Protected hinge in the immunoglobulin G2-A2 disulfide isoform

Area of Science:

• Immunology
• Structural Biology
• Protein Chemistry

Background:

• Human IgG2 antibodies exist as structural isoforms based on Fab arm disulfide linkages to the hinge region.
• These isoforms include IgG2-B (both Fab arms linked), IgG2-A (neither Fab arm linked), and IgG2-A/B (one Fab arm linked).
• Subtle differences in disulfide linkages define subtypes within these major isoforms.

Purpose of the Study:

• To investigate the structural basis for the distinct behaviors of IgG2-A1 and IgG2-A2 isoform subtypes.
• To understand why the IgG2-A1 isoform converts to other isoforms under mild redox conditions, while IgG2-A2 does not.

Main Methods:

• Characterization of disulfide connectivities in IgG2-A2.
• Redox condition experiments to assess isoform stability and conversion.
• Thermal treatment to induce disulfide reduction.
• Insertion mutagenesis to disrupt the upper hinge region.
• (1)H NMR studies to analyze Fc glycan dynamics and conformational differences.

Main Results:

• IgG2-A2 isoform possesses disulfide linkages in the hinge region, similar to IgG2-A1, but these are resistant to reduction under mild conditions.
• Reduction of IgG2-A2 hinge disulfides requires thermal treatment (>55 °C).
• Protected disulfides are localized to cysteines 219 and 220 in the upper hinge region.
• Disruption of the upper hinge via insertion mutagenesis abolished the characteristic IgG2-A2 behavior.
• (1)H NMR revealed greater Fc glycan dynamism in IgG2-A1 compared to IgG2-A2.

Conclusions:

• The IgG2-A2 upper hinge region exhibits a more rigid, globular protein-like structure due to restricted disulfide bonds.
• This structural rigidity explains the resistance of IgG2-A2 to redox-induced conversion observed in other IgG2 isoforms.
• The findings highlight significant structural heterogeneity within human IgG2, impacting its functional properties.