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

Abstract

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.

Structure of the Murine Unglycosylated IgG1 Fc Fragment

“…The Fc fragment was refolded from inclusion bodies in an established manner. 22 Its secondary and tertiary structures in solution, based on far-UV and near-UV CD spectra, respectively, were highly similar to that of the authentic glycosylated Fc fragment and the enzymatically deglycosylated Fc fragment produced in eukaryotic cell cultures (data not shown).…”

“…Recombinant Fc comprised MAK33 residues Glu239-Glu450 and was purified as described previously. 22 All proteins used in this study were devoid of hinge disulfide bridges.…”

Systematic Single-Cell Analysis of Pichia pastoris Reveals Secretory Capacity Limits Productivity

“…We selected enhanced green fluorescent protein (eGFP), which is known to mature rapidly (∼30 min) and spontaneously [24], to enable monitoring of intracellular, folded protein in relation to secreted, folded protein. For comparison, we also chose to examine both glycosylated and aglycosylated versions of a human Fc fragment, a dimeric protein that requires foldases and chaperones for proper folding [25]. To control for variations in coarse transcriptional activities, all strains used the same locus (GAPDH) for insertion of the gene of interest.…”

Diselenides as universal oxidative folding catalysts of diverse proteins

“…The folding of antibodies is difficult, as it requires the formation of both intra-and interchain disulfide bonds (Glockshuber et al, 1992). The oxidative folding pathway has been studied in some detail (Goto and Hamaguchi, 1981;Mayer et al, 2000), and presumably proceeds sequentially (Vinci et al, 2004). In the cell, binding immunoglobulin protein (BiP) and protein disulfide isomerase (PDI) are required for efficient folding of antibodies.…”