Dissecting the Alternatively Folded State of the Antibody Fab Fragment

Feige, Matthias J.; Simpson, Emma R.; Herold, Eva Maria; Bepperling, Alexander; Heger, Klaus; Büchner, Johannes

doi:10.1016/j.jmb.2010.04.032

Cited by 20 publications

(31 citation statements)

References 42 publications

(71 reference statements)

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“…The mean diameter of the particles was 9.9 ± 1.5 nm, suggesting in average 30 monomers per oligomer (S4 Fig). This is in excellent agreement with previous studies using analytical ultracentrifugation, which yielded 17 to 42 monomers per oligomer [22]. Upon shaking at 37°C, the oligomers transform to well-structured fibrils within 24 hours (Fig 3C).…”

Section: Resultssupporting

confidence: 92%

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MAK33 antibody light chain amyloid fibrils are similar to oligomeric precursors

et al. 2017

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Little structural information is available so far on amyloid fibrils consisting of immunoglobulin light chains. It is not understood which features of the primary sequence of the protein result in fibril formation. We report here MAS solid-state NMR studies to identify the structured core of κ-type variable domain light chain fibrils. The core contains residues of the CDR2 and the β-strands D, E, F and G of the native immunoglobulin fold. The assigned core region of the fibril is distinct in comparison to the core identified in a previous solid-state NMR study on AL-09 by Piehl at. al, suggesting that VL fibrils can adopt different topologies. In addition, we investigated a soluble oligomeric intermediate state, previously termed the alternatively folded state (AFS), using NMR and FTIR spectroscopy. The NMR oligomer spectra display a high degree of similarity when compared to the fibril spectra, indicating a high structural similarity of the two aggregation states. Based on comparison to the native state NMR chemical shifts, we suggest that fibril formation via domain-swapping seems unlikely. Moreover, we used our results to test the quality of different amyloid prediction algorithms.

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Section: Resultssupporting

confidence: 92%

“…The murine κ-type V L domain of MAK33 has been studied extensively regarding its native structure [20], biophysical properties and folding pathways [21,22]. Several point mutations facilitate fibril formation [23–26].…”

Section: Introductionmentioning

confidence: 99%

MAK33 antibody light chain amyloid fibrils are similar to oligomeric precursors

et al. 2017

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“…Feige et al reported that murine CH1 formed oligomers at pH 2, 100 mM NaCl. 18 However, CH1-CL dimer did not form oligomers in the present experiments. The higher colloidal stability of CH1-CL dimer may arise from the close proximity of CH1 and CL, since the two domains are covalently connected through their C terminal disulfide bond, allowing CL to function as a "solubility tag" and improve the solubility of CH1.…”

Section: Molecular Pharmaceuticscontrasting

confidence: 54%

“…Feige et al showed that all murine Fab domains (VH, VL, CH1, and CL) also adopted the AFS. 18 In their study, although CL globally unfolded at pH 2, 0.1 M NaCl, a considerable amount of secondary structure was observed at pH 2 and NaCl concentrations of 175 mM and higher, but CL remained monomeric. The physicochemical characteristics of murine and human CL would also be very similar.…”

Section: Time-dependent CD and Dls Measurements Of Conformational Chamentioning

confidence: 89%

Conformational and Colloidal Stabilities of Isolated Constant Domains of Human Immunoglobulin G and Their Impact on Antibody Aggregation under Acidic Conditions

et al. 2015

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Antibody therapeutics are now in widespread use and provide a new approach for treating serious diseases such as rheumatic diseases and cancer. Monoclonal antibodies used as therapeutic agents must be of high quality, and their safety must be guaranteed. Aggregated antibody is a degradation product that may be generated during the manufacturing process. To maintain the high quality and safety of antibody therapeutics, it is necessary to understand the mechanism of aggregation and to develop technologies to strictly control aggregate formation. Here, we extensively investigated the conformational and colloidal characteristics of isolated antibody constant domains, and provided insights into the molecular mechanism of antibody aggregation. Isolated domains (CH2, CH3, CL, and CH1-CL dimer) of human immunoglobulin G were synthesized, solubilized using 49 sets of solution conditions (pH 2-8 and 0-300 mM NaCl), and characterized using circular dichroism, intrinsic tryptophan fluorescence, and dynamic light scattering. Salt-induced conformational changes and oligomer formation were kinetically analyzed by NaCl-jump measurements (from 0 to 300 mM at pH 3). Phase diagrams revealed that the domains have different conformational and colloidal stabilities. The unfolded fractions of CH3 and CH2 at pH 3 were larger than that of CL and CH1-CL dimer. The secondary and tertiary structures and particle sizes of CH3 and CH2 showed that, in non-native states, these domains were sensitive to salt concentration. Kinetic analyses suggest that oligomer formation by CH3 and CH2 proceeds through partially refolded conformations. The colloidal stability of CH3 in non-native states is the lowest of the four domains under the conditions tested. We propose that the impact of IgG constant domains on aggregation follows the order CH3 > CH2 > CH1-CL dimer > CL; furthermore, we suggest that CH3 plays the most critical role in driving intact antibody aggregation under acidic conditions.

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“…Plasmid DNA from a single colony was isolated with the Wizard Plus SV Miniprep kit (Promega) and sequenced using the T7 forward or pET-RP sequencing primer by Eurofins MWG Operon to verify the desired mutation. All MAK33 V L , 1OPG V L , 1AQK V L , and 1VGE V H variants were expressed and purified as described previously (15,16). In brief, the plasmid was transformed into E. coli BL21(DE3)-star cells (for V L and variants) or in E. coli JM109 cells (for V H variants) for expression at 37°C.…”

Section: Methodsmentioning

confidence: 99%

A Residue-specific Shift in Stability and Amyloidogenicity of Antibody Variable Domains

Nokwe

Zacharias

Yagi

et al. 2014

Journal of Biological Chemistry

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Background: Amyloid fibrils of variable (V L ) domains are the main cause of death in light chain amyloidosis. Results: Conserved V L residue 2 is crucial for structural integrity and amyloidogenicity of V L domains. Conclusion: Our data reveal novel insights into the architecture of variable domains in general and the prerequisite for fibrillation. Significance: This is important for understanding the principles of antibody structure and amyloidogenicity.

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Dissecting the Alternatively Folded State of the Antibody Fab Fragment

Cited by 20 publications

References 42 publications

MAK33 antibody light chain amyloid fibrils are similar to oligomeric precursors

MAK33 antibody light chain amyloid fibrils are similar to oligomeric precursors

Conformational and Colloidal Stabilities of Isolated Constant Domains of Human Immunoglobulin G and Their Impact on Antibody Aggregation under Acidic Conditions

A Residue-specific Shift in Stability and Amyloidogenicity of Antibody Variable Domains

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