Comparing domain interactions within antibody Fabs with kappa and lambda light chains.

ABSTRACT: IgG antibodies are multi-domain proteins with complex inter-domain interactions. Human IgG heavy chains (HCs) associate with light chains (LCs) of the ? or ? isotype to form mature antibodies capable of binding antigen. The HC/LC interaction involves 4 domains: VH and CH1 from the HC and VL and CL from the LC. Human Fabs with ? LCs have been well characterized for their unfolding behaviors and demonstrate a significant level of cooperativity and stabilization when all 4 domains are intact. Very little is known regarding the thermodynamic properties of human Fabs with ? LCs. Here, we dissect the domain contributions to Fab stability for both ? and ? LC-containing Fabs. We find the cooperativity of unfolding between the constant domains, CH1/C?, and variable domains, VH/V?, within ? LC-containing Fabs is significantly weaker than that of ? LC-containing Fabs. The data suggests there may not be an evolutionary necessity for strong variable/constant domain cooperativity within ? LC-containing Fabs. After investigating the biophysical properties of Fabs with mismatched variable and constant domain subunits (e.g., VH/V? paired with CH1/C? or T cell receptor C?/C?), the major role of the constant domains for both ?- and ?-containing Fabs may be to reduce the hydrophobic exposure at the VH/VL interface. Even though Fabs with these non-native pairings were thermodynamically less stable, they secreted well from mammalian cells as well behaved monodisperse proteins, which was in contrast to what was observed with the VH/V? and VH/V? scFvs that secreted as a mixture of monomer and aggregates.

SUBMITTER: Toughiri R 

PROVIDER: S-EPMC5058631 | biostudies-literature | 2016 Oct

REPOSITORIES: biostudies-literature