Dynamic fluctuations of protein-carbohydrate interactions promote aggregation

Protein-carbohydrate interactions are important for glycoprotein structure and function. Antibodies of the IgG class, with increasing significance as therapeutics, are glycosylated at a conserved site in the constant Fc region. We hypothesized that disruption of protein-carbohydrate interactions in the glycosylated domain of antibodies leads to the exposure of aggregation-prone motifs. Aggregation is one of the main problems in protein-based therapeutics because of immunogenicity concerns and decreased efficacy. To explore the significance of intramolecular interactions between aromatic amino acids and carbohydrates in the IgG glycosylated domain, we utilized computer simulations, fluorescence analysis, and site-directed mutagenesis. We find that the surface exposure of one aromatic amino acid increases due to dynamic fluctuations. Moreover, protein-carbohydrate interactions decrease upon stress, while protein-protein and carbohydrate-carbohydrate interactions increase. Substitution of the carbohydrate-interacting aromatic amino acids with non-aromatic residues leads to a significantly lower stability than wild type, and to compromised binding to Fc receptors. Our results support a mechanism for antibody aggregation via decreased protein-carbohydrate interactions, leading to the exposure of aggregation-prone regions, and to aggregation. Citation: Voynov V, Chennamsetty N, Kayser V, Helk B, Forrer K, et al. (2009) Dynamic Fluctuations of Protein-Carbohydrate Interactions Promote Protein Aggregation. PLoS ONE 4(12): e8425. doi:10.1371/journal.pone.0008425 Editor: Laurent Kreplak, Dalhousie University, Canada Received September 7, 2009; Accepted November 30, 2009; Published December 23, 2009 Copyright: 2009 Voynov et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by Novartis Pharma AG. Employees of Novartis participated in study design, data collection and analysis, and preparation of the manuscript. Competing Interests: The authors declare competing financial interests. The authors Bernhard Helk, Kurt Forrer, Heidi Zhang, Cornelius Fritsch, and Holger Heine are current employees of Novartis Pharma AG. This work was supported by Novartis Pharma AG. * E-mail: [email protected]