Contribution of the intramolecular disulfide bridge to the folding stability of REIv, the variable domain of a human immunoglobulin κ light chain

Conformation, structure, and oligomeric state of immunoglobulins not only control quality and functional properties of antibodies but are also critical for immunoglobulins secretion. Unassembled immunoglobulin heavy chains are retained intracellularly by delayed folding of the C H 1 domain and irreversible interaction of BiP with this domain. Here we show that the three C H 1 cysteines play a central role in immunoglobulin folding, assembly, and secretion. Remarkably, ablating all three C H 1 cysteines negates retention and enables BiP cycling and non-canonical folding and assembly. This phenomenon is explained by interdependent formation of intradomain and interchain disulfides, although both bonds are dispensable for secretion. Substituting Cys-195 prevents formation not only of the intradomain disulfide, but also of the interchain disulfide bond with light chain, BiP displacement, and secretion. Mutating the light chain-interacting Cys-128 hinders disulfide bonding of intradomain cysteines, allowing their opportunistic bonding with light chain, without hampering secretion. We propose that the role of C H 1 cysteines in immunoglobulin assembly and secretion is not simply to engage in disulfide bridges, but to direct proper folding and interact with the retention machinery.

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“…Reverse primer [25][26][27][28][29][30][31][32][33][34][35][36]. The data are representative of seven independent experiments.…”

Section: Forward Primermentioning

confidence: 99%

Cysteines in CH1 Underlie Retention of Unassembled Ig Heavy Chains

Elkabetz

Argon

Bar-Nun

2005

Journal of Biological Chemistry

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“…A characteristic feature of antibodies is that the intradomain disulfide bond, which connects residues far apart in sequence, is completely buried in the core of the protein. These proteins provide an excellent system to study the folding and assembly of all ␤-sheet proteins and to elucidate the hierarchy of intra/interchain disulfide bond formation during the folding process of multimeric and multidomain proteins (11)(12)(13)(14)(15). The ␤-sheet folding is usually much slower than the ␣-helix formation because amino acid residues, which are far apart in the polypeptide chain, must interact correctly in the three-dimensional space to form stabilizing interactions (16).…”

mentioning

confidence: 99%

Hierarchical Formation of Disulfide Bonds in the Immunoglobulin Fc Fragment Is Assisted by Protein-disulfide Isomerase

Vinci

Catharino

Frey

et al. 2004

Journal of Biological Chemistry

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Antibodies provide an excellent system to study the folding and assembly of all ␤-sheet proteins and to elucidate the hierarchy of intra/inter chain disulfide bonds formation during the folding process of multimeric and multidomain proteins. Here, the folding process of the Fc fragment of the heavy chain of the antibody MAK33 was investigated. The Fc fragment consists of the C H 3 and C H 2 domains of the immunoglobulin heavy chain, both containing a single S-S bond. The folding process was investigated both in the absence and presence of the folding catalyst protein-disulfide isomerase (PDI), monitoring the evolution of intermediates by electrospray mass spectrometry. Moreover, the disulfide bonds present at different times in the folding mixture were identified by mass mapping to determine the hierarchy of disulfide bond formation. The analysis of the uncatalyzed folding showed that the species containing one intramolecular disulfide predominated throughout the entire process, whereas the fully oxidized Fc fragment never accumulated in significant amounts. This result suggests the presence of a kinetic trap during the Fc folding, preventing the one-disulfide-containing species (1S2H) to reach the fully oxidized protein (2S). The assignment of disulfide bonds revealed that 1S2H is a homogeneous species characterized by the presence of a single disulfide bond (Cys-130 -Cys-188) belonging to the C H 3 domain. When the folding experiments were carried out in the presence of PDI, the completely oxidized species accumulated and predominated at later stages of the process. This species contained the two native S-S bonds of the Fc protein. Our results indicate that the two domains of the Fc fragment fold independently, with a precise hierarchy of disulfide formation in which the disulfide bond, especially, of the C H 2 domain requires catalysis by PDI.

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“…Indeed, it was found that scFv fragments expressed cytoplasmically in COS cells do not form the disulfide bonds (10). The intradomain disulfide contributes about 4 -5 kcal/mol to the stability of antibody domains (11,12). Therefore, antibody fragments expressed in a reducing environment are strongly destabilized, compared with the same molecules containing disulfides, and a smaller fraction of these fragments is likely to fold to the correct native structure.…”

mentioning

confidence: 99%

Correlation between in Vitro Stability and in Vivo Performance of Anti-GCN4 Intrabodies as Cytoplasmic Inhibitors

Wörn¹,

Maur²,

Escher³

et al. 2000

Journal of Biological Chemistry

128

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A cellular assay system for measuring the activity of cytoplasmically expressed anti-GCN4 scFv fragments directed against the Gcn4p dimerization domain was established in the budding yeast Saccharomyces cerevisiae. The inhibitory potential of different constitutively expressed anti-GCN4 scFv intrabodies was monitored by measuring the activity of ␤-galactosidase expressed from a GCN4-dependent reporter gene. The in vivo performance of these scFv intrabodies in specifically decreasing reporter gene activity was related to their in vitro stability, measured by denaturant-induced equilibrium unfolding. A framework-engineered stabilized version showed significantly improved activity, while a destabilized point mutant of the anti-GCN4 wild-type showed decreased effects in vivo. These results indicate that stability engineering can result in improved performance of scFv fragments as intrabodies. Increasing the thermodynamic stability appears to be an essential factor for improving the yield of functional scFv in the reducing environment of the cytoplasm, where the conserved intradomain disulfides of antibody fragments cannot form.

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Contribution of the intramolecular disulfide bridge to the folding stability of REIv, the variable domain of a human immunoglobulin κ light chain

Cited by 63 publications

References 32 publications

Cysteines in CH1 Underlie Retention of Unassembled Ig Heavy Chains

Cysteines in CH1 Underlie Retention of Unassembled Ig Heavy Chains

Hierarchical Formation of Disulfide Bonds in the Immunoglobulin Fc Fragment Is Assisted by Protein-disulfide Isomerase

Correlation between in Vitro Stability and in Vivo Performance of Anti-GCN4 Intrabodies as Cytoplasmic Inhibitors

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