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1.
The disulphide bridges of a mouse immunoglobulin G1 protein 总被引:7,自引:3,他引:4
[(35)S]Cystine-labelled immunoglobulin MOPC21 (IgG1) was prepared from myeloma cells in tissue culture. Carrier myeloma protein was added and the protein was digested with pepsin. The digest was fractionated on Sephadex G-50 into two fractions, further digested with trypsin and again fractionated on Sephadex. Disulphide-bridge peptides were purified by electrophoresis and chromatography and identified by radioautography. A peptide of 96 residues was isolated, which contains both the heavy-light interchain disulphide bridge and all the inter-heavy-chain disulphide bridges. Other peptides were isolated, accounting for all the intrachain disulphide bridges (which could be placed by homology with proteins of other species), except for the variable section of the light chain. Sequences describing this missing disulphide bridge were obtained from totally reduced and alkylated light chains. Peptides related to the interchain disulphide-bridge peptide were isolated from partially reduced and alkylated myeloma protein and from totally reduced heavy chain. The interchain disulphide-bridge peptide was placed at the C-terminal position of the F(ab')(2) fragment, prepared by digestion of the protein with pepsin at pH4.0. Sequences from the heavy-chain intrachain disulphide bridges of MOPC 21 immunoglobulin are compared with homologous sequences from mouse myeloma proteins of other subclasses and proteins of other species. 相似文献
2.
The relative lability of the interchain disulphide bonds of mouse G2a-myeloma protein 5563 was studied as a function of 2-mercaptoethanol concentration. Analysis of partial-reduction mixtures by polyacrylamide-gel electrophoresis and microdensitometry showed that the disulphide bonds between light and heavy chains are much more susceptible to reduction than the bonds between heavy chains. At a low concentration of 2-mercaptoethanol (10mm) the major dissociable products of mouse immunoglobulin G are heavy-chain dimers and free light chains. These findings contrast with the reported behaviour of rabbit immunoglobulin G, for which the lability of inter-heavy-chain bonds was found to exceed that of the bonds linking light and heavy chains (Hong & Nisonoff, 1965); the relative stability of rabbit immunoglobulin G interchain bonds was confirmed in the present study. Examination of human immunoglobulin G and an immunoglobulin G (γ2) of guinea pig showed that at least in the majority of molecules, as with mouse immunoglobulin G, the disulphide bonds between light and heavy chains are more susceptible to reduction than the inter-heavy-chain bonds. 相似文献
3.
Intrachain disulphide bridges in immunoglobulin G heavy chains. The Fc fragment 总被引:5,自引:2,他引:3
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The disulphide bridges of the Fc fragment (C-terminal half of the heavy chain) have been studied in several human immunoglobulins, containing heavy chains of different antigenic types (gamma1, gamma2, gamma3 and gamma4), and in heavy-chain-disease proteins. Two intrachain disulphide bridges were found to be present. The sequences appear to be identical in the Fc fragments of two types of chain studied (gamma1 and gamma3), and very similar to corresponding sequences of the Fc fragment in rabbit. These results suggest that the C-terminal half of the heavy chains is covalently folded (in a similar fashion to the light chains) with a C-terminal loop and an N-terminal loop. The similarity is emphasized by comparison of the sequence and location of the disulphide-bridged peptides of the C-terminal loop of heavy and light chains. The N-terminal loop, on the other hand, appears to be very different in Fc fragments and light chains. The C-terminal loop is the only one present in the F'c fragment. 相似文献
4.
To reveal non-covalent interactions between the Fab and Fc regions of IgG molecules the average conformational free-energy change (delta Go), associated with reversible micro-unfoldings, was measured by hydrogen-deuterium exchange for the Fab and Fc fragments and the complete molecule. Human monoclonal IgG1 and pooled IgG samples were used in these experiments. Hydrogen-deuterium exchange data were summarized and compared in the form of exchange relaxation spectra. The experimentally observed relaxation spectrum of intact IgG could not be deduced by weighted summation of spectra measured for Fab and Fc fragments. A comparison of the measured and calculated data revealed a 5-kJ/mol increase in the conformational free energy upon splitting the IgG molecule into two Fab and Fc pieces, i.e. an increase of conformational mobility occurred. This change can be explained either by related fluctuation patterns of the Fab and Fc pieces in the intact molecule or by a shielding effect on the contact surfaces. Both interpretations suppose non-covalent interactions between Fab and Fc that can be a means of information transduction between recognition and effector sites. The pH dependence of the hydrogen-deuterium exchange also indicates interactions between the Fab and Fc regions. A shift in the relaxation spectra of the Fab fragment was observed between pH 8.2 and 7.3 revealing destabilization of the structure at lower pH. This effect is absent in the intact molecule, reflecting interactions that stabilize the Fab structure. Comparison of the relaxation spectra of Fab and Fc shows a difference of about 10 kJ/mol in the microstability of these fragments: the Fab part possesses more conformational flexibility (i.e. its microstability is smaller) than the Fc part. 相似文献
5.
The disulphide bridges of immunoglobulin κ-chains 总被引:5,自引:5,他引:0
C. Milstein 《The Biochemical journal》1966,101(2):338-351
The arrangement of the disulphide bridges of the major component of the light chains of immunoglobulins (kappa-chains) has been studied in the Bence-Jones proteins. Three disulphide bridges have been found. An interchain bridge at the C-terminus has been shown to occur in the dimers of all the proteins studied and was characterized by symmetrical peptides. In the monomer form, the C-terminal half-cystine of the corresponding peptides was linked to a lone half-cystine residue. A second common disulphide-bridge peptide in which a single amino acid difference could be related to the Inv factors of the individual proteins was found in Bence-Jones proteins and in the kappa-chains of normal and abnormal immunoglobulins. Peptides characteristic of a third disulphide bridge studied in three specimens were found to have differences in some residues, but also striking similarities. A methionine peptide has also been characterized in two specimens as a by-product of the technique employed. It is suggested that a general manner of folding may be a common feature of the heterogeneous population of kappa-chains: one bridge which folds an invariable stretch of the chain, another bridge which folds a stretch that varies from protein to protein, and a bridge at the C-terminus which is the interchain link. 相似文献
6.
Mouse IgM (immunoglobulin M) was selectively and partially reduced and treated with iodo[2-14C]acetate to label the interchain disulphide bridges. The carboxymethylation was studied in some detail. The labelled peptides were purified, sequenced and positioned by homology with human IgM. Only peptides originating from three interchain disulphide bridges were labelled, in contrast with the four labelled bridges obtained in human IgM under the same conditions. These peptides are homologous to human bridge peptides forming the heavy-light bridge and two inter-heavy bridges, one present in the CMU2 region and the other in the C-terminal region. The inter-heavy bridge in the Cmu2 region was alone cleaved and radioactively labelled in selectively reduced IgM held together as a pentamer by non-covalen interactions. The same bridge was the only one to be totally cleaved in subunits released after more extensive, though still selective, reduction. In the light of these results a possible arrangement of the disulphide bridges of the mouse IgM. 相似文献
7.
Occurrence, isolation and interchain bridges of natural 7-S immunoglobulin M in human serum 总被引:3,自引:0,他引:3
F Dolder 《Biochimica et biophysica acta》1971,236(3):675-685
8.
J Kellermann C Thelen F Lottspeich A Henschen R Vogel W Müller-Esterl 《The Biochemical journal》1987,247(1):15-21
Transposon mutant strains which were affected in bile acid catabolism were isolated from four Pseudomonas spp. Two of the mutant groups isolated were found to accumulate 12 alpha-hydroxyandrosta-1,4-diene-3,17-dione as the major product from deoxycholic acid. Strains in one of these two groups were able to grow on steroids such as chenodeoxycholic acid, which lacks a 12 alpha-hydroxy function, whereas the one member of the second group could not. With chenodeoxycholic acid, this latter strain accumulated a yellow muconic-like derivative, tentatively identified as 3,7-dihydroxy-5,9,17-trioxo-4(5),9(10)-disecoandrosta-1(10)2 -dien-4-oic acid. Members of two further mutant groups accumulated either 12 beta-hydroxyandrosta-1,4-diene-3,17-dione or 3,12 beta-dihydroxy-9(10)-secoandrosta-1,3,5(10)-triene-9,17-dione as the major product from deoxycholic acid. The relationship between the catabolism of m- and p-cresol, 3-ethylphenol and the bile acids was also examined. 相似文献
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11.
Disulphide bridges of the heavy chain of human immunoglobulin G2 总被引:2,自引:0,他引:2
Amino acid sequences around the disulphide bridges of the heavy chain of an immunoglobulin of the gamma2 subclass have been studied. The protein was digested with pepsin and the digest fractionated by Sephadex. Screening of the eluate by one-dimensional electrophoresis of oxidized and unoxidized samples was used as an assay and pools of fractions were prepared. Identification by diagonal electrophoresis of several inter- and intra-chain disulphide bridges was done on the pooled fractions. The inter-heavy-chain bridged peptide included four cystine residues. Comparison with proteins of other human subclasses indicated that the intrachain bridges identified are the bridges of the invariable section of gamma2 heavy chains. The amino acid sequence of one cysteic acid peptide that may have been derived from the variable part of the molecule was determined. Partial reduction followed by carboxymethylation with radioactive iodoacetate of two proteins of the gamma2 class showed a number of labelled peptides that could be identified as being related to the inter-chain bonded cystine residues. 相似文献
12.
Six peptides containing eight half-cystine residues were isolated in good yield, after either oxidation or reduction and carboxymethylation of fragment C-1, which contains the N-terminal half of the heavy chain of rabbit immunoglobulin G. The sequences of five of these peptides had been reported previously (Cebra, Steiner & Porter, 1968b; Wilkinson, 1969) and that of the sixth was established. Other peptides containing half-cystine residues were isolated in much lower yield and are presumed to be derived from minor sequence variants. The cystine-containing peptides from enzymic digests of whole immunoglobulin G and Fc fraction were studied by several techniques and the results obtained enable us to put forward a scheme of the arrangement of the inter- and intra-chain disulphide bonds. 相似文献
13.
The interchain disulfide bonds of a human pathological immunoglobulin 总被引:12,自引:0,他引:12
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15.
Yamaguchi Y Nishimura M Nagano M Yagi H Sasakawa H Uchida K Shitara K Kato K 《Biochimica et biophysica acta》2006,1760(4):693-700
The Fc portion of immunoglobulin G (IgG) expresses the biantennary complex type oligosaccharides at Asn297 of the C(H)2 domain of each heavy chain with microheterogeneities depending on physiological and pathological states. These N-glycans are known to be essential for promotion of proper effector functions of IgG such as complement activation and Fcgamma receptor (FcgammaR)-mediated activities. To gain a better understanding of the role of Fc glycosylation, we prepared a series of truncated glycoforms of human IgG1-Fc and analyzed their interactions with human soluble FcgammaRIIIa (sFcgammaRIIIa) and with staphylococcal protein A by surface plasmon resonance and nuclear magnetic resonance (NMR) methods. Progressive but less pronounced reductions in the affinity for sFcgammaRIIIa were observed as a result of the galactosidase and subsequent N-acetylhexosaminidase treatments of IgG1-Fc. The following endoglycosidase D treatment, giving rise to a disaccharide structure composed of a fucosylated GlcNAc, abrogated the affinity of IgG1-Fc for sFcgammaRIIIa. On the other hand, those glycosidase treatments did not significantly affect the affinity of IgG1-Fc for protein A. Inspection of stable-isotope-assisted NMR data of a series of Fc glycoforms indicates that the stepwise trimming out of the carbohydrate residues results in concomitant increase in the number of amino acid residues perturbed thereby in the C(H)2 domains. Furthermore, the cleavage at the GlcNAcbeta1-4GlcNAc glycosidic linkage induced the conformational alterations of part of the lower hinge region, which makes no direct contact with the carbohydrate moieties and forms the major FcgammaR-binding site, while the conformation of the C(H)2/C(H)3 interface was barely perturbed that is the protein A-binding site. These results indicate that the carbohydrate moieties are required for maintaining the structural integrity of the FcgammaR-binding site. 相似文献
16.
The proton magnetic resonance spectrum of bovine alpha-lactalbumin has been observed, and three peaks assignable to the position-2 CH protons of the three histidine rpsidues (His 32, 68, and 107) of this protein have been subjected to detailed examination. The assignments of these peaks to His 32, 68, and 107 were made on the basis of the difference in their reactivities with iodoacetic acid. The rate constants of the hydrogen-deuterium exchange reactions were found to be 8.0 X 10(-5), 2.6 X 10(-4), and 8.0 X 10(-5) min-1, respectively, at pH 8.5 and at 35 degrees, while at 62 degrees all three were found to be 0.84 approximately 1.1 X 10(-2) min-1. On the basis of these data, it has been shown that, in the native form of this protein, His 68 is the most exposed to the solvent while His 32 and His 107 are buried slightly deeper in the surface of the molecule. The fluctuation amplitudes gamma, or the effective chances of His 32, 68, and 107 to be fully exposed to the solvent, were found to be 0.4, 1.3, and 0.4, respectively. 相似文献
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The clinical severity of Osteogenesis Imperfecta (OI), also known as the brittle bone disease, relates to the extent of conformational changes in the collagen triple helix induced by Gly substitution mutations. The lingering question is why Gly substitutions at different locations of collagen cause different disruptions of the triple helix. Here, we describe markedly different conformational changes of the triple helix induced by two Gly substitution mutations placed only 12 residues apart. The effects of the Gly substitutions were characterized using a recombinant collagen fragment modeling the 63-residue segment of the alpha1 chain of type I collagen containing no Hyp (residues 877-939) obtained from Escherichia coli. Two Gly --> Ser substitutions at Gly-901 and Gly-913 associated with, respectively, mild and severe OI variants were introduced by site-directed mutagenesis. Biophysical characterization and limited protease digestion experiments revealed that while the substitution at Gly-901 causes relatively minor destabilization of the triple helix, the substitution at Gly-913 induces large scale unfolding of an unstable region C-terminal to the mutation site. This extensive unfolding is caused by the intrinsic low stability of the C-terminal region of the helix and the mutation induced disruption of a set of salt bridges, which functions to lock this unstable region into the triple helical conformation. The extensive conformational changes associated with the loss of the salt bridges highlight the long range impact of the local interactions of triple helix and suggest a new mechanism by which OI mutations cause severe conformational damages in collagen. 相似文献
19.
The reduction by dithiothreitol (DTT) of the four interchain disulfides of a human IgGlkappa immunoglobulin has been studied by two methods: variation of the concentration of DTT relative to the protein concentration (incremental reduction); and variation of the time of reduction at fixed levels of DTT and protein (kinetic reduction). In both cases, the results depend on whether the reduction is carried out aerobically or anaerobically. Under aerobic conditions, the relative levels of intermediates (HL, H2, and H2L) which are generated as native molecules (H2L2) are converted to reduced heavy (H) and light (L) chains depend on the concentrations of protein and DTT as well as on the exposure time to DTT; no stable equilibrium is reached between reduced and oxidized states and conditions gradually revert from those favoring reduction to those favoring reoxidation. By contrast, anaerobic reduction is independent of protein concentration or time of exposure to DTT, beyond about 30 min, indicating that an equilibrium between partially reduced and oxidized states is achieved. The distribution of intermediates observed under anaerobic conditions has been analyzed according to theoretical models (Sears, D.W., and Beychok, S. (1977), Biochemistry 16 (second in a series of three articles in this issue)). Within experimental error, both kinds of anaerobic experiments resemble a random reduction process wherein the four disulfides are equivalent and independent of each other with respect to rate and extent of reduction by D. It is concluded that there are no readily detected pathways in the process, as would occur if the intrinsic reactivities of the bonds were distinct, and no marked cooperatively between the four reaction sites, as would be observed if reduction of one bond materially facilitated or hampered reactivity at another site. Both of these characteristics of the reduction are in direct contrast to those of the reoxidative process, which is marked by the initial preference for formation of a bond between heavy and light chains, and by kinetic cooperativity in bond formation during the course of the reaction (Sears, D.W., et al. (1977), Biochemistry 16 (first in a series of three articles in this issue); Sears, D.W., and Beychok, S. (1977), Biochemistry 16 (second in this series)). 相似文献