Determination of the Primary Structure of a Mouse γG2a Immunoglobulin Identification of the Disulfide Bridges

Préval, C. de; Fouegreau, Michel

doi:10.1111/j.1432-1033.1972.tb02117.x

Cited by 7 publications

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“…A complete identity was found with the constant region of the light chain of MOPC 21 from residue 97 to residue 214.We have undertaken the determination of the complete amino acid sequence of the murine MOPC 173 immunoglobulin molecule, which is a monoclonal protein of the IgG2a (x) class [I]. A topological model of the molecule has been proposed, on the grounds of results derived from the isolation and characterization of the CNBr fragments of the entire molecule [2], and from the identification of the interchain and intrachain disulfide bridges [3,4]. The sequence of the CNBr fragments H1, H2, H3 (covering most of the V, region), and that of fragments H5 to H10, accounting for the hinge region and the Fc fragment have already been published [5 -71.We report in the present paper the amino acid sequence of the light chain of this molecule.…”

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Determination of the Primary Structure of a Mouse IgG2a Immunoglobulin

Schiff¹,

Fougereau²

1975

European Journal of Biochemistry

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The amino acid sequence of the light chain of the mouse monoclonal MOPC 173 immunoglobulin molecule (IgG2a,x) is presented. This kappa chain contains 214 residues. Comparisons of this sequence with murine kappa chains already published by other workers bring a confirmation of the large size of the murine Vx chain pool. A complete identity was found with the constant region of the light chain of MOPC 21 from residue 97 to residue 214.We have undertaken the determination of the complete amino acid sequence of the murine MOPC 173 immunoglobulin molecule, which is a monoclonal protein of the IgG2a (x) class [I]. A topological model of the molecule has been proposed, on the grounds of results derived from the isolation and characterization of the CNBr fragments of the entire molecule [2], and from the identification of the interchain and intrachain disulfide bridges [3,4]. The sequence of the CNBr fragments H1, H2, H3 (covering most of the V, region), and that of fragments H5 to H10, accounting for the hinge region and the Fc fragment have already been published [5 -71.We report in the present paper the amino acid sequence of the light chain of this molecule. MATERIALS AND METHODS Isolation of the MOPC 173 ProteinPlasmacytomas which originated from Dr Potter's laboratory, were serially transplanted in Balb/c mice, in solid form, by subcutaneous injection of small tumor fragments. On the average, mice were bled three weeks after the transplantation. The immunoglobulin fraction of pooled sera was precipitated with ammonium sulfate (33 % saturation, final concentration), and was further purified by chromatography on DEAE cellulose (DE 52, Whatman) equilibrated with 0.017 M phosphate buffer (pH 7.3). Purity was checked by immunoelectrophoresis [8], using a rabbit antiserum against whole Balb/c mouse serum. Isolation of the Light ChainsThe MOPC 173 immunoglobulin molecule (500mg) was mildly reduced in 0.1 M Tris buffer, 0.15 M NaC1, 0.002 M EDTA, pH 8.2, with dithiothreitol (Calbiochem), according to Frangione et al. [9] (except that the molarity of the reagent was 8 mM) and alkylated with a 2.5 molar excess of iodoacetamide. Chains were then separated by gel-filtration on a Sephadex G-100 superfine column ( 5 x 100 cm) equilibrated in 1.0 M propionic acid. Purity of the light chains was tested by polyacrylamide (7.5 %) gel electrophoresis according to Shapiro and Maize1 [lo].Complete reduction and alkylation of the isolated light chains was made according to Waxdal et al.[ 1 1 1.except that 8 M urea was used instead of guanidine. Whenever labelled peptides were required, alkylation was performed, according to Frangione et al. [9], by making the reduced solution 0.02 M with iodo['"C]acetic acid (Amersham), using a 0.1 M stock solution (specific activity 83 pCi/ml). Radioactivity was counted in a Packard spectrometer 3380. Isolation of the CNBr FragmentsCNBr fragments were prepared by direct attack of the whole MOPC 173 molecule. Fragments L1 and L3 were isolated by successive steps of gel filtration, as previously descri...

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Determination of the Primary Structure of a Mouse IgG2a Immunoglobulin

Schiff¹,

Fougereau²

1975

European Journal of Biochemistry

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“…This parallelism in evolution is discussed.Murine MOPC 173 immunoglobulin is a monoclonal protein of the IgG2a class [l]. Isolation and characterization of t'he CNBr fragments [2] and localization of the disulfide bridges [3,4] have led to the proposals of a topological model [4]. Cleavage of the heavy chain with CNBr allows isolation of ten fragments, which have been designated H1 to H10[2].…”

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Determination of the Primary Structure of a Mouse IgG2a Immunoglobulin: Amino‐Acid Sequence of the Fc Fragment

Bourgois

Fougereau

Rocca-Serra

1974

European Journal of Biochemistry

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The amino-acid sequence of the Fc fragment of a mouse monoclonal IgG2a molecule is presented. With the exception of one deletion, this region possesses the same length as that of the human y l chain or that of the rabbit y chain. Identities between the Fc fragments of 3 animal species (human, mouse and rabbit) average 50°/, and are markedly more pronounced for the c~2 domain than for the C H~ domain, observation which is in agreement with an independent evolution of each homology region. Strikingly, the degree of conservation of the VH region is of the same osder of magnitude as that observed for the Fc fragment. This parallelism in evolution is discussed. [2]. Fragments HI to H3 cover residues 3 to 104, the sequence of which has been reported [5]. Fragment H4 covers the entire constant section of Fd, the hinge region and the beginning of the Fc fragment. We report in the present paper, the amino-acid sequence of the last 18 COOH-terminal residues of H4 and that of the remaining fragments H5 to H10, thus accounting for the entire Fc fragment. MATERIALS AND METHODSCNRr fragments were prepared by direct attack of the whole molecule, essentially as previously described [2], with the minor following modifications.The H4 fragment was separated from the L chain derived L2 fragment by ion-exchange chromatography on DEAE-cellnlose (Whatman DE 52), equilibrated in 6 M urea, pH 10.0, under which conditions H4 was not retained, as opposed to L2.Abbreviations. Ala, alanine; Arg, arginine; Asn, asparagine; Asp, aspartic acid; Cys, cysteine; Glu, glutamic acid; Gln, glutamine; Gly, glycine; His, histidine; Ile, iaoleucine; Leu, leucine; Lys, lysine; Met, methionine; Phe, phenyl alanine; Pro, proline; Ser, serine; Thr, threonine; Trp,tryptophan ; Tyr, tyrosine; Val, d i n e ; Cmc, carboxamidomethylcysteine ; Hsr, homoserine.Fragments H5 and H6-7 were separated by ionexchange chromatography on a column of SE-Sephadex equilibrated in acetic acid 0.05 M made 6 M in urea (pH 5.2), and using a stepwise elution with 0.5 M NaOH containing 6 M urea (pH 13.0). Eluted material was desalted on a column of Sephadex G 25 coarse in 1.0 M propionic acid.Enzymatic hydrolyses with trypsin [treated with L-( l-tosyl-amido-2-pheny1)ethyl chloromethyl ketone (i.e. TPCK-treated, Calbiochem)], chymotrypsin and subtilisin (N.B. Co), were performed as previously described [5]. Incubation times, however, were lowered to 20 or 30 min (at 37 "C) in most instances, with the exception of H4 and H9C peptides, for which a 4-h hydrolysis was used. Occasionally, tryptic hydrolysis, was used after fragments had been citraconylated according to Gibbons and Perham [6]. For acid hydrolysis, a 1 solution of substrate (fragment HlO) was prepared in 6 N HC1. Incubation proceeded a t 37 "C for 18 h. Peptides were separated by ionexchange chromatography on a column (55 x 0.9 cm) of either Dowex 5 0 x 2 (Biorad) or cation exchanger resin P (Technicon). Elution was obtained by applying a gradient of pH linear between 3.0 and 6.0 (first buffer: acetic acid-pyridine-water,...

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The idiotypy of the MOPC 173 (IgG_2a) mouse myeloma protein: Characterization of syngeneic, allogeneic and xenogeneic anti‐idiotypic antibodies. Contribution of the H and L chains to the idiotypic determinants

et al. 1979

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Anti‐idiotypic antibodies were raised against the mouse (γ2a, κ) myeloma protein MOPC 173 in syngeneic, allogeneic and xenogeneic conditions. Syngeneic immunization resulted in individual hemagglutination titers ranging from 1:40 to 1:160000 with about 30% of nonresponders, whereas upon allogeneic immunization (in A/J mice) almost all animals responded, with titers ranging from 1:1250 to 1:320000. Anti‐idiotypic antisera were also obtained in the rabbit. Inhibition of a radioimmunoassay using 125I‐labeled MOPC 173 Fab fragments as the reference antigen indicated that the native Ig and the Fab competed on a similar molar basis. No inhibition was observed with the Fc or with other mouse monoclonal Ig, of the same or different classes or subclasses. Standard BALB/c serum did not inhibit either. Whatever the anti‐idiotypic antiserum used, idiotypic determinants were not found on isolated heavy (H) and light (L) chains provided they were carefully purified. Reoccurrence of idiotypic determinants was obtained in high yield whenever Ig molecules were reformed from the homologous H and L chains, whereas no potentiation of the low inhibition of isolated chains was observed when hybrid molecules were formed using heterologous complementary chains isolated from the MOPC 21A (γl, ϰ) or the LPC 1 (γ2a, ϰ) protein. These results suggest that MOPC 173 idiotypic determinants rely on the specific H‐L interaction and that they must be largely conformational in nature. Heterogeneity of the anti‐idiotypic antibodies was analyzed by isoelectric focusing. A major pattern was observed for the A/J antisera, whereas, in the case of BALB/c, expression of a few clones, characteristic of each “high responder” animal, was strongly suggested, superimposed to faint production of antibodies by clones that were common to all mice.

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Determination of the Primary Structure of a Mouse γG2a Immunoglobulin Identification of the Disulfide Bridges

Cited by 7 publications

References 45 publications

Determination of the Primary Structure of a Mouse IgG2a Immunoglobulin

Determination of the Primary Structure of a Mouse IgG2a Immunoglobulin

Determination of the Primary Structure of a Mouse IgG2a Immunoglobulin: Amino‐Acid Sequence of the Fc Fragment

The idiotypy of the MOPC 173 (IgG_2a) mouse myeloma protein: Characterization of syngeneic, allogeneic and xenogeneic anti‐idiotypic antibodies. Contribution of the H and L chains to the idiotypic determinants

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Determination of the Primary Structure of a Mouse γG2a Immunoglobulin Identification of the Disulfide Bridges

Cited by 7 publications

References 45 publications

Determination of the Primary Structure of a Mouse IgG2a Immunoglobulin

Determination of the Primary Structure of a Mouse IgG2a Immunoglobulin

Determination of the Primary Structure of a Mouse IgG2a Immunoglobulin: Amino‐Acid Sequence of the Fc Fragment

The idiotypy of the MOPC 173 (IgG2a) mouse myeloma protein: Characterization of syngeneic, allogeneic and xenogeneic anti‐idiotypic antibodies. Contribution of the H and L chains to the idiotypic determinants

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The idiotypy of the MOPC 173 (IgG_2a) mouse myeloma protein: Characterization of syngeneic, allogeneic and xenogeneic anti‐idiotypic antibodies. Contribution of the H and L chains to the idiotypic determinants