Thermal Induction of an Alternatively Folded State in Human IgG-Fc

Abstract

We report the formation of a non-native, folded state of human IgG4-Fc induced by a high temperature at neutral pH and at a physiological salt concentration. This structure is similar to the molten globule state in that it displays a high degree of secondary structure content and surface-exposed hydrophobic residues. However, it is highly resistant to chemical denaturation. The thermally induced state of human IgG4-Fc is thus associated with typical properties of the so-called alternatively folded state previously described for murine IgG, IgG-Fab, and individual antibody domains (V(L), V(H), C(H)1, and C(H)3) under acidic conditions in the presence of anions. Like some of these molecules, human IgG4-Fc in its alternative fold exists as a mixture of different oligomeric structures, dominated by an equilibrium between monomeric and heptameric species. Heating further induces the formation of fibrous structures in the micrometer range.

Tracing Primordial Protein Evolution through Structurally Guided Stepwise Segment Elongation

“…AF.2A1 bound to all of those treated IgG and Fc regions. These results imply that AF.2A1 did not recognize natively formed IgG nor the Fc region, but rather non-native conformers caused upon acidification or heat treatment (25)(26)(27). Despite such a unique binding mode, the recognition ability of AF.2A1 was highly specific enough to distinguish the Fc region from crude cell lysate (Fig.…”

One-Step Identification of Antibody Degradation Pathways Using Fluorescence Signatures Generated by Cross-Reactive DNA-Based Arrays

“…These are typical signatures of the so-called "alternatively folded state (AFS)". 34 Further heat-treatment induced a gradual increase in optical density due to the formation of visible large aggregates. Thus, Oma solutions after incubation for 5 and 15 min were used as denatured and visibly aggregated states, respectively.…”

“…To test these hypotheses, we selected two different human antibodies as target analytes: (i) polyclonal immunoglobulin G (IgG, p I ≈ 7.3, a mixture of IgG subtypes), and (ii) monoclonal omalizumab [Oma, p I ≈ 7.6, IgG1κ subtype with approval from the Food and Drug Administration (FDA) for the treatment of allergic asthma]. Irreversibly degraded antibodies were prepared upon thermal treatment at 75 °C (IgG) or 80 °C (Oma) in phosphate-buffered saline (PBS) at pH = 7.4 according to previously reported procedures. − In the case of Oma, an increase in negative ellipticity at 216 nm (θ 216 ), i.e., an increase in the proportion of β-sheet secondary structure, occurred within several minutes (Figure , red circles), although no substantial increase in optical density at 400 nm (OD 400 ) was observed (Figure , black circles). These are typical signatures of the so-called “alternatively folded state (AFS)” .…”

“…Irreversibly degraded antibodies were prepared upon thermal treatment at 75 °C (IgG) or 80 °C (Oma) in phosphate-buffered saline (PBS) at pH = 7.4 according to previously reported procedures. − In the case of Oma, an increase in negative ellipticity at 216 nm (θ 216 ), i.e., an increase in the proportion of β-sheet secondary structure, occurred within several minutes (Figure , red circles), although no substantial increase in optical density at 400 nm (OD 400 ) was observed (Figure , black circles). These are typical signatures of the so-called “alternatively folded state (AFS)” . Further heat-treatment induced a gradual increase in optical density due to the formation of visible large aggregates.…”

IgG Rheumatoid Factors Against the Four Human Fc‐gamma Subclasses in Early Rheumatoid Arthritis (The Swedish TIRA Project)

“…All serum samples were analysed in triplicate, and the individual results expressed as mean values. The RF target antigens, hIgG‐Fc subclasses 1–4, respectively, were expressed in a eukaryotic host ( Pichia pastoris ) and purified as previously described [18]. Microtitre plate wells (Corning Costar 96‐well half area; Corning Inc., Corning, NY, USA) were incubated over night with the different subclasses of hIgG‐Fc (5 μg/ml) prepared in 50 m m carbonate‐bicarbonate buffer, pH 9.6.…”