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Protected hinge in the immunoglobulin G2‐A 2 disulfide isoform
Author(s) -
Liu Yaoqing Diana,
Chou Robert Y.T.,
Dillon Thomas M.,
Poppe Leszek,
Spahr Chris,
Shi Stone D. H.,
Flynn Gregory C.
Publication year - 2014
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1002/pro.2557
Subject(s) - gene isoform , hinge , chemistry , disulfide bond , biochemistry , mutagenesis , stereochemistry , mutation , gene , physics , classical mechanics
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 A 1 and A 2 isoform subtypes. Whereas A 1 isoform converts into the A/B and B isoforms under mild redox conditions, A 2 does not. Characterization of the disulfide connectivities of A 2 isoform revealed a similar structure to A 1 isoform, with parallel inter heavy chain disulfide linkages in the hinge region. However, the hinge disulfides in A 2 isoform were resistant to reduction under conditions where A 1 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 A 2 isoform behavior. 1 H NMR studies showed that the A 1 isoform Fc glycan was more dynamic than that on A 2 isoform and showed some other conformational differences. Results point to an IgG2‐A 2 upper hinge region that is more akin to the interior of a globular protein than the flexible hinge region expected on an IgG.