title: Dynamic fluctuations of protein-carbohydrate interactions promote protein aggregation.
abstract: 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.
authors.affiliation: Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.
authors.forename: Vladimir
authors.initials: V
authors.lastname: Voynov
authors.forename: Naresh
authors.initials: N
authors.lastname: Chennamsetty
authors.forename: Veysel
authors.initials: V
authors.lastname: Kayser
authors.forename: Bernhard
authors.initials: B
authors.lastname: Helk
authors.forename: Kurt
authors.initials: K
authors.lastname: Forrer
authors.forename: Heidi
authors.initials: H
authors.lastname: Zhang
authors.forename: Cornelius
authors.initials: C
authors.lastname: Fritsch
authors.forename: Holger
authors.initials: H
authors.lastname: Heine
authors.forename: Bernhardt L
authors.initials: BL
authors.lastname: Trout
mesh_terms: D000596:Amino Acids / Q000378:metabolism; D050260:Carbohydrate Metabolism; D002240:Carbohydrate Sequence; D002241:Carbohydrates / Q000737:chemistry; D002460:Cell Line; D003198:Computer Simulation; D018869:Heat-Shock Response; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D007074:Immunoglobulin G / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D010455:Peptides / Q000378:metabolism; D011485:Protein Binding; D055550:Protein Stability; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D013499:Surface Properties
chemical_list: D000596:Amino Acids; D002241:Carbohydrates; D007074:Immunoglobulin G; D050505:Mutant Proteins; D010455:Peptides