Mouse Myeloma Protein Research Articles

The isolation and characterization of a mouse myeloma protein with anti-dextran activity

A myeloma protein in ascitic fluid from BALB/c mice bearing W3129 plasma cell tumors was isolated by affinity chromatography. This protein exhibits anti-dextran activity and has been obtained in highly purified form by selective adsorption on isomaltosyl-Sepharose and elution with isomaltose solution. The isomaltosyl-Sepharose was synthesized from maltose, p-aminophenyl glucoside and cyanogen bromide-activated Sepharose by a new procedure utilizing glucosyltransferase and chemical coupling reactions. Results of gel electrophoresis, isoelectrofocusing and agar diffusion experiments showed that the purified myeloma protein consisted of 6 isomeric proteins with each isomer possessing anti-dextran activity. Data from hapten inhibition studies were interpreted to show that the W3129 myeloma protein combines with terminal isomaltosyl units of branched dextrans and oligosaccharides.

Helper T cell recognition of the variable domains of a mouse myeloma protein (315). Effect of the major histocompatibility complex and domain conformation.

We have examined the recognition of the variable (V) domain of the heavy (VH) and light (V lambda 2) chains of mouse myeloma protein 315 by helper T cells. Mice were primed with the isolated V domain in complete Freund's adjuvant, and carrier (V domain)-primed spleen cells were transferred together with hapten (NIP)-primed spleen cells to recipient mice that were boosted with NIP3-Fab-315. The helper cell response to both domains was governed by H-2-linked immune response (Ir) genes, and VH-315 and V lambda 2 displayed different Ir phenotypes. H-2k conferred high responsiveness to VH on three different genetic backgrounds, BALB/c, C3H, and B10; mice of the d and b haplotypes were low responders. Conversely, H-2d conferred high responsiveness to V lambda 2 on two backgrounds, BALB/c and C3H, whereas mice of the k haplotype were low responders to this domain. Non-H-2 genes of the B10 background extinguished the helper cell response to V lambda 2 in animals with the high responder d haplotype. The VH Ir gene mapped to the K-A interval of the H-2 complex. Unfolded (completely reduced and alkylated) V domains primed helper cells as efficiently as folded domains for responses to NIP3-Fab-315, indicating that the helper cells recognized an antigenic determinant that was not conformation-dependent. The data indicate that there exists helper T cells which recognize each member of the M315 pair of V domains independent of the other, and that these V domains are recognized like conventional extrinsic protein antigens.

Solid phase synthesis of the protected 43-55 tridecapeptide of the heavy chain of myeloma immunoglobin M603, employing cyclohexyl ester protection for glutamic acid.

A protected tridecapeptide of the sequence Boc-Lys(2CIZ)-Arg(Tos)-Leu-Glu (OcHex)-Trp(For)-Ile-Ala-Ala-Ser(Bzl)-Arg(Tos)-Asn-Lys(2CIZ)-Gly-OH, representing residues 43-55 of the variable region of the heavy chain of mouse myeloma protein M603, was synthesized. It was assembled by a stepwise solid phase method designed to give a fully protected peptide in high yield and purity with minimal side reactions. Thus, the peptide chain was attached as an alpha-methyl phenacyl ester to a 2-bromopropionyl-resin. After the synthesis the protected peptide fragment was obtained in 89% yield by photolytic cleavage from the resin. The peptide was purified by multiple precipitation and column chromatography. It was shown to be homogeneous by reverse phase high pressure liquid chromatography, and it had the correct amino acid composition and sequence. In the course of this work it was shown that tert.-butyloxycarbonyl-amino acids caused the formation of significant amounts of pyrrolidone carboxylic acid residues during the coupling reaction when a gamma-benzyl glutamyl residue was NH2-terminal. Other weak-acid additives also caused this chain terminating side reaction. The cyclization was markedly suppressed by protection of the glutamyl side chain as a cyclohexyl ester. With this protecting group, no evidence of pyrrolidone carboxylic acid formation could be detected in the tridecapeptide 43-55.

Biosynthesis of Arabinogalactan-Protein in Lolium multiflorum (Ryegrass) Endosperm Cells. II. In vitro Incorporation of Galactosyl Residues From UDPgalactose Into Polymeric Products

When mixed-membrane fractions from suspension-cultured Lolium multiflorum endosperm cells are incubated in vitro with UDP-[14C]galactose, 66% ethanol-insoluble products of apparent molecular weight greater than 60 000 are labelled in both galactosyl and glucosyl residues, suggesting that an active UDPgalactose 4-epimerase is present on the membrane fraction. The epimerase can be inhibited with ADPribose, to produce polymeric material in which [14C]galactosyl residues pre-dominate. While some of these residues appear to be associated with glycoproteins, affinity chromatography of the products on mouse myeloma protein J539-Sepharose provides evidence that β-galactans containing 1,6-linkages are amongst the products. Monosaccharide analyses and methylation data indicate that the mixed-membrane preparations contain associated polysaccharide of structure analogous to the 1,3;1,4-β-glucans, arabinoxylans and arabino-3,6-galactans normally found in cell walls or secreted into the medium.

Mouse myeloma proteins with lambda 2 and lambda 3 light chains.

Serum and ascites fluid from mice bearing 260 different myeloma tumors were screened serologically for myeloma proteins having light chains like that of L315, the lambda 2 chain produced by myeloma MOPC-315. Four proteins, made by myelomas TEPC-952, CBPC-49, ABPC-72, and SAPC-15, were identified. Their light chains were essentially indistinguishable serologically from L315, and each of them also yielded the characteristic C-terminal amino acid (leucine) and C-terminal tryptic peptide of L315. However, amino acid sequences and other findings reported elsewhere have revealed that although the light chain of TEPC-952 is indeed a lambda 2 chain, the light chains of the others (CBPC-49, ABPC-72, and SAPC-15) represent another, slightly different type of light chain, which has been designated lambda 3.

A monoclonal antibody that detects a V kappa-TEPC15 idiotypic determinant cross-reactive with a Thy-1 determinant.

To identify T lymphocyte antigens with immunoglobulin-like determinants, we prepared rat anti-mouse T cell monoclonal antibodies and screened them against a panel of purified mouse myeloma proteins representing all isotypes of immunoglobulin. One hybridoma, designated 42-21, was found to detect a novel antigenic determinant shared by V kappa-TEPC15 and the Thy-1 molecule on all T lymphocytes. Although several explanations for this unusual phenomenon exist, it may imply some role for the Thy-1 molecule in antigen and/or mitogen recognition. In any event, future studies of idiotypes on T lymphocytes must consider the possibility that anti-idiotypic sera detect cell surface molecules unrelated to classical immunoglobulin.

Small-angle neutron scattering studies of the conformation of myeloma protein MOPC315 and its Fab fragment, and the interaction with a monovalent dinitrophenyl hapten.

The first small-angle scattering study of an immunoglobulin A is reported. Neutron measurements have been made to determine conformational parameters of the mouse myeloma protein MOPC315 and to relate these to previous immunoglobulin G results. Use of the contrast method shows that the MOPC315 IgA molecule is not simply globular, that it has a dry volume of 220.0 +/- 4.5 nm3 corresponding to a mass density of 1.275 +/- 0.025 g cm-3 and that its full and cross-sectional radii of gyration, corrected for concentration dependence, are 7.97 +/- 0.07 nm, 2.40 +/- 0.08 nm and 1.33 +/- 0.07 nm respectively. Similar study of its Fab fragment gives a dry molecular volume of 69.0 +/- 0.7 nm3, a mass density of 1.285 +/- 0.015 g cm-3 and uncorrected radii of gyration that are consistent with those of the parent and support an overall "T" or "Y" conformation in solution. Addition to saturation of a small monovalent dinitrophenyl hapten leaves the dry volume of the whole molecule unaltered, but may slightly lower one or more of its radii of gyration. The significance of this finding is discussed. Comparative studies with rabbit anti-dinitrophenyl immunoglobulin G antibody suggest a different initial conformation but similar consequences of hapten binding, which, if real, are probably unrelated to classical complement fixation.

Structural basis for M-173 idiotypic determinants distinctively recognized in syngeneic and allogeneic immunization: Contribution of DH, JH and Jκ regions to an idiotope recognized by allogeneic antisera

Antiidiotypic antibodies directed against the M-173 (IgG2a) mouse myeloma protein have been raised in syngeneic and allogeneic conditions. The antiidiotypic repertoires of several strains of mice have been compared by isoelectrofocusing, and a major idiotope has been identified by several antisera raised in allogeneic conditions in strains of mice which did not express the Igh-C a allotype of the BALB background. Since this idiotope could be reformed in hybrid molecules containing the M-173 heavy chains and light chains which contained the Jκ2 region, we propose that this determinant is dependent upon the Jκ2, DH and JH regions, in addition, most probably, to a specific contribution of residues 45 and 54 of the heavy chains.

Structural evidence for distinct IgG subclass-specific Fc receptors on mouse peritoneal macrophages.

Membrane proteins which selectively bind to the Fc portion of IgG were identified in the Nonidet P-40 extracts of radiolabeled thioglycollate- elicited mouse peritoneal macrophages. Affinity columns of various IgG preparations coupled to Sepharose 4B were used to absorb the Fc-binding proteins. Analysis of the acetic acid or sodium dodecyl sulfate (SDS) eluates from aggregated human IgG or antigen-complexed rabbit IgG columns revealed two Fc(gamma)/-specific proteins with apparent 67,000 and 52,000 mol wt. These proteins were not detected in acid or SDS eluates from F(ab')(2) columns or in eluates from IgG column, over which were passed lysates of Fc receptor-negative cells. With the use of affinity columns that contained aggregated mouse myeloma proteins of different IgG subclasses, we found that the 67,000-dahon protein selectively binds to IgG2a, whereas the 52,000-dalton protein binds to IgG1 and IgG2b. Neither protein was found in SDS eluates from IgG3 columns. Trypsin treatment of the macrophages before detergent lysis removed the 67,000-dalton protein, although it leaves intact the 52,000-dalton protein. These results provide structural confirmation for the existence of separate Fc receptors on mouse macrophages and indicate that the two Fc-binding proteins identified in this study represent all or part of the trypsin- sensitive Fc receptor which binds IgG2a and the trypsin-resistant Fc receptor which binds IgG2b and IgG1.

Chemical properties of functional groups of mouse immunoglobulins of the IgA, IgG2a, and IgM classes.

Chemical properties of functional groups of mouse immunoglobulins of the IgA, IgG2a, and IgM classes.

Antibodies to immunoglobulin heavy chain variable regions protect helper cells from specific suicide by radiolabeled antigen.

The antigen-binding receptor of helper T cells was studied by radioactive antigen-caused suicide in vitro. Purified antibodies to immunoglobulin variable regions, obtained from sera of rabbits immunized with isolated VH and VL fragments of mouse myeloma proteins (MOPC 315, XRPC 25), were used to inhibit the binding of radiotoxic antigen. Anti-VH, but not anti-V lambda or anti-V chi inhibited suicide of carrier-primed cells.

Anti-idiotypic IgM antibodies produced in carps with BALB/c mouse myeloma protein S117

Carps are able to synthesize xenogeneic anti-idio-typic antibodies against a mouse myeloma protein (S117) with specificity for N-acetyl-D-glucosamine. The properties of carp anti-idiotype (IgM) are compared to anti-idiotypic antisera from guinea pig (mainly IgG) elicited against the same idiotype. By radioprecipitin inhibition tests and passive hemagglutination inhibition tests, the specificity of carp and guinea pig anti-idiotypic antibodies is closely related. Furthermore, inhibition experiments with amino sugars suggest that at least some determinants localized in the antigen combining site of the S117 antibody are recognized by both carp and guinea pig anti-idiotypes. These results should contribute to the phylogenetic aspect of idiotype anti-idiotype interaction and possibly also of idiotype regulation.

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.

AbstractAnti‐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.

On the role of Tyr 34 L in the antibody combining site of the dinitrophenyl binding protein 315

The role of Tyr 34 L in the combining site of the dinitrophenyl binding mouse myeloma protein 315 has been studied by chemical modification. Tyr 34 L was specifically nitrated by separating the peptide chains of the Fv fragment intoV L and V H (Hochman et al., 1973), nitrating V L with tetranitromethane and reassociating the modified V L with unmodified V H Comparison of the binding properties of NO 2 Fv and Fv, and of NO 2V L and V L was performed by equilibrium dialysis with 3H-Dnp-lysine, dinitrophenol, Tnpamino caproic acid and Tnp-amino ethylamine at pH 5.0 and pH 8.0. Since the p K a of the NO 2 Tyr in the above fragments is 6.2–6.6. these binding studies were designed to investigate the possibility of a steric effect of introduction of the nitro group, and to demonstrate the presence or absence of a hydrogen bond between Tyr 34 L and the ligand suggested in a recent paper (Dower et al., 1977), by observing the effect of ionization of the phenol group. Both the affinities and numbers of sites of the modified and unmodified fragments were found to be very similar. Nuclear magnetic resonance studies showed that the hapten occupied the same position in the combining sites of the modified and unmodified Fv fragments. Hence it is concluded that Tyr 34 L does not form a hydrogen bond to nitrophenyl ligands.

T helper lymphocytes recognize the VL domain of the isologous mouse myeloma protein 315.

The localization of a major determinant on an isologous myeloma protein (M315) which stimulates BALB/c helper T cells was investigated. Augmentation of the adoptive secondary antibody response to NIP-M315 and the idiotype of M315 (Id315) was used as an indicator of helper effects. Spleen cells primed with the light chain of M315 (L315) and its V-domain (VL315) were highly efficient helpers; priming with the fragment containing the two V-domains of M315 (FV315) induced a somewhat weaker helper effect than L315 or VL315. The helper effect was abolished or markedly reduced by treating the primed cells with rabbit anti-brain theta + C. Cells primed with the heavy chain of M315 (H315) effected weak but significant help. The V-domain of H315 (VH315) was incapable of eliciting cells with detectable helper effect. The data indicate that the VL315 embodies a major determinant for T helper lymphocytes. This determinant is expressed on the free VL315 as well as on the complete M315. In contrast, previous studies have shown that BALB/c antibodies produced against Id315 recognize antigenic sites that are only displayed on associated (VL315 + VH315) domains.

Crystallization of the Fv fragment of mouse myeloma protein M315.

The Fv fragment of mouse myeloma protein M313 was crystallized from poly(ethylene glycol) solution in the form of monoclinic crystals, space group C2 and unit cell dimensions a = 5.96 nm (59.6 A), b = 5.66 nm (56.6 A), c = 13.79 nm (13.9 A) and beta = 99.7 degrees. Some unusual effects of poly(ethylene glycol)on protein crystals were noted and are discussed.

The location and expression of idiotypic determinants in the immunoglobulin variable region—II.: Chain location of variable region determinants

Both myeloma protein 460 and myeloma protein 315 each exhibit two sets of idiotypic determinants. A ‘private’ determinant set which distinguishes protein 460 and protein 315 from each other, and a common or ‘public’ set of idiotypic determinants which are shared by proteins 315 and 460 and which distinguish these two proteins from many other mouse IgA myeloma proteins. One other mouse myeloma protein which shares the ‘public’ idiotype is myeloma protein S.23 which has heavy chain amino acid sequences which resemble in part those of protein 460. Heavy-light chain recombinant molecules of these three proteins were made. The amino acids constituting both the public and private idiotypic markers were located on the heavy chains. The public idiotypic is expressed in recombinatant molecules no matter which light chains were associated with the heavy chains. Antigenic expression of private idiotypes, however, depended on the type of light chain which was present. Modulation of antigenic expression of structural idiotypic determinants by associated chains must be considered when idiotypic determinants are used as genetic markers.

Preparation and characterization of anti-framework antibodies to the heavy chain variable region (VH) of mouse immunoglobulins.

AbstractThe heavy chain variable portion of mouse myeloma protein MOPC‐315 (α, λ2) was obtained from its Fv fragment (J. Hochman, D. Inbar and D. Givol, Biochemistry 1973. 12: 1130.) and was used to immunize rabbits. Rabbit anti‐VH antibodies precipitated VH, Fv and the intact protein 315. The VH region specificity of these antibodies was evident from the line of identity in agar gel diffusion between Fv and M315, and from the lack of precipitation with another a chain of XRPC‐25 or with light (L) chains. Radioimmunoassay binding and inhibition studies demonstrated that anti‐VH 315 antibodies cross‐react with heavy (H) chains derived from various mouse myeloma proteins (MOPC‐460, XRPC‐25, MOPC‐104E, UPC‐10, TEPC‐15, HOPC‐8, MOPC‐167) as well as with H chains derived from mouse IgG. These H chains can inhibit 90–100% of the binding of VH 315 by anti‐VH indicating extensive sharing of antigenic determinants by various VH regions. The relative affinity of anti‐VH for various H chains was 20–1000‐fold weaker than its affinity for the MOPC‐315 H chain. The cross‐reaction of the various H chains was independent of antibody specificity, subclass, subgroup or CH allotype. This suggests that anti‐VH may be considered as anti‐V framework antibody.Anti‐VH 315 cross‐reacts with isolated H chains but not with intact molecules and can therefore be used to detect VH determinants only in the absence of chains.

Resonance Raman-spectroscopic studies of the hapten features involved in the binding of 2,4-dinitrophenyl haptens by the mouse myeloma proteins MOPC 315 and MOPC 460.

The binding of four dinitrophenyl haptens to the mouse myeloma proteins MOPC 315 IgA (immunoglobulin A) and MOPC 460IgA was studied by resonance Raman spectroscopy. Isotopic substitution with 15N and 2H was used to assign features in the resonance Raman spectra of the free haptens. Changes in each of these features on binding to the proteins could then be attributed to interactions of the proteins' binding sites with either the p-NO2 or the o-NO2/amine regions of the haptens. The interactions between a given hapten and MOPC 315 IgA are often quite distinct from those between the same hapten and MOPC 460 IgA. Moreover, for both antibodies the nature of the R side chain in a Dnp-NHR (Dnp, 2,4-dinitrophenyl) compound appears to modify the interactions between the Dnp chromophore and the protein. Thus, with the haptens studied, there is no unique set of contacts between the Dnp group and the binding site. The contacts expected between epsilon-2,4-dinitrophenyl-L-lysine and the site on MOPC 315 IgA, on the basis of a recent model for this site [Dwek, Wain-Hobson, Dower, Gettins, Sutton, Perkins & Givol (1977) Nature (London) 266, 31--37] were not detected. However, the contacts between this hapten and the site on MOPC 460 IgA were closer to those predicted by the model for MOPC 315 IgA.

Preparation and characterization of antibodies to the lambda chain variable region (Vlambda) of mouse immunoglobulins.

AbstractThe variable portion (VL) of the mouse myeloma protein MOPC‐315 (α, λ2) was obtained from its Fv fragment and was used to immunize rabbits. Anti‐VL antibodies were found to be specific to the VL region of protein 315 and to precipitate VL, Fv or light chain but not Fab' or intact M315. Anti‐VL 315 precipitates also the light chain of MOPC‐104E (μ, λ1) but not the intact protein. Intact immunoglobulins (Ig) containing λ chains, although they do not precipitate with anti‐VL, inhibit the binding of anti‐VL to VL in the radioimmunoassay. Radioimmunoassay inhibition studies demonstrated that Vλ1 and Vλ2 cross‐react extensively and that anti‐VL 315 can be used as a general anti‐Vλ reagent to detect molecules containing λ chains in mouse serum. Analysis of sera from several mouse strains indicated that Vλ‐containing molecules are present in approximately 3% of the Ig population, whereas the SJL strain has no detectable Vλ‐bearing molecules. In some of the antisera, anti‐VL 315 slightly cross‐reacted with VK‐bearing molecules. Anti‐VL 315 antibodies are not anti‐idiotypes since they react with myeloma proteins M315 (anti‐2,4‐dinitrophenyl MOPC‐104E (anti‐dextran) and HOPC‐1 (specificity unknown) and with most serum Ig containing λ chains. In view of the similarity of mouse λ chain sequences, it is suggested that most anti‐VL 315 is “anti‐framework” antibody.