title: 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 beta-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.
authors.affiliation: Dipartimento di Chimica Organica e Biochimica, Complesso Universitario di Monte S. Angelo, Università degli Studi di Napoli Federico II, Via Cinthia, 80126 Napoli, Italy.
authors.forename: Floriana
authors.initials: F
authors.lastname: Vinci
authors.forename: Silvia
authors.initials: S
authors.lastname: Catharino
authors.forename: Stephen
authors.initials: S
authors.lastname: Frey
authors.forename: Johannes
authors.initials: J
authors.lastname: Buchner
authors.forename: Gennaro
authors.initials: G
authors.lastname: Marino
authors.forename: Piero
authors.initials: P
authors.lastname: Pucci
authors.forename: Margherita
authors.initials: M
authors.lastname: Ruoppolo
mesh_terms: D000818:Animals; D000906:Antibodies / Q000737:chemistry; D002384:Catalysis; D002850:Chromatography, Gel; D004220:Disulfides; D005978:Glutathione / Q000737:chemistry; D007141:Immunoglobulin Fc Fragments / Q000737:chemistry*; D007700:Kinetics; D051379:Mice; D010100:Oxygen / Q000378:metabolism; D019704:Protein Disulfide-Isomerases / Q000737:chemistry*; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013053:Spectrophotometry; D013997:Time Factors
chemical_list: D000906:Antibodies; D004220:Disulfides; D007141:Immunoglobulin Fc Fragments; D019704:Protein Disulfide-Isomerases; D005978:Glutathione; D010100:Oxygen