title: Protected hinge in the immunoglobulin G2-A2 disulfide isoform.
abstract: Human IgG2 consists of disulfide-mediated structural isoforms, classified by the number of Fab arms disulfide-linked to the heavy chain hinge. In the IgG2-B isoform, both Fab arms are linked to the hinge region, and in IgG2-A, neither Fab arm are linked to the hinge. IgG2-A/B is a hybrid between these two forms, with only one Fab arm disulfide-linked to the hinge. Within each of these isoform types are subtypes, with subtle disulfide-linkage differences. Here we explored the structural basis for the A1 and A2 isoform subtypes. Whereas A1 isoform converts into the A/B and B isoforms under mild redox conditions, A2 does not. Characterization of the disulfide connectivities of A2 isoform revealed a similar structure to A1 isoform, with parallel inter heavy chain disulfide linkages in the hinge region. However, the hinge disulfides in A2 isoform were resistant to reduction under conditions where A1 isoform hinge disulfides became reduced and they required thermal treatment (>55 °C) to obtain thiol-dependent disulfide reduction. Structural analysis of the hinge region indicated that the protected disulfides were restricted to cysteines 219 and 220 of the upper hinge. Disruption of the upper hinge through insertion mutagenesis eliminated A2 isoform behavior. (1)H NMR studies showed that the A1 isoform Fc glycan was more dynamic than that on A2 isoform and showed some other conformational differences. Results point to an IgG2-A2 upper hinge region that is more akin to the interior of a globular protein than the flexible hinge region expected on an IgG.
authors.affiliation: Department of Product Attribute Sciences, Amgen Inc, Thousand Oaks, California, 91320.
authors.forename: Yaoqing Diana
authors.initials: YD
authors.lastname: Liu
authors.forename: Robert Y-T
authors.initials: RY
authors.lastname: Chou
authors.forename: Thomas M
authors.initials: TM
authors.lastname: Dillon
authors.forename: Leszek
authors.initials: L
authors.lastname: Poppe
authors.forename: Chris
authors.initials: C
authors.lastname: Spahr
authors.forename: Stone D H
authors.initials: SD
authors.lastname: Shi
authors.forename: Gregory C
authors.initials: GC
authors.lastname: Flynn
mesh_terms: D004220:Disulfides / Q000737:chemistry*; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry*; D019906:Nuclear Magnetic Resonance, Biomolecular; D020033:Protein Isoforms / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry
chemical_list: D004220:Disulfides; D007074:Immunoglobulin G; D020033:Protein Isoforms; D011994:Recombinant Proteins
keywords: NMR; disulfide reduction; monoclonal antibodies; protein dynamics