Assembly and plasma membrane targeting of recombinant immunoglobulin chains in plants with a murine immunoglobulin transmembrane sequence

The cDNA encoding a full-length murine immunoglobulin 1 heavy chain with its native leader sequence, transmembrane and intracellular domains was introduced into transgenic plants. Transformed plants expressed the recombinant polypeptide, but, in contrast to plants expressing the heavy chain without transmembrane sequence, the protein appeared to be associated with a plant cell membrane. Extraction of the membrane-associated heavy chain required the presence of a non-ionic detergent, and immunofluorescence studies of protoplasts demonstrated surface expression of membrane Ig heavy chain on up to 40% of the cells from a transgenic leaf. In plants expressing both the membrane Ig heavy chain and its partner light chain, functional antibody was also localised to the plant cell membrane and retention of the heavy chain at this site appeared to have no effect on the efficiency of antibody assembly. This approach of localising and accumulating recombinant antibody in cell membranes may have a number of applications, including passive immunisation against plant pathogens.     

High-throughput screening and quantitative structure-efficacy relationship models of potential displacer molecules for ion-exchange systems

The stability and structure of protein-containing water-in-oil (w/o) microemulsions were investigated by using the large protein immunoglobulin G (IgG, MW 155,000) in a mixture comprised of brine, sulfosuccinic acid bis [2-ethylhexyl]ester (sodium salt), and isooctane. We explored factors affecting the initial uptake of IgG into the w/o microemulsion and its subsequent release to a solid (precipitate) phase, and the kinetics of the latter process. Influences of such parameters as pH, ionic strength, and protein concentration on the solubilization and precipitation of bovine IgG in the organic phase are described. The structure and dynamics in microemulsions containing bovine IgG were probed by using dynamic light scattering, and it was found that the presence of IgG in the microemulsion induced strong attractive forces between the droplets. Based on results obtained by using these various experimental approaches, a model for protein solubilization and release is proposed. In this model, we propose the formation of clusters within which bovine IgG resides and which substantially slow the kinetics of protein release from the droplets to the precipitate phase.     

Spectroimmunochemistry using colloidal gold bioconjugates

Using surface-enhanced infrared absorption (SEIRA) spectroscopy of dry films of colloidal gold (CG) bioconjugates with protein A, it is shown that certain characteristic bands of the protein (e.g., amide I, amide II and some other vibration modes) are essentially affected by the metal surface. Thus, the method may be used for controlling the quality of such bioconjugates. Moreover, it is demonstrated that the biospecific reaction of protein A attached to CG particles with human immunoglobulin G (IgG) results in further essential changes in SEIRA spectra, providing a means for an easy and rapid IR spectroscopic detection of biospecific immunochemical interactions (i.e., spectroimmunochemistry). The results obtained can form a basis for developing test systems for detecting various biospecific interactions.     

Folding and oxidation of the antibody domain C(H)3

The non-covalent homodimer formed by the C-terminal domains of the IgG1 heavy chains (C(H)3) is the simplest naturally occurring model system for studying immunoglobulin folding and assembly. In the native state, the intrachain disulfide bridge, which connects a three-stranded and a four-stranded beta-sheet is buried in the hydrophobic core of the protein. Here, we show that the disulfide bridge is not required for folding and association, since the reduced C(H)3 domain folds to a dimer with defined secondary and tertiary structure. However, the thermodynamic stability of the reduced C(H)3 dimer is much lower than that of the oxidized state. This allows the formation of disulfide bonds either concomitant with folding (starting from the reduced, denatured state) or after folding (starting from the reduced dimer). The analysis of the two processes revealed that, under all conditions investigated, one of the cysteine residues, Cys 86, reacts preferentially with oxidized glutathione to a mixed disulfide that subsequently interacts with the less-reactive second thiol group of the intra-molecular disulfide bond. For folded C(H)3, the second step in the oxidation process is slow. In contrast, starting from the unfolded and reduced protein, the oxidation reaction is faster. However, the overall folding reaction of C(H)3 during oxidative folding is a slow process. Especially, dimerization is slow, compared to the association starting from the denatured oxidized state. This deceleration may be due to misfolded conformations trapped by the disulfide bridge.     

Restriction fragment alleles of the rabbit IGHG genes with reference to the rabbit IGHGCH2 or e locus polymorphism

Among domesticated mammals, rabbit (Oryctolagus cuniculus) is the only species possessing not more than one subclass of immunoglobulin (IgG) antibodies. The rabbit IGHGCH2 or e locus presents two serologically defined alleles, the e14 and e15 allotypes, which are correlated with amino acid variation at the IgG CH2-CH3 interface. Genetic studies, while revealing the adaptive value of this polymorphism, have relied so far entirely upon allo-antisera. Here we show how these alleles can be distinguished by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) methods. The proposed PCR-RFLP approach allows the monitoring of IGHG locus diversity in rabbit.     

Estimating domain orientation of two human antibody IgG4 chimeras by crystallohydrodynamics

A modified crystallohydrodynamic approach introduced in 2001 is applied to two human IgG4 constructs from mouse IgG1. The constructs were point mutants of the chimeric antibody molecule cB72.3(4): cB72.3(4A), devoid of inter-chain disulfide bridging, and cB72.3(4P), which has full inter-chain bridging. As before, the known crystallographic structures for the Fab and Fc domains were combined with the measured translational frictional ratios to obtain an estimate for the apparent time-averaged hydration of the domains and hence for that of the intact molecule. The original approach was modified with the hydrated dimensions of the domains being applied, rather than the anhydrous crystallographic dimensions, for assessing the inter-domain orientations using the algorithms HYDROSUB and SOLPRO. Both chimeric IgG4 molecules were found to have open, rather than compact, structures, in agreement with the previous study on wild-type human IgG4. The insertion of a frictionless connector between the domains was necessary, however, for representing the cB72.3(4A) chimera. It therefore appears that the inter-chain disulfide bonds act as physical constraints in the cB72.3(4P) chimera, forcing the antibody domains together and producing a less elongated structure than that of cB72.3(4A). The open structures produced for the two IgG4 chimeras showed similarity to those structures identified for murine IgG1 and IgG2a molecules through X-ray crystallography.Presented at the conference for Advances in Analytical Ultracentrifugation and Hydrodynamics, 8–11 June 2002, Grenoble, France     

No effects of pulsed radio frequency electromagnetic fields on melatonin, cortisol, and selected markers of the immune system in man

There is growing public concern that radio frequency electromagnetic fields may have adverse biological effects. In the present study eight healthy male students were tested to see whether or not radio frequency electromagnetic fields as used in modern digital wireless telecommunication (GSM standard) have noticeable effects on salivary melatonin, cortisol, neopterin, and immunoglobulin A (sIgA) levels during and several hours after exposure. In a specifically designed, shielded experimental chamber, the circularly polarized electromagnetic field applied was transmitted by an antenna positioned 10 cm behind the head of upright sitting test persons. The carrier frequency of 900 MHz was pulsed with 217 Hz (average power flux density 1 W/m2). In double blind trials, each test person underwent a total of 20 randomly allotted 4 hour periods of exposure and sham exposure, equally distributed at day and night. The results obtained show that the salivary concentrations of melatonin, cortisol, neopterin and sIgA did not differ significantly between exposure and sham exposure.     

Inducible resistance to Fas—mediated apoptosis in B cells

Apoptosis produced in B cells through Fas(APO-1,CD95) triggering is regulated by signals derived from other surface receptors:CD40 engagement produces upregulation of Fas expression and marked susceptibility to Fas-induced cell death,whereas antigen receptor engagement,or IL-4R engagement,inhibits Fas killing and in so doing induces a state of Fas-resistance,even in otherwise sensitive,CD40-stimulated targets.Surface immunoglobulin and IL-4R utilize at least partially distinct path ways to produce Fas-resistance that differentially depend on PKC and STAT6,respectively.Further,surface immunoglobulin signaling for inducible Fas-resistance bypasses Btk,requires NF-κB,and entails new macromolecular synthesis.Terminal effectors of B cell Fas-resistance include the known anti-apoptotic gene products,Bcl-XL and FLIP,and a novel anti-apoptotic gene that encodes FAIM (Fas Apoptosis Inhibitory Molecule).faim was identified by differential display and exists in two alternatively spliced forms;faim-S is broadly expressed,but faim-L expression is tissue-specific.The FAIM sequence is highly evolu tionarily conserved,suggesting an important role for this molecule throughout phylogeny.Inducible resistance to Fas killing is hypothesized to protect foreign antigen-specific B cells during potentially hazardous interactions with FasL-bearing T cells,whereas autoreactive B cells fail to become Fas-resistant and are deleted via Fas-dependent cytotoxicity.Inadvertent or aberrant acquisition of Fas-resistance may permit autoreactive B cells to escape Fas deletion,and malignant lymphocytes to impede anti-tumor immunity.     

Hydrogen exchange mass spectrometry reveals protein interfaces and distant dynamic coupling effects during the reversible self-association of an IgG1 monoclonal antibody

There is a need for new analytical approaches to better characterize the nature of the concentration-dependent, reversible self-association (RSA) of monoclonal antibodies (mAbs) directly, and with high resolution, when these proteins are formulated as highly concentrated solutions. In the work reported here, hydrogen exchange mass spectrometry (HX-MS) was used to define the concentration-dependent RSA interface, and to characterize the effects of association on the backbone dynamics of an IgG1 mAb (mAb-C). Dynamic light scattering, chemical cross-linking, and solution viscosity measurements were used to determine conditions that caused the RSA of mAb-C. A novel HX-MS experimental approach was then applied to directly monitor differences in local flexibility of mAb-C due to RSA at different protein concentrations in deuterated buffers. First, a stable formulation containing lyoprotectants that permitted freeze-drying of mAb-C at both 5 and 60 mg/mL was identified. Upon reconstitution with RSA-promoting deuterated solutions, the low vs. high protein concentration samples displayed different levels of solution viscosity (i.e., approx. 1 to 75 mPa.s). The reconstituted mAb-C samples were then analyzed by HX-MS. Two specific sequences covering complementarity-determining regions CDR2H and CDR2L (in the variable heavy and light chains, respectively) showed significant protection against deuterium uptake (i.e., decreased hydrogen exchange). These results define the major protein-protein interfaces associated with the concentration-dependent RSA of mAb-C. Surprisingly, certain peptide segments in the V_H domain, the constant domain (C_H2), and the hinge region (C_H1-C_H2 interface) concomitantly showed significant increases in local flexibility at high vs. low protein concentrations. These results indicate the presence of longer-range, distant dynamic coupling effects within mAb-C occurring upon RSA.     

Therapeutic antibody engineering by high efficiency cell screening

In recent years, several cell-based screening technologies for the isolation of antibodies with prescribed properties emerged. They rely on the multi-copy display of antibodies or antibody fragments on a cell surface in functional form followed by high through put screening and isolation of cell clones that carry an antibody variant with the desired affinity, specificity, and stability. Particularly yeast surface display in combination with high-throughput fluorescence-activated cell sorting has proven successful in the last fifteen years as a very powerful technology that has some advantages over classical generation of monoclonals using the hybridoma technology or bacteriophage-based antibody display and screening. Cell-based screening harbours the benefit of single-cell online and real-time analysis and characterisation of individual library candidates. Moreover, when using eukaryotic expression hosts, intrinsic quality control machineries for proper protein folding and stability exist that allow for co-selection of high-level expression and stability simultaneously to the binding functionality. Recently, promising technologies emerged that directly rely on antibody display on higher eukaryotic cell lines using lentiviral transfection or direct screening on B-cells. The combination of immunisation, B-cell screening and next generation sequencing may open new avenues for the isolation of therapeutic antibodies with prescribed physicochemical and functional characteristics.