Abstract
In this work, we present studies of the covalent structure of human IgG2 molecules. Detailed analysis showed that recombinant human IgG2 monoclonal antibody could be partially resolved into structurally distinct forms caused by multiple disulfide bond structures. In addition to the presently accepted structure for the human IgG2 subclass, we also found major structures that differ from those documented in the current literature. These novel structural isoforms are defined by the light chain constant domain (C(L)) and the heavy chain C(H)1 domain covalently linked via disulfide bonds to the hinge region of the molecule. Our results demonstrate the presence of three main types of structures within the human IgG2 subclass, and we have named these structures IgG2-A, -B, and -A/B. IgG2-A is the known classic structure for the IgG2 subclass defined by structurally independent Fab domains and hinge region. IgG2-B is a structure defined by a symmetrical arrangement of a (C(H)1-C(L)-hinge)(2) complex with both Fab regions covalently linked to the hinge. IgG2-A/B represents an intermediate form, defined by an asymmetrical arrangement involving one Fab arm covalently linked to the hinge through disulfide bonds. The newly discovered structural isoforms are present in native human IgG2 antibodies isolated from myeloma plasma and from normal serum. Furthermore, the isoforms are present in native human IgG2 with either kappa or lambda light chains, although the ratios differ between the light chain classes. These findings indicate that disulfide structural heterogeneity is a naturally occurring feature of antibodies belonging to the human IgG2 subclass.
Highlights
In IgG1, the C-terminal Cys residue of the light chain (LC) connects to the first Cys residue in the genetic hinge of the heavy chain (HC), whereas in IgG2, IgG3, and IgG4 subtypes, the LC is disulfide-bonded to the Cys residue in the Fab portion of the antibody immediately N-terminal of the CH1 disulfide loop, which is spatially close in the folded antibody structure [8, 9]
We extended our structural studies to human myeloma-derived IgG2 with both and LC as well as to polyclonal IgG purified from normal human serum, and we found that the observed alternate structural isoforms exist in immunoglobulins from natural sources
The data presented here reveal the existence of disulfide structural isoforms in human IgG2, and we use a nomenclature to identify those that are reflective of the symmetry of the forms: IgG2-A, IgG2-B, and IgG2-A/B
Summary
Structural Isoforms of IgG2 cules (Fig. 1), and the HC-HC disulfide bridges were hypothesized to exist as parallel bonds. These original IgG2 structural studies, which were performed with human myeloma-derived IgG2 with a LC, detected some additional low abundance disulfide-linked peptides that were not further characterized. Observations of heterogeneous behavior of IgG2 recombinant mAbs when applying independent separation techniques, CE-SDS, CEX-HPLC, and RP-HPLC under nonreducing conditions, led us to perform detailed structural studies. We extended our structural studies to human myeloma-derived IgG2 with both and LC as well as to polyclonal IgG purified from normal human serum, and we found that the observed alternate structural isoforms exist in immunoglobulins from natural sources. Our studies provide evidence that the disulfide-mediated structural heterogeneity is an important and intrinsic feature of the IgG2 subclass
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