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MULTIPLE MYELOMA, GAMMOPATHIES

An N-glycosylation hotspot in immunoglobulin κ light chains is associated with AL amyloidosis

Abstract

Immunoglobulin light chain (AL) amyloidosis is caused by a small, minimally proliferating B-cell/plasma-cell clone secreting a patient-unique, aggregation-prone, toxic light chain (LC). The pathogenicity of LCs is encrypted in their sequence, yet molecular determinants of amyloidogenesis are poorly understood. Higher rates of N-glycosylation among clonal κ LCs from patients with AL amyloidosis compared to other monoclonal gammopathies indicate that this post-translational modification is associated with a higher risk of developing AL amyloidosis. Here, we exploited LC sequence information from previously published amyloidogenic and control clonal LCs and from a series of 220 patients with AL amyloidosis or multiple myeloma followed at our Institutions to define sequence and spatial features of N-glycosylation, combining bioinformatics, biochemical, proteomics, structural and genetic analyses. We found peculiar sequence and spatial pattern of N-glycosylation in amyloidogenic κ LCs, with most of the N-glycosylation sites laying in the framework region 3, particularly within the E strand, and consisting mainly of the NFT sequon, setting them apart with respect to non-amyloidogenic clonal LCs. Our data further support a potential role of N-glycosylation in determining the pathogenic behavior of a subset of amyloidogenic LCs and may help refine current N-glycosylation-based prognostic assessments for patients with monoclonal gammopathies.

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Fig. 1: Sequence and spatial features of predicted N-glycosylation of amyloidogenic and non-amyloidogenic clonal κ light chains.
Fig. 2: Biochemical and proteomics confirmation of N-glycosylation prediction in amyloidogenic κ light chains.
Fig. 3: Genomic versus somatic origin of the predicted N-glycosylation site in amyloidogenic κ light chains.
Fig. 4: Secondary structure analysis and structural mapping of N-glycosylation sites in amyloidogenic κ light chains.
Fig. 5: Prognostic significance of N-glycosylation site mapping within clonal κ light chains.

Data availability

The data generated in this study are available within the article and its Supplementary Data Files. LC sequences have been deposited to GenBank (MZ595009-MZ595094, OM885091-OM885224).

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Acknowledgements

We thank Andrea Patrignani and staff of the Functional Genomic Center Zurich for PacBio library preparation and sequencing. This work was supported by grants from the Amyloidosis Foundation (MNu), the Italian Ministry of Health (Ricerca Finalizzata, grant #GR-2018-12368387) (MNu), the Italian Ministry of Research and Education (PRIN 20207XLJB2) (SR, GP), the CARIPLO Foundation (grant #2018-0257) (MNu), Fondazione ARISLA (project TDP-43-STRUCT) (SR), Cancer Research UK [C355/A26819], FC AECC and AIRC under the Accelerator Award Program (BP, GM, GP, MNu). We would like to acknowledge the use of the Boston University AL-Base, supported by HL68705, in this work.

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Contributions

Conceived, designed, and supervised the project: MNu. Provided financial support to the project: SR, BP, GMe, GP, MNu. Performed clinical evaluations, provided patients’ samples and/or collected clinical data: SM, BP, PM, CCS, PB, IG, FF, MB, AF, MNa, MTP, LA, GMe, GP, MNu. Maintained biorepository: AN, MG, PC, SCam, MAS, SCas. Curated clinical database: AN, PB, FF, MNu. Retrieved published sequences and performed in silico analyses: AN, MG, PC, MP, PR, MNu. Processed bone-marrow samples, prepared sequencing libraries and analyzed LC sequences: AN, MG, PC, MP, MNu. Performed PNGase F digestion and Western blotting: MG, PC, MP. Performed mass spectrometry analyses: GMa, SCam, FL. Performed genomic analyses and Sanger sequencing: AN, MG, PC, MP, SCas. Performed molecular modeling and structural analyses: VS, SR. Performed statistical analyses: PPO. Prepared figures: AN, PC, MP, VS, SR, MNu. Wrote the paper: AN, MNu. Read, edited and approved the paper: All authors.

Corresponding authors

Correspondence to Giovanni Palladini or Mario Nuvolone.

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Competing interests

PC, GP, and MNu are inventors on a patent application related to immunoglobulin sequencing.

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Clinical records and biological samples were from subjects referred to the Italian Amyloid Center or to the Department of Hematology, Fondazione IRCCS Policlinico San Matteo, Pavia, Italy and to the University Hospital of Navarra, Pamplona, Spain for a diagnostic workout in the suspicion of systemic AL amyloidosis or MM. Per the Declaration of Helsinki, all patients gave their written informed consent for the use of their clinical data and biological samples for research purposes, and this study was approved by the local Institutional Review Board.

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Nevone, A., Girelli, M., Mangiacavalli, S. et al. An N-glycosylation hotspot in immunoglobulin κ light chains is associated with AL amyloidosis. Leukemia (2022). https://doi.org/10.1038/s41375-022-01599-w

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