Domain Interactions Determine the Amyloidogenicity of Antibody Light Chain Mutants

Weber, Benedikt; Hora, Manuel; Kazman, Pamina; Pradhan, Tejaswini; Rührnößl, Florian; Reif, Bernd; Büchner, Johannes

doi:10.1016/j.jmb.2020.10.005

Cited by 15 publications

(14 citation statements)

References 37 publications

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“…In fact, it is known that the salt bridge R60-D81 is crucial for protein stability. The mutated V L protein R60A has a dramatically reduced stability and readily forms brils (Nokwe et al, 2016;Weber et al, 2020). This is re ected as well in our MD simulations (Fig.…”

Section: Discussionsupporting

confidence: 83%

“…We assume that competition for the salt bridge R60-D80/E81 by R49 results in a destabilization of the protein. In fact, it has been shown experimentally that the mutation R60A has a dramatic impact on the bril formation kinetics and the thermodynamic stability of the V L domain (Weber et al, 2020). For the patient protein FOR005, high RMSF uctuations are found in particular for CDR-3 (residues 89-95).…”

Section: S5mentioning

confidence: 99%

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Mechanistic insight into the aggregation pathway of an immunoglobulin light chain protein

Reif¹,

Pradhan²,

Sarkar

et al. 2022

Preprint

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Systemic antibody light chains (AL) amyloidosis is characterized by deposition of amyloid fibrils derived from a particular antibody light chain protein. In addition, soluble oligomeric AL protein has a direct cytotoxic effect on cardiomyocytes prior to organ malfunction and amyloid formation. The structure of these oligomers and the mechanism of toxicity is, however, not understood. Using solution-state NMR spectroscopy, we are able to follow the individual steps involved in protein misfolding from the native state to the amyloid fibril structure at atomic resolution for the first time. We show that unfavorable mutations in the complementary determining regions introduce a strain in the protein structure which induces partial unfolding of the native state. Subsequently, the protein converts into a high molecular weight molten globule like structure. We demonstrate that this transition is driven by electrostatic interactions. The high local concentration of aggregation prone regions in the oligomer finally catalyzes the conversion into amyloid fibrils. We find that the topology of the aggregated state is determined by balanced electrostatic interactions in the core of the fibril, resulting in an anti-parallel arrangement of the beta-sheets around the conserved disulfide bond.

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Section: Discussionsupporting

confidence: 83%

Section: S5mentioning

confidence: 99%

Mechanistic insight into the aggregation pathway of an immunoglobulin light chain protein

Reif¹,

Pradhan²,

Sarkar

et al. 2022

Preprint

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“…The general view is that CL domains exert a stabilizing effect on VL domains [ 19 , 31 , 33 , 34 ]. Nevertheless, recent data indicate that full‐length LCs with destabilized CL domains or nonoptimal interdomain interactions are highly amyloidogenic [ 31 , 35 , 36 ]. It is possible that initial proteolytic events on exposed loops (aa 120–131 and 181–190) destabilize the CL domain, which in turn renders the whole native LC more aggressively amyloidogenic.…”

Section: Discussionmentioning

confidence: 99%

Protease‐sensitive regions in amyloid light chains: what a common pattern of fragmentation across organs suggests about aggregation

et al. 2021

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Light-chain (AL) amyloidosis is characterized by deposition of immunoglobulin light chains (LC) as fibrils in target organs. Alongside the full-length protein, abundant LC fragments are always present in AL deposits. Herein, by combining gel-based and mass spectrometry analyses, we identified and compared the fragmentation sites of amyloid LCs from multiple organs of an AL k amyloidosis patient (AL-55). The positions pinpointed here in kidney and subcutaneous fat, alongside those previously detected in heart of the same patient, were aligned and mapped on the LC's dimeric and fibrillar states. All tissues contain fragmented LCs along with the full-length protein; the fragment pattern is coincident across organs, although microheterogeneity exists. Multiple cleavage positions were detected; some are shared, whereas some are organ-specific, likely due to a complex of proteases. Cleavage sites are concentrated in 'proteolysisprone' regions, common to all tissues. Several proteolytic sites are not accessible on native dimers, while they are compatible with fibrils. Overall, data suggest that the heterogeneous ensemble of LC fragments originates in tissues and is consistent with digestion of preformed fibrils, or with the hypothesis that initial proteolytic cleavage of the constant domain triggers the amyloidogenic potential of LCs, followed by subsequent proteolytic degradation. This work provides a unique set of molecular data on proteolysis from ex vivo amyloid, which allows discussing hypotheses on role and timing of proteolytic events occurring along amyloid formation and accumulation in AL patients. Abbreviations 2D-PAGE, two-dimensional polyacrylamide gel electrophoresis; ACN, acetonitrile; AL amyloidosis, immunoglobulin light-chain amyloidosis; CL, light chain's constant domain; Cryo-EM, cryo-electron microscopy; DTT, dithiothreitol; EA, ethanolamine; FA, formic acid; HCD, higher energy collisional dissociation; IEF, isoelectrofocusing; LCs, immunoglobulin light chains (note, LC in the standard hyphenated abbreviation LC-MS/MS indicates liquid chromatography); PTM, post-translational modifications; VL, light chain's variable domain.

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“…Growing experimental evidence indicates that specific biochemical properties play a relevant role or at least strongly correlate with LCs amyloidogenicity. Thermodynamic and kinetic stability are lower in amyloidogenic LCs compared to control non-amyloidogenic LCs, whereas protein flexibility and dynamics are typically increased in AL LCs compared to non-AL ones [15,[23][24][25][26][27][28][29][30][31][32][33][34][35]. Specific LC germlines are overrepresented among AL sequences, and recent algorithms tend to predict more accurately the AL propensity of LC sequences [36].…”

Section: Introductionmentioning

confidence: 99%

“…The four available Cryo-EM structures of the ex vivo fibrils from AL patients show independent assemblies [37][38][39][40]. The ex vivo amyloid deposits show a large group of proteoforms caused by proteolytic events [41][42][43]; whether such proteolysis is a trigger for LCs aggregation or is the result of post-aggregation processes is still under debate [30,31,43,44].…”

Section: Introductionmentioning

confidence: 99%

Cu(II) Binding Increases the Soluble Toxicity of Amyloidogenic Light Chains

Russo

Romeo

Schulte

et al. 2022

IJMS

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Light chain amyloidosis (AL) is caused by the aberrant overproduction of immunoglobulin light chains (LCs). The resulting abnormally high LC concentrations in blood lead to deposit formation in the heart and other target organs. Organ damage is caused not only by the accumulation of bulky amyloid deposits, but extensive clinical data indicate that circulating soluble LCs also exert cardiotoxic effects. The nematode C. elegans has been validated to recapitulate LC soluble toxicity in vivo, and in such a model a role for copper ions in increasing LC soluble toxicity has been reported. Here, we applied microscale thermophoresis, isothermal calorimetry and thermal melting to demonstrate the specific binding of Cu2+ to the variable domain of amyloidogenic H7 with a sub-micromolar affinity. Histidine residues present in the LC sequence are not involved in the binding, and yet their mutation to Ala reduces the soluble toxicity of H7. Copper ions bind to and destabilize the variable domains and induce a limited stabilization in this domain. In summary, the data reported here, elucidate the biochemical bases of the Cu2+-induced toxicity; moreover, they also show that copper binding is just one of the several biochemical traits contributing to LC soluble in vivo toxicity.

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Domain Interactions Determine the Amyloidogenicity of Antibody Light Chain Mutants

Cited by 15 publications

References 37 publications

Mechanistic insight into the aggregation pathway of an immunoglobulin light chain protein

Mechanistic insight into the aggregation pathway of an immunoglobulin light chain protein

Protease‐sensitive regions in amyloid light chains: what a common pattern of fragmentation across organs suggests about aggregation

Cu(II) Binding Increases the Soluble Toxicity of Amyloidogenic Light Chains

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