Three-Dimensional Structure of the Human Myeloma IgG2

Abstract

Human immunoglobulin G, subclass 2 (hIgG2), plays an important role in immunity to bacterial pathogens and in numerous pathological conditions. However, there is a lack of information regarding the three-dimensional (3D) structure of the hIgG2 molecule. We used electron microscopy (EM), differential scanning microcalorimetry (DSC) and fluorescence for structural analysis of the hIgG2. DSC and fluorescence indicated two types of interaction between CH1 domain of Fab (antigen-binding fragment/subunit) and CH2 domain of Fc (complement fixation fragment/subunit) simultaneously present in the sample: close interaction, which increases the thermostability of both, CH1 and CH2 domains, and weak (or no) interaction, which is typical for most IgGs but not hIgG2. Thermodynamics could not determine if both types of interactions are present within a single molecule. To address this question, EM was used. We employed a single-particle reconstruction and negative staining approach to reveal the three-dimensional structure of the hIgG2. A three-dimensional model of hIgG2 was created at 1.78 nm resolution. The hIgG2 is asymmetrical: one Fab subunit is in close proximity to the upper portion of the Fc subunit (CH2 domain) and the other Fab is distant from Fc. The plane of Fab subunits is nearly perpendicular to Fc. EM structure of the hIgG2 is in good agreement with thermodynamic data: a Fab distant from Fc should exhibit a lower melting temperature while a Fab interacting with Fc should exhibit a higher melting temperature. Both types of Fab subunits exist within one molecule resembling an A/B hIgG2 isoform introduced earlier on physicochemical level by Dillon et al. (2008). In such an arrangement, the access to the upper portion of Fc subunit is partially blocked by a Fab subunit. That might explain for instance why hIgG2 mildly activates complement and binds poorly to Fc receptors. Understanding of the three-dimensional structure of the hIgG2 should lead to better design of antibody-based therapeutics.

Evidence of disulfide bond scrambling during production of an antibody-drug conjugate

“…Detection of these species after disruption of all hinge disulfides indicates the presence of strong noncovalent forces between the HCs, in alignment with the extensive Fab-Fab and Fab-C H 2 interactions previously reported for the B isoform. 26,51 Our observed disulfide isoform conversion directionality is in agreement with previous studies that have reported conversion of the A → A/B → B forms in a reducing environment similar to physiological conditions. 24,25 The higher thermal stability of the IgG2-B mAb and ADCs observed in this study provides further evidence that IgG2 disulfide scrambling favors the thermodynamic product.…”

Thymus cDNA library survey uncovers novel features of immune molecules in Chinese giant salamander Andrias davidianus

“…Prior to this study, only a small number of ESTs (307 ESTs) in NCBI were available for this species, thus our data enrich the existing genetic resources. A total of 137 candidate genes were potentially involved in immune responses, a high ratio most likely due to an effective stimulation with ranavirus, since ranavirus infection can induce robust immune response in amphibians (Grayfer et al, 2014;Ryazantsev et al, 2013). It has long been recognized that amphibian thymus is the primary lymphoid organ (Chen and Robert, 2011).…”

“…To our knowledge, it represents the first hinge-containing IgD in nonmammalian tetrapods, since no hinges have been reported in the lower vertebrate IgD sequenced to date. The hinge region is critical for Ig function: it confers conformational flexibility, thus facilitating antigen binding and triggering effector functions (Ryazantsev et al, 2013). This segment is generally present in mammalian Igs, but it is assumed to be evolved in lower vertebrates.…”

Probing the extracellular diffusion of antibodies in brain using in vivo integrative optical imaging and ex vivo fluorescence imaging

“…Space-filled rendering of murine IgG in Jmol (PDB ID: 1IGY [24]) with the Fab and Fc domains outlined. Mammalian IgG has two long axes (with typical lengths of approximately 10–15 nm[24, 25]) and one short axis with a unique Y- or T-shaped geometry.…”