Influence of a Single Point Mutation in the Constant Domain of the Bence-Jones Protein BIF on Its Aggregation Properties
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Multiple myeloma nephropathy occurs due to the aggregate formation by monoclonal immunoglobulin light chains (Bence-Jones proteins) in kidneys of patients with multiple myeloma. The mechanism of amyloid deposit formation is still unclear. Earlier, the key role in the fibril formation has been assigned to the variable domains that acquired amyloidogenic properties as a result of somatic mutations. However, fibril formation by the Bence-Jones protein BIF was found to be the function of its constant domain. The substitution of Ser177 by Asn in the constant domain of the BIF protein is most likely an inherited than a somatic mutation. To study the role of this mutation in amyloidogenesis, the recombinant Bence-Jones protein BIF and its mutant with the N177S substitution typical for the known immunoglobulin Cκ allotypes Km1, Km1,2, and Km3 were isolated. The morphology of aggregates formed by the recombinant proteins under conditions similar to those occurring during the protein transport in bloodstream and its filtration into the renal glomerulus, in the distal tubules, and in the proximal renal tubules was analyzed by atomic force microscopy. The nature of the aggregates formed by BIF and its N177S mutant during incubation for 14 days at 37°C strongly differed and depended on both pH and the presence of a reducing agent. BIF formed fibrils at pH 7.2, 6.5, and 10.1, while the N177S mutant formed fibrils only at alkaline pH 10.1. The refolding of both proteins in the presence of 5 mM dithiothreitol resulted in the formation of branched structures.
KeywordsBence-Jones proteins myeloma aggregation atomic force microscopy
atomic force microscopy
- BJ myeloma
Bence-Jones proteins (Ig light chains)
constant domain of Ig light chain
variable domain of Ig light chain.
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- 4.Mukherjee, S., Pondaven, S. P., and Jaroniec, C. P. (2011) Conformational flexibility of a human immunoglobulin light chain variable domain by relaxation dispersion nuclear magnetic resonance spectroscopy: implications for protein misfolding and amyloid assembly, Biochemistry, 50, 5845–5857.CrossRefPubMedGoogle Scholar
- 5.Wilkins-Stevens, P., Raffen, R., Hanson, D. K., Deng, Y. L., Berrios-Hammond, M., Westholm, F. A., Murphy, C., Eulitz, M., Wetzel, R., Solomon, A., Schiffer, M., and Stevens, F. J. (1995) Recombinant immunoglobulin variable domains generated from synthetic genes provide a system for in vitro characterization of light-chain amyloid proteins, Protein Sci., 4, 421–432.CrossRefGoogle Scholar
- 6.Maniatis, T., Fritisch, E. F., and Sambrok, J. (1982) in Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, N. Y.Google Scholar
- 8.Dubnovitsky, A. P., Kravchuk, Z. I., Chumanevich, A. A., Cozzi, A., Arosio, P., and Martsev, S. P. (2000) Expression, refolding, and ferritin-binding activity of the isolated VL-domain of monoclonal antibody F11, Biochemistry (Moscow), 65, 1011–1018.Google Scholar
- 14.Timchenko, A., Timchenko, M., Shinjo, M., and Kihara, H. (2015) SAXS study of N177S mutant of Bence-Jones protein BIF, Photon Factory Activity Report, 2014, 32, 366.Google Scholar
- 17.Myatt, E. A., Westholm, F. A., Weiss, D. T., Solomon, A., Schiffer, M., and Stevens, F. J. (1994) Pathogenic potential of human monoclonal immunoglobulin light chains: relationship of in vitro aggregation to in vivo organ deposition, Proc. Natl. Acad. Sci. USA, 91, 3034–3038.CrossRefPubMedPubMedCentralGoogle Scholar
- 20.Davis, D. P., Gallo, G., Vogen, S. M., Dul, J. L., Sciarretta, K. L., Kumar, A., Raffen, R., Stevens, F. J., and Argon, Y. (2001) Both the environment and somatic mutations govern the aggregation pathway of pathogenic immunoglobulin light chain, J. Mol. Biol., 313, 1021–1034.CrossRefPubMedGoogle Scholar