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Query: UNIPROT:P06889 (Mol)
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The hinge region of the immunoglobulin molecule is responsible for antigen-binding and cross-linking reactions, varying the distance between the two antigen-binding sites. As the amino acid sequence of the hinge region is identical among immunoglobulin molecules of the same (sub)class, it has been regarded as a constant region. By comparison of the nucleotide sequences among primate C alpha genes, it is clear that there is a wide variety of length among the hinge regions of hominoid C alpha genes, which basically consist of tandem repeats of a 15 base-pair sequence. This reiterated structure probably facilitates rapid evolutionary changes in the length of the hinge region. The hinge region of the Old World monkey C alpha gene has a non-reiterated structure whose nucleotide sequence is quite different from those of the hominoid C alpha genes, although its surrounding region is conserved during evolution. This unusual hypervariability reveals that the hinge region has evolved as a semi-variable region in contrast to its constant character from an ontogenic viewpoint.
J Mol Biol 1990 Sep 20
PMID:Evolutionary hypervariability in the hinge region of the immunoglobulin alpha gene. 212 Apr 47

Evidence from several experimental approaches allows us to conclude that the primary amino acid sequence of the lower hinge region (residues 234-237) of human IgG molecules determines recognition by human Fc gamma RI, Fc gamma RII and Fc gamma RIII. Glycosylation of the CH2 domain is also essential, although the carbohydrate is not accessible for direct interaction with ligands. The role of the carbohydrate moiety may be to maintain a protein conformation that allows accessibility to amino acid side chains essential for ligand recognition and binding. It appears logical that the evolutionarily-related Fc gamma R molecules should interact with overlapping non-identical sites on the IgG molecule.
Mol Immunol 1990 Dec
PMID:Molecular definition of interaction sites on human IgG for Fc receptors (huFc gamma R). 214 8

We have characterized the activities of the cytochrome bc1 complex in mitochondrial membranes from a yeast strain in which we deleted the nuclear gene (QCR6, COR3) which codes for the highly acidic subunit 6 of the bc1 complex. The chromosomal copy of QCR6 was replaced with a plasmid derived copy of QCR6, in which the entire coding region of QCR6 was replaced with the yeast LEU2 gene. The resulting deletion strain, MES8, contained no detectable mRNA for QCR6, and the cytochrome bc1 complex purified from the deletion strain lacked subunit 6. The deletion strain respired and grew on nonfermentable carbon sources such as ethanol and glycerol. Ubiquinol-cytochrome c reductase activity of mitochondria from the deletion strain was decreased 50% under conditions where the activity is zero order with respect to cytochrome c, and there was a similar decrease in the first-order rate constant for cytochrome c reduction. The loss of bc1 complex activities, observed at physiological ionic strengths, was reversible. Both the zero order rate and the first-order rate constant for cytochrome c reduction could be recovered to those of the parental strain by measuring these activities in mitochondrial membranes under conditions of low ionic strength. The zero order rate and first-order rate constant for cytochrome c reduction in membranes from the parent, wild-type yeast showed essentially no change coincident with this change in ionic strength. The 50% drop in both turnover number and first-order rate constant of ubiquinol-cytochrome c reductase activity indicates that half of the cytochrome bc1 complexes are inactive in the deletion strain at physiological ionic strengths. Inhibition by myxothiazol of cytochrome c reductase activity of mitochondrial membranes from the deletion strain showed an ionic strength-dependent lag in the titration curve that extended to the point where half of the inhibitor sites are filled. This lag was not observed with membranes from the wild-type, parent strain. This response to the inhibitor is consistent with half of the cytochrome bc1 complexes being inactive in mitochondria from the deletion strain at physiological ionic strength, but with both active and inactive complexes still able to bind inhibitor. The reversible, half-of-the-sites reactivity indicates that the bc1 complex must be dimeric in situ, in agreement with previous findings that the complexes isolated from fungal (Leonard, K., Wingfield, P., Arad, T., and Weiss, H. (1981) J. Mol. Biol. 149, 259-274) and mammalian (Nalecz, M. J., Bolli, R., and Azzi, A. (1985) Arch. Biochem. Biophys. 236, 619-628) mitochondria are structural dimers.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Subunit 6 regulates half-of-the-sites reactivity of the dimeric cytochrome bc1 complex in Saccharomyces cerevisiae. 217 Mar 63

Glycosylated chimeric mouse-human anti-NIP IgG3 antibody produced by growth of the J558L mouse B cell plasmacytoma is characterised with respect to the single carbohydrate chain at Asn-297 in the CH2 domain indicating that the mouse cell glycosyl transferases dictate the pattern of glycosylation rather than the human CH region of the heavy chain. Additionally, three unusual alpha-galactose-containing oligosaccharides are reported. Only the Fc region has detectable carbohydrate. Aglycosylated anti-NIP IgG3 antibody has been produced by cell growth in the presence of the antibiotic tunicamycin. Functionally, whilst the glycosylated intact IgG3 interacts with human Fc gamma R111 expressed on human killer (K) cells to trigger antibody-dependent cellular cytotoxicity the aglycosylated intact IgG3 fails to trigger cell lysis, localising the site on IgG for triggering human Fc gamma R111 mediated functions to the CH2 domain. The monomeric aglycosylated trypsin Fc fragment inhibits human Fc gamma R1 recognition by U937 cells 115-fold less well (K50 = 2 microM) than does glycosylated Fc (K50 = 17 nM), confirming that aglycosylation disrupts the site for human Fc gamma R1 within the CH2 domain and indicating that the trypsin Fc fragments reflect the functional properties of the intact IgG glycoforms. Structurally, 1H NMR shows that the absence of carbohydrate at Asn-297 results in a small and localised protein structural change in the vicinity of the reporter group His-268 within the CH2 domain. The site on IgG for triggering human Fc gamma R111 mediated functions is then localised to the vicinity of His-268. The profound impact of aglycosylation on human Fc gamma R1 recognition implies structural disruption of the proposed site for human Fc gamma R1 in the lower hinge region of IgG (residues 234-239), proximal to His-268.
Mol Immunol 1990 Nov
PMID:A protein structural change in aglycosylated IgG3 correlates with loss of huFc gamma R1 and huFc gamma R111 binding and/or activation. 217 19

The proteolytic Fab fragment of the well characterized antibody McPC603 was compared to the recombinant Fab fragment, which was obtained in functional form from an Escherichia coli expression system [(1989) Methods Enzymol. 178, 497-515]. We found evidence that the proteolytic fragment is glycosylated at Asn H160 in the CH1 domain, where additional electron density had been observed in the crystal structure [J. Mol. Biol. 190, 593-604]. In addition, its heavy chain is about 30 amino acids longer than visible in the electron density and thus contains the complete hinge region. These structural differences between the recombinant Fab fragment, which had been designed exactly according to the defined electron density, and the proteolytic Fab fragment of McPC603 had no effect on the hapten binding properties of these antigen binding fragments. Yet, it may be important to be aware of these structural features of McPC603 in folding studies and some comparative analyses of antibody structures.
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PMID:Structural features of the McPC603 Fab fragment not defined in the X-ray structure. 222 4

Cleavage of mouse IgA T15 with papain yielded (a) a glycosylated Fab fragment, (b) a non-glycosylated Fc fragment and (c) a glycosylated C-terminal peptide. The cleavage sites at the hinge and at the end of the C alpha 3 domain were located by sequencing. The two glycopeptides were prepared from the Fab and C-terminal fragments by pronase digestion. The C alpha 1 glycopeptide at Asn 155 was complex type with alpha (1-3)galactose terminal groups, and closely resembled the Asn 171 glycopeptide of mouse IgM (Anderson et al. (1985) Arch. Biochem. Biophys. 243, 605-618). In contrast, the C-terminal glycopeptide at Asn 446 was entirely different from the corresponding IgM glycopeptide, being complex rather than high-mannose type.
Mol Immunol 1990 Nov
PMID:The glycopeptides of the mouse immunoglobulin A T15. 224 87

We have studied by electron microscopy a fascinating series of antidansyl monoclonal antibodies developed by Dangl et al. (Cytometry 2, 395-401, 1982) which have the same variable domain but different constant domains. Three of the four subclasses of mouse IgG were represented, IgG1, IgG2a and IgG2b. Previously, Oi et al. (Nature 307, 136-140, 1984) had examined the flexibilities of these antibodies by time-resolved fluorescence depolarization and found that IgG1 was least flexible, IgG2a was intermediate and IgG2b was the most flexible. In this communication we examine the conformations of circular complexes formed between these antibodies and a bivalent hapten, bis-dansyl cadaverine. The circular complexes were predominantly composed of two antibodies linked into a ring by two bivalent haptens, and are referred to as dimers, since only the antibody molecules are seen with the electron microscope. A few trimers and an occasional tetramer were also present in these preparations. For the least flexible IgG1, almost all (greater than 99%) of the circular dimers were "open-hinge" complexes with a hinge angle between the Fab arms of 100-120 degrees. For the intermediate IgG2a, most of the dimers were "open-hinge" complexes, but a larger percentage, 4 to 5%, had closed hinges with a hinge angle approaching 0 degrees. For the most flexible IgG2b, over 40% of the dimers were "closed-hinge" complexes. A model is proposed to explain these differences based upon orientation of the hapten in the combining site and differences in hinge structure.
Mol Immunol 1990 Feb
PMID:Electron microscopic study of ring-shaped, bivalent hapten, bivalent antidansyl monoclonal antibody complexes with identical variable domains but IgG1, IgG2a and IgG2b constant domains. 232 11

A reproducible method has been developed for the negative staining of myosin molecules. The dimensions of stained molecules are in close agreement with those obtained by metal shadowing. Sharp bends in the tail, indicative of hinge regions, were observed at two positions 44 nm and 76 nm from the head-tail junction. The tail was often ill-defined at the position of the first (44 nm) bend. The bend positions may be sites of proteolytic cleavage that result in the production of long and short myosin subfragment S2. About half the molecules exhibited bending to various degrees at one or both of these positions, but cases where the tail folded back on itself in a 180 degrees bend were comparatively rare (approximately equal to 10%). However, in the absence of EGTA, a large fraction of the molecules (approximately equal to 80%) exhibited 180 degrees bends. A small region, approximately 20 nm long, at the tip of the tail often appears to be significantly different from the rest. The heads are about 19 nm long and roughly pear-shaped. Although sometimes straight, more often they show a pronounced curvature. Both senses of curvature were observed, but those curved in a clockwise manner were the most common, indicating preferential binding of one side of the head to the carbon substrate. An analysis of the different combinations of head shapes in individual molecules indicates that each head can rotate independently around its long axis. No preferred angle of orientation between the two heads in a molecule, or between either head and the tail could be found. Substructure has been observed within the heads.
J Mol Biol 1985 Aug 05
PMID:Negative staining of myosin molecules. 241 17

We have analysed and compared the fine specificity and behavior in various immunoassays of 10 mouse monoclonal antibodies, from three independent laboratories, directed against IgA1, IgA2 or non-IgA2m(2). The following observations were made. (1) Although all of the monoclonal antibodies were specific for a particular IgA subclass or isoallotype in a radioimmunoassay, three of them were not specific when tested in indirect immunofluorescence on plasma cells derived from pokeweed-activated peripheral blood lymphocytes. In this highly sensitive system, contrary to direct immunofluorescence previously performed using formalin-fixed lymphoid tissue, the anti-IgA1 69.114 reacted with some of the IgA2 plasma cells, the anti-IgA2 DLDB7 reacted with some of the IgA1 plasma cells and the anti-IgA2 16.512 dimly reacted with all IgM plasma cells. (2) Among the eight anti-IgA subclass antibodies, seven were directed against the CH2 domain of IgA whereas the anti-IgA1 1-155-1 recognised an epitope destroyed by Streptococcus sanguis IgA1 protease and localised in the hinge region of IgA1. The two anti-isoallotype antibodies were directed against epitope(s) probably localised in the 65 C-terminal amino acid residues of the alpha-CH3 domain. All of the 10 antibodies were able to react with endogeneously produced surface IgA on B-cells. (3) Using monoclonal anti-IgA subclass antibodies in radioimmunoassay may be hazardous in the absence of knowledge of their affinity constants and of careful control experiments: some of the antibodies were not sensitive in radioimmunoassays designed to measure the serum titer of specific IgA1 and IgA2 antibodies. Moreover, major differences were observed between the different monoclonal reagents with respect to the influence of the size of IgA on a solid-phase sandwich radioimmunoassay. While three of the anti-IgA1 underestimated dimeric IgA relative to monomeric IgA, the fourth anti-IgA1 and all the anti-IgA2 overestimated dimeric IgA relative to monomeric IgA, by a factor sometimes close to 7.
Mol Immunol 1986 Apr
PMID:Monoclonal antibodies against isotypic and isoallotypic determinants of human IgA1 and IgA2: fine specificities and binding properties. 242 48

It has previously been demonstrated that exposure of polyclonal IgG to free radicals results in denaturation evidenced by aggregation, auto-fluorescence and destruction of cysteine, proline and aromatic amino acids. In the present study we have used a panel of monoclonal antibodies (McAb) to epitopes expressed on the IgG3 heavy chain to detect changes in antigenicity. When IgG3 was exposed to u.v. irradiation, as a source of free radicals, subclass specific epitopes were rapidly lost whilst other epitopes were unaffected. Prolonged exposure resulted in further denaturation and a progressive loss of expression of further epitopes. The IgG3 subclass specific McAb are specific to epitopes localized to the hinge region of IgG3. Thus, this exposed cysteine and proline rich region is shown to be particularly vulnerable to free radical attack; however, prolonged exposure results in structural alterations throughout the heavy chain.
Mol Immunol 1987 Nov
PMID:Immunogenic and antigenic epitopes of immunoglobulin--XX. Denaturation of human IgG3 by free radicals. 244 91


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