Originally published In Press as doi:10.1074/jbc.M311480200 on January 17, 2004
J. Biol. Chem., Vol. 279, Issue 15, 15059-15066, April 9, 2004
Hierarchical Formation of Disulfide Bonds in the Immunoglobulin Fc Fragment Is Assisted by Protein-disulfide Isomerase*
Floriana Vinci
,
Silvia Catharino
,
Stephen Frey,
Johannes Buchner,
Gennaro Marino¶||,
Piero Pucci¶, and
Margherita Ruoppolo¶**
From the
Dipartimento di Chimica Organica e Biochimica, Complesso Universitario di Monte S. Angelo, Università degli Studi di Napoli Federico II, Via Cinthia, 80126 Napoli, Italy, Institut für Organische Chemie und Biochemie Technische Universität München, D-85747 Germany, ¶CEINGE, Biotecnologie Avanzate, 80131 Napoli, Italy, ||School of Biotechnology Sciences, Federico II, University of Naples, 80126 Italy, and **Dipartimento di Biochimica e Biotecnologie Mediche, Università degli Studi di Napoli Federico II, 80131 Napoli, Italy
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 CH3 and CH2 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 CH3 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 CH2 domain requires catalysis by PDI.
Received for publication, October 20, 2003
, and in revised form, January 15, 2004.
* The work was supported by European Community Grant BIO4-CT96-0436 and Progetto Finalizzato Consiglio Nazionale delle Ricerche, "Folding of recombinant immunoglobulins", FIRB 2002 "Folding di proteine, l'altra metà del codice genetico", and MIUR_Rome PRIN 2001-COFINLAB2001. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dipartimento di Biochimica e Biotecnologie Mediche, Facoltà di Medicina e Chirurgia, Università degli Studi di Napoli Federico II, Via Pansini 5, 80131 Napoli, Italy. Tel.: 39-81-7462426; Fax: 39-81-7462404; E-mail: ruoppolo{at}dbbm.unina.it.
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Copyright © 2004 by the American Society for Biochemistry and Molecular Biology.
