F. Checkpoints that control B cell development. J. Clin. Invest. 125, 2203–2210 (2015).
Tonegawa, S. Somatic generation of antibody diversity. Nature 302, 575–581 (1983).
Vettermann, C., Herrmann, K. & Jäck, H.-M. Powered by pairing: the surrogate light chain amplifies immunoglobulin heavy chain signaling and pre-selects the antibody repertoire. Semin. Immunol. 18, 44–55 (2006).
Burrows, P. D., Lejeune, M. & Kearney, J. F. Evidence that murine pre-B cells synthesize mu heavy chains but no light chains. Nature 280, 838–840 (1979).
Rolink, A. & Melchers, F. Molecular and cellular origins of B lymphocyte diversity. Cell 66, 1081–1094 (1991).
Kawano, Y., Yoshikawa, S., Minegishi, Y. & Karasuyama, H. Pre-B cell receptor assesses the quality of IgH chains and tunes the pre-B cell repertoire by delivering differential signals. J. Immunol. 177, 2242–2249 (2006).
Kline, G. H. et al. Pre-B cell receptor-mediated selection of pre-B cells synthesizing functional µ heavy chains. J. Immunol. 161, 1608–1618 (1998).
Cherayil, B. J. & Pillai, S. The w/l5 surrogate immunoglobulin light chain is expressed on the surface of transitional B lymphocytes in murine bone marrow. J. Exp. Med. 173, 111–116 (1991).
Pillai, S. & Baltimore, D. Formation of disulphide-linked µ2 w2 tetramers in pre-B cells by the 18K w-immunoglobulin light chain. Nature 329, 172–174 (1987).
Wang, Y.-H., Nomura, J., Faye-Petersen, O. M. & Cooper, M. D. Surrogate light chain production during B cell differentiation. J. Immunol. 161, 1132–1139 (1998).
Reth, M. G., Petrac, E., Wiese, P., Lobel, L. & Alt, F. W. Activation of Vk gene rearrangement in pre-B cells follows the expression of membrane-bound immunoglobulin heavy chains. EMBO J. 6, 3299–3305 (1987).
Guelpa-Fonlupt, V., Tonnelle, C., Blaise, D., Fougereau, M. & Fumoux, F. Discrete early pro-B and pre-B stages in normal human bone marrow as defined by surface pseudo-light chain expression. Eur. J. Immunol. 24, 257–264 (1994).
Schlissel, M. S. & Morrow, T. Ig heavy chain protein controls B cell development by regulating germ-line transcription and retargeting V(D)J recombination. J. Immunol. 153, 1645–1657 (1994).
Brouns, G. S., de Vries, E., Neefjes, J. J. & Borst, J. Assembled pre-B cell receptor complexes are retained in the endoplasmic reticulum by a mechanism that is not selective for the pseudo-light chain. J. Biol. 271, 19272–19278 (1996).
Wang, Y.-H. et al. Differential surrogate light chain expression governs B-cell differentiation. Blood 99, 2459–2467 (2002).
Mundt, C., Licence, S., Shimizu, T., Melchers, F. & Martensson, I.-L. Loss of precursor B cell expansion but not allelic exclusion in VpreB1/VpreB2 double-deficient mice. J. Exp. Med. 193, 435–445 (2001).
Kudo, A. et al. The expression of the mouse VpreB/l5 locus in transformed cell lines and tumors of the B lineage differentiation pathway. Int. Immunol. 4, 831–840 (1992).
Melchers, F. et al. Repertoire selection by pre-B-cell receptors and B-cell receptors, and genetic control of B-cell development from immature to mature B cells. Immunol. Rev. 175, 33–46 (2000).
Melchers, F. Fit for life in the immune system? Surrogate L chain tests H chains that test L chains. Proc. Natl. Acad. Sci. USA 96, 2571–2573 (1999).
Lanig, H., Bradl, H. & Jäck, H.-M. Three-dimensional modeling of a pre-B-cell receptor. Mol. Immunol. 40, 1263–1272 (2004). 1.
Bankovich, A. J. et al. Structural insight into pre-B cell receptor function. Science 316, 291–294 (2007).
Guelpa-Fonlupt, V. et al. The human pre-B cell receptor: structural constraints for a tentative model of the pseudo-light (YL) chain. Mol. Immunol. 31, 1099–1108 (1994).
Melchers, F. et al. The surrogate light chain in B-cell development. Immunol. Today 14, 60–68 (1993).
Karasuyama, H., Kudo, A. & Melchers, F. The proteins encoded by the VpreB and l5 pre-B cell-specific genes can associate with each other and with m heavy chain. J. Exp. Med. 172, 969–972 (1990). 1.
Tsubata, T. & Reth, M. G. The products of pre-B cell-specific genes (l5 and VpreB) and the immunoglobulin µ chain form a complex that is transported onto the cell surface. J. Exp. Med. 172, 973–976 (1990).
Bossy, D., Salamero, J., Olive, D., Fougereau, M. & Schiff, C. Structure, biosynthesis, and transduction properties of the human µ-fL complex: similar behavior of preB and intermediate preB - B cells in transducing ability. Int. Immunol. 5, 467–478 (1993).
Schiff, C. et al. l-Like and V pre-B genes expression: an early B-lineage marker of human leukemias. Blood 78, 1516–1526 (1991).
Jasper, P. J., Zhai, S.-K., Kalis, S. L., Kingzette, M. & Knight, K. L. B lymphocyte development in rabbit: progenitor B cells and waning of B lymphopoiesis. J. Immunol. 171, 6372–6380 (2003).
Minegishi, Y., Hendershot, L. M. & Conley, M. E. Novel mechanisms control the folding and assembly of l5/14.1 and VpreB to produce an intact surrogate light chain. Proc. Natl. Acad. Sci. USA 96, 3041–3046 (1999).
Morstadt, L. et al. Engineering and characterization of a single chain surrogate light chain variable domain. Protein Sci. 17, 458–465 (2008).
Gauthier, L., Lemmers, B., Guelpa-Fonlupt, V., Fougereau, M. & Schiff, C. -Surrogate light chain physicochemical interactions of the human preB cell receptor: implications for VH repertoire selection and cell signaling at the preB cell stage. J. Immunol. 162, 41–50 (1999).
Hirabayashi, Y., Lecerf, J.-M., Dong, Z. & Stollar, B. D. Kinetic analysis of the interactions of recombinant human VpreB and Ig V domains. J. Immunol. 155, 1218–1228 (1995).
Hendershot, L. M., Bole, D. G., Köhler, G. & Kearney, J. F. Assembly and secretion of heavy chains that do not associate posttranslationally with immunoglobulin heavy chain-binding protein. J. Cell Biol. 104, 761–767 (1987).
Feige, M. J. et al. An unfolded CH1 domain controls the assembly and secretion of IgG antibodies. Mol. Cell 34, 569–579 (2009).
Übelhart, R. et al. N-linked glycosylation selectively regulates autonomous precursor BCR function. Nat. Immunol. 11, 759–765 (2010).
Buchner, J. & Rudolph, R. Renaturation, purification and characterization of recombinant Fab-fragments produced in Escherichia coli. Biotechnology 9, 157–162 (1991).
Feige, M. J., Hendershot, L. M. & Buchner, J. How antibodies fold. Trends Biochem. Sci. 35, 189–198 (2010).
Hildenbrand, K., Aschenbrenner, I., Franke, F. C., Devergne, O. & Feige, M. J. Biogenesis and engineering of interleukin 12 family cytokines. Trends Biochem. Sci. 47, 936–949 (2022).
Gauthier, L., Rossi, B., Roux, F., Termine, E. & Schiff, C. Galectin-1 is a stromal cell ligand of the pre-B cell receptor (BCR) implicated in synapse formation between pre-B and stromal cells and in pre-BCR triggering. Proc. Natl. Acad. Sci. USA 99, 13014–13019 (2002).
Fang, T., Smith, B. P. & Roman, C. A. J. Conventional and surrogate light chain differentially regulate Ig µ and Dµ heavy chain maturation and surface expression. J. Immunol. 167, 3846–3857 (2001).
Ohnishi, K. & Melchers, F. The nonimmunoglobulin portion of l5 mediates cell-autonomous pre-B cell receptor signaling. Nat. Immunol. 4, 849–856 (2003).
Knoll, M. et al. The non-Ig parts of the VpreB and λ5 proteins of the surrogate light chain play opposite roles in the surface representation of the precursor B cell receptor. J. Immunol. 188, 6010–6017 (2012).
Schneider, M. et al. BiPPred: combined sequence- and structure-based prediction of peptide binding to the Hsp70 chaperone BiP. Proteins 84, 1390–1407 (2016).
Rossi, B., Espeli, M., Schiff, C. & Gauthier, L. Clustering of pre-B cell integrins induces galectin-1-dependent pre-B cell receptor relocalization and activation. J. Immunol. 177, 796–803 (2006).
Haslbeck, M., Weinkauf, S. & Buchner, J. Small heat shock proteins: simplicity meets complexity. J. Biol. Chem. 294, 2121–2132 (2018).
Janowska, M. K., Baughman, H. E. R., Woods, C. N. & Klevit, R. E. Mechanisms of small heat shock proteins. Cold Spring Harb. Perspect. Biol. 11, a034025 (2019).
Jeong, J.-Y. et al. One-step sequence- and ligation-independent cloning as a rapid and versatile cloning method for functional genomics studies. Appl. Environ. Microbiol. 78, 5440 (2012).
Li, M. Z. & Elledge, S. J. SLIC: a method for sequence- and ligation-independent cloning. Methods Mol. Biol. 852, 51–59 (2012). 1.
Brown, P. H. & Schuck, P. Macromolecular size-and-shape distributions by sedimentation velocity analytical ultracentrifugation. Biophys. J. 90, 4651–4661 (2006).
Schuck, P. Size-distribution analysis of macromolecules by sedimentation velocity ultracentrifugation and Lamm equation modeling. Biophys. J. 78, 1606–1619 (2000).
Lau, A. M., Claesen, J., Hansen, K. & Politis, A. Deuteros 2.0: peptide-level significance testing of data from hydrogen deuterium exchange mass spectrometer. Bioinformatics 37, 270–272 (2020).
Acknowledgements
This work was supported by grants from the Deutsche Forschungsgemeinschaft to J.B. (BU 836/6-3) and M.J.F. (B11, SFB 1035). We thank Liane Gretschel and Daniel Weinfurtner (Roche Diagnostics, Penzberg) for providing CK-MM.
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Natalia Catalina Sarmiento Alam
Present address: Sanofi, Technologiepark 21 9052 Ghent/zwijnaarde, Ghent, Belgium
Authors and Affiliations
Department Bioscience, School of Natural Sciences, Technical University Munich, Garching, Germany
Jasmin König, Natalia Catalina Sarmiento Alam, Ruiming He, Nicolas Blömeke, Olga Sieluzycka, Florian Rührnößl, Maximilian Riedl, Bernd Reif, Matthias J. Feige & Johannes Buchner 1.
Center for Functional Protein Assemblies, Technical University Munich, Ernst-Otto-Fischer-Strasse 8, Garching, Germany
Jasmin König, Natalia Catalina Sarmiento Alam, Ruiming He, Nicolas Blömeke, Florian Rührnößl, Maximilian Riedl, Matthias J. Feige & Johannes Buchner 1.
Institute of Structural Biology (STB), Helmholtz-Zentrum München (HMGU), Ingolstädter Landstr. 1, Neuherberg, Germany
Bernd Reif
Authors
- Jasmin König
- Natalia Catalina Sarmiento Alam
- Ruiming He
- Nicolas Blömeke
- Olga Sieluzycka
- Florian Rührnößl
- Maximilian Riedl
- Bernd Reif
- Matthias J. Feige
- Johannes Buchner
Contributions
Experiments were designed and performed by J.K., N.C.S.A., N.B., O.S., F.R., and M.R. The manuscript was written by J.B., J.K., M.J.F., R.H., and B.R. Figures were prepared by J.K., R.H., O.S., B.R., M.J.F., and J.B.
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Correspondence to Johannes Buchner.
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König, J., Sarmiento Alam, N.C., He, R. et al. Association-induced folding governs surrogate light chain and pre-B cell receptor core assembly. Nat Commun (2026). https://doi.org/10.1038/s41467-026-68965-5
Received: 21 December 2023
Accepted: 20 January 2026
Published: 30 January 2026
DOI: https://doi.org/10.1038/s41467-026-68965-5