Fc intermediate (FcI), a papain-generated fragment of human IgG, intermediate in charge, molecular weight and cleavage between the Fc and Fc' fragments of IgG (original) (raw)
Related papers
Journal of The American Society for Mass Spectrometry
Immunoglobulins, such as immunoglobulin G (IgG), are of prime importance in the immune system. Polyclonal human IgG comprises four subclasses, of which IgG1 and IgG2 are the most abundant in healthy individuals. In an effort to develop an absolute MALDI-ToF-MS quantitative method for these subclasses and their Fc N-glycoforms, (glyco)peptides were synthesized using a solid-phase approach and used as internal standards. Tryptic digest glycopeptides from monoclonal IgG1 and IgG2 samples were first quantified using EEQYN(GlcNAc)STYR and EEQFN(GlcNAc)STFR standards, respectively. For IgG1, a similar glycopeptide where tyrosine (Y) was isotopically labelled was used to quantify monoclonal IgG1 that had been treated with the enzyme Endo-F2, i.e., yielding tryptic glycopeptide EEQYN(GlcNAc)STYR. The next step was to quantify single subclasses within polyclonal human IgG samples. Although ion abundances in the MALDI spectra often showed higher signals for IgG2 than IgG1, depending on the spotting solvent used, determination of amounts using the newly developed quantitative method allowed to obtain accurate concentrations where IgG1 species were predominant. It was observed that simultaneous analysis of IgG1 and IgG2 yielded non-quantitative results and that more success was obtained when subclasses were quantified one by one. More experiments served to assess the respective extraction and ionization efficiencies of EEQYNSTYR/EEQFNSTFR and EEQYN(GlcNAc)STYR/ EEQFN(GlcNAc)STFR mixtures under different solvent and concentration conditions.
Isolation of a Fragment, Fh, Corresponding to the Hinge Region of Human IgG3
Scandinavian Journal of Immunology, 1974
An Fh fragment corresponding to the hinge region of IgG3 has been isolated by a single 7.'Chymotrypsin treatment of whole IgG3. Various combinations of two enzymes have also been used, but with somewhat lower yield and less purity. The isolated Fh fragment has features similar to those of the bound hinge region in the intact IgG3 and its F(ab')2 and Fch fragments. The Fh fragment ha.s a characteristically high content of cystine and proline and seems to lack aromatic amino acids.
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.
The role of carbohydrate in the assembly and function of polymeric IgG
Molecular Immunology, 2000
The carbohydrate present on glycoprotein can influence their biologic and functional properties. In the present paper we have assessed the role of oligosaccharides in the polymerization and effector functions of IgG with the 18 amino acid extension of IgM added to its carboxy terminus (IgGmtp). We found that IgG1mtp and IgG3mtp lacking the carbohydrate addition site in C H 2, in the tail-piece or both assembled into polymers as well as the glycosylated versions. Aglycosylated polymers retained the ability to activate complement as assayed by C1q binding and hemolysis, although they were not as effective as their wild type polymer counterparts. Although IgGmtp lacking the carbohydrate in the tail-piece was able to bind to FcgRII, completely aglycosylated polymers lost the ability to bind to both FcgRI and FcgRII, suggesting a critical role for the C H 2 sugar in FcR binding. Absence of the mtp carbohydrate increased the half life of polymeric IgG1, whereas absence of the carbohydrate in C H 2 accelerated the clearance rate.
Biochemistry, 1984
The carbohydrate attached at Asn-107 of the light chain of a human myeloma IgGIK (Hom) was isolated and the structure determined by 'H NMR. Two oligosaccharides were found corresponding to mono-and disialylated forms of the bisected biantennary class of glycopeptides. Both structures had F u c a l d linked to the GlcNAc residue attached to Asn and NeuNAc residues linked a2-6. Because of the unusual nature of these structures, the Asn-297 oligosaccharides of the same IgG were prepared from Fc fragments and heavy chains. Comparison of the structures of the latter glycopeptides with structures from the same site on a second human myeloma IgGIK (Tem) showed them to be quite similar in that the majority of the structures were biantennary but not bisected. We suggest that the completely bisected nature of the lightchain oligosaccharides comes from a high level of activity of I n an earlier paper , a human IgGlk monoclonal protein (Hom) was described in which some of the L chains were glycosylated at Asn-107 within the J region. The absence of carbohydrate on a proportion of the chains was not due to a lack of the appropriate acceptor sequence but may have reflected a competition between protein folding and the transfer of core sugars from dolichol pyrophosphate oligosaccharide. In this paper, we describe the determination .of the structure of the carbohydrate of the L chain and a comparison with the structure of the carbohydrate present on the CYz domain from the same protein. The latter structures were found to be similar to those determined for human myeloma IgG(Tem) , whereas the carbohydrate from the L chain of IgG(Hom) was found to have a relatively rare structure belonging to the sialylated bisected biantennary class.
Glycobiology, 2000
We have now produced mouse-human chimeric IgG1 in wild-type Chinese hamster ovary (CHO) cell lines Pro-5 as well as in the glycosylation mutants Lec 2, Lec 8, and Lec 1. Analysis of the attached carbohydrates shows those present on IgG1-Lec 1 were mannose terminated. Carbohydrate present on IgG1-Lec8 was uniformly biantennary terminating in N-acetylglucosamine. The glycosylation profiles of IgG1-Lec 2 and IgG1-Pro-5 were heterogeneous. Only IgG1-Pro-5 was sialylated with sialic acid present on only a small percentage of the carbohydrate structures. When the in vivo fate of antibodies labeled with 125 I-lactotyramine was determined, it was found that the majority of all of the antibodies, irrespective of the structure of their attached carbohydrate, is catabolized in the skin and muscle. However, the attached carbohydrate structure does influence the amount that is catabolized in the liver and the liver serves as a major site for the catabolism of proteins bearing carbohydrate with the Lec2 (with terminal galactose) or Lec1(with terminal mannose) structure.
Fc fragment from human IgG induces PGE2 secretion
Cellular Immunology, 1989
In humans, in vitro, Fc fragment of IgG at a low concentration induces plasma cell generation. However, Fc fragment at a high concentration induces PGEr release of monocyte activation capable of inhibiting this differentiation. The levels of PGEz in the suspematant culture from mononuclear cells from normal donors were examined as a function of culture duration and concentration of Fc, Fab fragments and IgG. Plasma cells containing intracytoplasmic Ig were demonstrated by immunofluorescence with a polyvalent antiserum to human immunoglobulin(s). PGE2 levels, from mononuclear cell cultures, were analyzed by the RIA test. The results indicated that the Fc fragments are able to induce PGE2 secretion. The maximal release of PGE2 occurs after 24 hr of culture; this level is proportionate to the quantity of Fc fragments introduced. The addition of indomethacin in the cell culture stimulated by a high concentration of Fc fragments reestablishes the percentage of plasma cells. These results suggest the regulatory role of Fc fragment by PGE, secretion in B cell differentiation. o 1989 Academic press, IDC.
Electrophoretic evidence for the presence of structural isoforms specific for the IgG2 isotype
ELECTROPHORESIS, 2008
Recombinant monoclonal antibodies of therapeutic interest were analyzed by a nonreduced CE-SDS (nrCE-SDS) method developed for the evaluation of size-based variants. We found that immunoglobulins analyzed by this technique exhibited different behavior depending on their subclasses. Under nrCE-SDS conditions, IgG1 molecules were separated in a wellresolved, single peak, whereas IgG2 molecules were consistently separated as a doublet. Investigation of these isoforms showed that they were structurally different, and that the difference was not caused by cell culture condition, glycosylation structure, or recombinant expression system. Commercially available IgG2 affinity-purified from human plasma also showed the presence of structural isoforms. The structural isoforms remained present under pH-and temperature-stressed conditions. Application of a mild cysteine/cystine redox potential converted the main peak doublet into a single peak, indicating that these isoforms were disulfide bond-related species. Bioactivity measured before and after application of a redox potential gave similar values, indicating that the structural isoforms have comparable potency. The nrCE-SDS technique described here demonstrated a unique capability to resolve IgGs, leading to the discovery of novel structural isoforms specific to the IgG2 isotype.
Molecular Immunology, 1996
Fibronectin (Fn), a mosaic protein composed of multiple copies of three different module types (Fl, F2 and F3), has been found associated with circulating immune complexes (ICs) and immunoglobulin (Ig) aggregates in a variety of IC diseases and myeloproliferative disorders. We have previously shown that a proteolytic fragment of M,=25,900 Da, from the NH,-terminal domain of Fn, composed of five type 1 modules ('Fl-'Fl) binds to the major Ig classes under physiologic conditions, suggesting that the presence of Fn in ICs and cryoglobulins results from a physicochemical binding interaction between these two molecules. Using an ELISA, we now show that the interaction between Fn and IgG is: (1) not influenced by any other constituent of plasma; (2) unaffected by temperature;