Post-translational Modifications Differentially Affect IgG1 Conformation and Receptor Binding

Post-translational modifications (PTMs) can have profound effects on protein structure and protein dynamics and thereby can influence protein function. To understand and connect PTM-induced functional differences with any resulting conformational changes, the conformational changes must be detected and localized to specific parts of the protein. We illustrate these principles here with a study of the functional and conformational changes that accompany modifications to a monoclonal immunoglobulin γ1 (IgG1) antibody. IgG1s are large and heterogeneous proteins capable of incorporating a multiplicity of PTMs both in vivo and in vitro. For many IgG1s, these PTMs can play a critical role in affecting conformation, biological function, and the ability of the antibody to initiate a potential adverse biological response. We investigated the impact of differential galactosylation, methionine oxidation, and fucosylation on solution conformation using hydrogen/deuterium exchange mass spectrometry and probed the effects of IgG1 binding to the FcγRIIIa receptor. The results showed that methionine oxidation and galactosylation both impact IgG1 conformation, whereas fucosylation appears to have little or no impact to the conformation. FcγRIIIa binding was strongly influenced by both the glycan structure/composition (namely galactose and fucose) and conformational changes that were induced by some of the modifications.The structure of many proteins can be altered by post-translational modifications (1). Although the impact of post-translational modifications (PTMs)¹ on protein structure is more understood for some modifications (e.g. phosphorylation; see Ref. 2), it is less defined for other PTMs and in many cases is protein-dependent. Because there are many important downstream effects of PTMs, including changes in protein localization, protein and cellular diversification, protein functionality, protein stability, protein life cycle, and so forth, understanding how PTMs alter protein structure for as many proteins as possible in a timely manner is a highly desirable goal. Furthermore, in an age where recombinant proteins are being used to treat disease, it becomes ever more important to understand how particular modifications may alter the structure and eventually the function of therapeutic proteins. To realize these goals, methods that permit access to conformational information for modified forms of therapeutic proteins must be developed and refined. In this report, we will illustrate how MS can contribute to structural proteomics by describing our recent work with a recombinant monoclonal antibody (an IgG1), which represents an important class of therapeutic proteins.Many biopharmaceutical companies are pursuing antibody drugs (3). In particular, the IgG1 subclass of antibodies has evolved into a commonly used therapeutic option for the treatment of a wide range of diseases. IgG1s consist of a dimer of identical heavy chains and light chains that fold to form (from N to C terminus) the variable, CL, CH1, CH2, and CH3 domains (as an example, see Ref. 4). Individual domains are structurally stable and are primarily composed of antiparallel β-sheets arranged in an immunoglobulin-like β-sandwich (5). The variable, CL, and CH1 domains are collectively referred to as the Fab (fragment antigen binding) portion of IgG1, which is responsible for recognizing a specific antigen. The CH2 and CH3 domains together are referred to as the Fc (fragment crystallizable) portion, which carries out effector functions such as binding to Fcγ receptors. These effector functions are essential to many therapeutic antibodies, especially when antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity are involved in the mechanisms of action (6).As a biopharmaceutical, IgG1 monoclonal antibodies are critically monitored throughout production (7). In many cases, the impact of structural modifications in these and other formulated versions of biopharmaceuticals are not well understood at a functional level. In the case of IgG1s, with over 1300 amino acid residues and a molecular mass approaching 150 kDa, a large array of PTMs can be incorporated both in vivo (during cellular synthesis) and in vitro (as a result of handling and processing steps that occur during purification, vialing, and storage). Commonly monitored PTMs on IgG1s include methionine oxidation, asparagine and glutamine deamidation, N-terminal acetylation or cyclization, glycation of lysine, and variable glycosylation (8). Some of these modifications affect only a small percentage of the protein product, and their presence may not change overall outcome. Others, however, can have significant impact on the structure, function, and biological activities of a protein that can involve self-association as well as interactions with other proteins (9). The same PTMs can affect different IgG1 molecules in different ways or have no effect(s) at all. Therefore assessing the presence of PTMs, determining the relative level of the modifications, and understanding the structural effects of PTMs are all important during development of protein biopharmaceuticals.Two commonly studied IgG1 modifications are methionine oxidation and glycosylation, each of which has been shown to affect biological function (6, 10). Methionine oxidation has been implicated in protein stability (inducing aggregation), and increased oxidation levels have been shown to provoke an immunogenic response (11–13). Elevated levels of methionine oxidation in an IgG1 were shown to impact neonatal Fc receptor (FcRn) and protein A binding (10). Variable glycosylation (i.e. different levels of sialic acid, galactose, fucose, or high mannose structures) is known to influence thermal stability and effector functions (14–16). Previous studies have shown that removal of fucose from the glycan present on the Fc portion of an IgG1 can greatly enhance Fc binding to FcγRIIIa, but removal of the entire glycan nearly abolishes FcγRIIIa binding (17). As oxidation and changes to the glycan are both common IgG1 modifications, we were interested in determining the conformational effects of oxidation, afucosylation, and galactosylation and correlating any conformational changes that were observed with changes of FcγRIIIa binding activity.Conformational analysis of large proteins like antibodies, however, is not trivial. Traditional biophysical techniques such as circular dichroism, DSC, and fluorescence provide useful information, but these techniques look at the entire protein and provide only a global view (18). NMR and x-ray crystallography can both provide high resolution structural analysis, but each is faced with limitations that often make the study of an intact IgG1 difficult or nearly impossible (19–21). Recently we described how hydrogen/deuterium exchange (H/DX) MS could be used to study the conformation and conformational dynamics of an intact IgG1 with resolution down to stretches of several amino acid residues (22). For the present work, we used H/DX MS to study the impact of galactosylation, oxidation, and afucosylation on the conformation and dynamics of an intact IgG1. We also studied the complex of IgG1 and FcγRIIIa to map the points of interaction and probe any changes in the dynamics of the IgG1 as a result of FcγRIIIa interaction. Finally, we correlated the functional activity of all the proteins that were studied by H/DX MS with the observed conformational disturbance(s). Such correlations are important to connect structure with function and to understand whether a particular PTM is something that may affect the therapeutic value of a recombinant protein.     

Workshop--Predicting the structure of biological molecules

This April, in Cambridge (UK), principal investigators from the Mathematical Biology Group of the Medical Research Council's National Institute of Medical Research organized a workshop in structural bioinformatics at the Centre for Mathematical Sciences. Bioinformatics researchers of several nationalities from labs around the country presented and discussed their computational work in biomolecular structure prediction and analysis, and in protein evolution. The meeting was intensive and lively and gave attendees an overview of the healthy state of protein bioinformatics in the UK.     

Virulence genotypes of Escherichia coli strains from bacteremia in relation to phylogeny

Bacteremia is the principal way of dissemination of local infections to distant organs. Escherichia coli bacteremia is almost always clinically significant, suggesting an increased risk of developing sepsis syndrome. Fifty-one E. coli bloodstream human isolates were analyzed using PCR technique for several molecular markers associated with extraintestinal virulence, and their phylogenetic group assignment, taking into account the link between the phylogenetic background and the intrinsic virulence of this species. Sixteen virulence genotypes have been identified, the majority of the blood isolates carrying the association of two genes. The genes encoding type 1 fimbria and aerobactin had the highest prevalence. As a confirmation of other studies, the strains assigned to E. coli phylogenetic group B2 exhibited the highest concentration of virulence genes, and represented almost half of the clinical blood isolates. The multifactorial virulence of E. coli strains isolated from invasive infections reflects a phylogenetic inheritance, and supports the concept of ExPEC pathotype as a subset of E. coli population involved in human infectious diseases. The surveillance of geographical variation of E. coli pathogenic clones is useful for epidemiological analysis.     

Vibratory Communication in the Jumping Spider Phidippus clarus: Substrate‐borne Courtship Signals are Important for Male Mating Success

Recently, work has shown that multimodal communication is common throughout the animal kingdom but the function of multimodal signals is still poorly understood. Phidippus clarus are jumping spiders in which males produce multimodal (visual and vibrational) signals in both male–male (aggressive) and male–female (courtship) contexts. The P. clarus mating system is complex, with sex ratios and the level of male competition changing over the course of the breeding season. Vibrational signal components have been shown to function in male aggressive contests but their role in courtship has not been investigated. Here, we performed an experiment to test the role of vibrational signaling in courtship by observing mating success for males that were experimentally muted. We show that vibratory courtship signals, and in particular signaling rate, is an important component of mating success and potentially a target of female choice. While the ability to produce vibratory signals significantly increased mating success, some muted males were still able to successfully mate. In these trials, signaling rate also predicted mating success suggesting that redundant signal components may compensate for errors and perturbations in signal transmission or that vibratory signals function to enhance the efficacy of visual signals.     

Time-resolved Raman spectroscopy with subpicosecond resolution: vibrational cooling and delocalization of strain energy in photodissociated (carbonmonoxy)hemoglobin

A Raman spectrometer that provides both subpicosecond resolution and independent, tunable pump and probe pulses is described. The spectrometer is employed to obtain time-resolved spectra of (carbonmonoxy)hemoglobin (HbCO) at times from 0.2 to 95 ps subsequent to ligand photodissociation. The spectra are interpreted in terms of a vibrationally hot heme that cools substantially in 10 ps. Concomitant with the proposed vibrational cooling is a slower relaxation, which we suggest results from a protein response to heme doming induced by ligand detachment. Results and interpretations are discussed in the context of current models of the heme photophysics and of hemoglobin reactivity.     

Distribution and range expansion of deer in Ireland

• 1 Throughout Europe, the range of many deer species is expanding. We provide current distribution maps for red deer Cervus elaphus, sika Cervus nippon, fallow deer Dama dama and muntjac deer Muntiacus sp. in Ireland, and estimates of range expansion rates for red deer, sika and fallow deer.
• 2 There was a considerable expansion in the ranges of red deer, sika and fallow deer between 1978 and 2008. The compound annual rate of expansion was 7% for red deer, 5% for sika and 3% for fallow deer. The total range increase was 565% for red deer, 353% for sika and 174% for fallow deer. The potential implications of these expansions are discussed.
• 3 There are unknown numbers of red‐sika hybrid deer in some parts of Ireland. Range expansion is likely to lead to further hybridizations with implications for the genetic integrity of deer stocks.
• 4 Sightings of free‐roaming muntjac deer were first recorded in 2007. The distribution of confirmed sightings of single and multiple animals in the eastern region of Ireland suggests multiple releases.
• 5 Deer are already impacting on both the economic and biodiversity values of habitats in Ireland, where, at present, no sustainable deer management policy exists.

     

Immunoendocrine interactions during chronic cysticercosis determine male mouse feminization: role of IL-6

Taenia crassiceps cysticercosis results in an impressive feminization in male mice during chronic infection, characterized by increased serum estradiol levels 100 times their normal values, while those of testosterone and dihydrotestosterone are decreased by 85 and 95% respectively. Concomitantly, the levels of follicle-stimulating hormone and IL-6 are increased 70 and 90 times their normal values in the infected male mice. Since a specific Th1/Th2 shift of the immune response has been previously reported during the chronic infection, and this shift may be associated with the feminization process, we proposed that this shift is induced by immunoendocrine interactions during the disease, and this gives way to a change in the initial resistance to the infection in the male mice, which become as susceptible as female mice. To confirm this hypothesis, we depleted immune system activity in two different ways: total body irradiation and neonatal thymectomy. Our results show that when immune system activity is depleted using either strategy, the male mice do not feminize, and the levels of follicle-stimulating hormone and IL-6 are inhibited. Depletion of IL-6 using IL-6(-/-) knockout mice does not produce the feminization process stated above, while restitution of the IL-6(-/-) knockout, irradiated, and thymectomized mice with murine recombinant IL-6 restores the feminization process. Expression of the IL-6 gene was found only in the testes and spleen of infected animals. Our results illustrate the importance of immunoendocrine interactions during a parasitic disease and show a possible new mechanism of parasite establishment in an initially resistant host.     

Dicyema shorti n. sp. (phylum Dicyemida) from Octopus burryi (Mollusca: Cephalopoda: Octopodidae) in the Gulf of Mexico

Nematogens and vermiform embryos of a new species of Dicyema are described from an octopus collected off Veracruz, Mexico. Dicyema shorti n. sp. is a small dicyemid species that rarely exceeds 500 microm in length. It is further characterized by the presence of 18 peripheral cells in the vermiform stages, a conical-shape calotte, and an axial cell that extends to the base of the propolar cells. Other stages in the life cycle of the parasite are not known. This is the first dicyemid to be described from Octopus burryi Voss. 1950, and also from both the southern Gulf of Mexico and the country of Mexico.     

How to grab a microtubule on the move

A recent paper in Cell highlights a second mechanism of regulation of separase in addition to the bound inhibitor securin. This second pathway involves separase phosphorylation and is dependent on CDC2.