Intracellular distribution and degradation of immunoglobulin G and immunoglobulin G fragments injected into HeLa cells (original) (raw)
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Conformation of human IgG subclasses in solution
European Journal …, 1985
The structure of six human myeloma proteins: IgGl(Bal), IgG2(Klu), IgG3(Bak), IgG3(Het), IgG4(Kov) and IgG4(Pol), was studied in solution using small-angle X-ray scattering and hydrodynamic methods. For IgGl (Bal) and IgG3(Het) the experimental data, including radius of gyration (R",), radii of gyration of the cross-section (Rql, RqJ, intrinsic viscosity [q], sedimentation coefficient SO^^,^) and molecular mass, were interpreted in terms of structural models based on the Fab and Fc conformations, observed in crystal, by varying the relative positions of the Fab and Fc parts, i.e. their relative angles and distances.
European Journal of Biochemistry, 1985
The structure of six human myeloma proteins: IgGl(Bal), IgG2(Klu), IgG3(Bak), IgG3(Het), IgG4(Kov) and IgG4(Pol), was studied in solution using small-angle X-ray scattering and hydrodynamic methods. For IgGl (Bal) and IgG3(Het) the experimental data, including radius of gyration (R",), radii of gyration of the cross-section (Rql, RqJ, intrinsic viscosity [q], sedimentation coefficient SO^^,^) and molecular mass, were interpreted in terms of structural models based on the Fab and Fc conformations, observed in crystal, by varying the relative positions of the Fab and Fc parts, i.e. their relative angles and distances.
European Journal of Nuclear Medicine, 1992
The binding parameters of iodine-125-1abelled intact monoclonal immunoglobulin G (IgG), F(ab')2 and Fab' fragments were compared. The study was carried out with the two monoclonal antibodies (MoAbs) K13 and K16 specific for human Ig light chains K and 2, respectively. When testing the 125I-MoAbs against monodisperse polymer particles coated with the specific antigens, the K a for the F(ab')2 fragments were similar to that for IgG, while the K a for the Fab / fragments were reduced to 10%-20% of that for IgG. The number N of effective target sites revealed with Fab' was higher than with F(ab')2 and IgG, presumably because less surface area is occupied by the small Fab' molecules. The immunoreactive fraction F ranged according to IgG> F(ab')2 >Fab I. The explanation of the moderate difference between the Ka of the monoclonal Fab' and the divalent IgG and F(ab')2 was that the divalent molecules were not divalently attached to the particles. When testing the same antibody preparations against human lymphoma cells producing Ig with light chains K or 2, the binding results were less reliable than when particles were utilised, presumably due to antigen shedding. Different MoAbs vary in their loss of immunoreactivity due to enzymatic degradation and the radiolabelling procedure. The preparation of the radiolabelled fragments should therefore be optimized for each MoAb, and evaluation is necessary before injection. Artificial targets with a low leakage of antigen, like the monodisperse polymer particles here applied, are recommended for the in vitro evaluation of the immunoreactivity of labelled MoAb preparations.
International Immunology, 1996
Murine mAb injected into patients behave as exogenous antigens and trigger a specific immune response characterized mainly by CD4 + T lymphocytes. They are recognized by T cells under a processed form and in a MHC class ll-restricted fashion. IgG degradation which occurs in antigenpresenting cells (APC) has been studied in vitro. We have shown that partial reduction of this antigen is an early event which is significant for the generation of class ll-restricted fragments presentable to antigen-specific T cells. Two different murine mAb were used as antigens and human monocytic U937 or antigen non-specific Epstein-Barr virus-transformed B cells as APC. Upon cellular internalization IgG are rapidly cleaved leading to 24-25 kDa fragments. One of the major and early events corresponds to partial reduction of IgG-the light chain is released from the intact molecule, heavy chains remaining bound together. Partial in vitro reduction of IgG followed by presentation by fixed B cells to specific T cells showed that only K light chain-specific T cell clones are stimulated, in contrast to heavy chain-specific T cell clones. The response to the K chains of specific T cells points to a significant role played by the early IgG partial reduction in the generation of K light chain class II binding fragments. In a previous study we have shown that protein C3 of the complement system, covalently linked to IgG, can up-regulate
Molecular Immunology, 1985
Fc intermediate (Fq) is a papain-generated fragment of human IgG which is intermediate in charge, mol. wt and state of cleavage between the Fc and Fc' fragments of IgG. It is composed of two polypeptide chains of unequal mol. wt held together by non-covalent bonds between the Cy 3 regions. The larger polypeptide chain has both a Cy2 and Cy3 domain and its N-terminus is at Leu 235 (60%) and Leu 234 (40%) (IgGl Eu numbering). The smaller polypeptide chain is composed of a Cy3 domain with its N-terminus at Gly 341. The carboxy-termini obtained by carboxypeptidase digestion and by a computer program which determined the most probable sequences by fitting the amino acid compositions to the sequence of IgG Eu Fc were heterogeneous involving residues 44&446 for the large polypeptide chain and 4299436 for the small one. The calculated mol. wt of the large polypeptide chain was 26,183, assuming the N-terminus at Leu 234 and C-terminus at 446 and including the carbohydrate moiety. The calculated mol. wt for the small polypeptide chain was 10,682, with the N-terminus at 341 and assuming the C-terminus at 434, for a combined mol. wt of 36,865 for the Fc, fragment. Sedimentation equilibrium ultracentrifugation of Fc, under non-dissociating conditions showed an M, of 36,200 + 1200, an M,.
Characterization of a heat-stable fraction of human IgG
Journal of Protein Chemistry, 1986
Heat aggregation of human IgG has been studied by photon correlation spectroscopy, ultracentrifugation, circular dichroism, and differential scanning calorimetry. It is found that pooled human IgG can be separated into two fractions of molecules, one that easily aggregates and one that is stable upon heating. In a buffer at pH = 7.6 and 0.2 M NaCl it is found that about half of the original monomeric molecules do not aggregate even after heating at 62°C for 24 h. No differences in the antigen binding capacity of the heat-stable fraction and normal lgG are observed. Heat-stable molecules can partially be transformed to heat-aggregating molecules by a rapid acid denaturation followed by neutralization. Differential scanning calorimetry shows that the major heat denaturation, which is a two-phase process at pH= 7.6, starts at about 63°C. Only minor differences between the heat-stable and the heat-labile fractions are observed in the thermograms. No differences are observed in the far-UV region of the CD spectra, indicating that the secondary structure of the heat-stable IgG does not differ from the native IgG molecule. While the aggregation of normal human IgG can be described by Smoluchowski kinetics, the heat-stable fraction follows another kinetics, which includes an activation step.
International Journal of Radiation Applications and Instrumentation. Part B. Nuclear Medicine and Biology, 1989
The monoclonal IgM 3G5, which reacts with the surface membranes of rat insulinoma cells RINmSF, was purified by HPLC and labeled with '*'I using Protag-125; bovine IgG (bIgG) was similarly radiolabeled, and used as a control. '*'1-3G5 was incubated with RINmSF cells either at 4°C or at 37°C. lZSI-3G5 bound onto RINmSF cells growing in Petri dishes remained approx. constant over 44 h when incubated at 4°C. whereas at 37°C radioactivity was released back in the medium starting at 3 h (plateau at approx. 20 h). At the end of incubation at 37°C. activity in the medium included a high percentage of free 12'1 (15.69 vs 2.62% for bIgG, and 1% for 3G5 at 4°C). In a cell suspension experiment, cell-bound '251-3GS also remained constant over a 24 h incubation at 4"C, whereas at 37°C it decreased to 37.5% of its initial value (64.1% at 4 h). Concomitant microautoradiography showed diffuse radioactive deposits within the RINmSF cells following incubation with '251-3G5 (but not '2SI-bIgG) at 37°C. These results indicate that 3G5-IgM reacts with a surface antigen on the RINmSF cells, but is rapidly internalized by the cells: within the cells, this antibody undergoes some metabolic processing which results in the release of free '25I outside the cells.