The combining site of the dinitrophenyl-binding immunoglobulin A myeloma protein MOPC 315

Magnetic-resonance techniques are used to refine the model of the combining site of the Fv fragment of the dinitrophenyl-binding mouse myeloma protein MOPC 315 constructed by Padlan, Davies, Pecht, Givol & Wright (1976) (Cold Spring Harbor Symp. Quant. Biol. 41, in the press). Light-absorption studies indicate a dinitrophenyl–tryptophan interaction in the Fv fragment of the type occurring in free solution. The Dnp-aspartate–tryptophan complex is therefore used as a starting point for the n.m.r. (nuclear-magnetic-resonance) analysis of the dinitrophenyl–Fv fragment interaction. Ring-current calculations are used to determine the geometry of the complex. The specificity of complex-formation between dinitrophenyl and tryptophan is confirmed by the lack of ring-current shifts of the dinitrophenyl resonances when tryptophan is replaced by any other aromatic amino acid. Proton n.m.r. difference spectra (at 270MHz), resulting from the addition of a variety of haptens to the Fv fragment, show that the combining site is highly aromatic in nature. Calculations on the basis of ring-current shifts define the geometry of the combining site, which involves a dinitrophenyl ring in van der Waals contact with four aromatic amino acid residues on the protein. The observation of a nuclear Overhauser effect on the H₍₃₎ resonance of the dinitrophenyl ring provides additional constraints on the relative geometry of the H₍₃₎ proton and an aromatic amino acid residue on the Fv fragment. The specificity of the Fv fragment for dinitrophenyl ligands arises from a stacking interaction of the dinitrophenyl ring with tryptophan-93_L, in an `aromatic box' of essentially tryptophan-93_L, phenylalanine-34_H and tyrosine-34_L; asparagine-36_L and tyrosine-34_L also contribute by forming hydrogen bonds with the nitro groups on the dinitrophenyl ring. The n.m.r. results also confirm that the antibody–hapten reaction may be visualized as a single encounter step. An Appendix shows the method of calculation of ring currents for the four aromatic amino acids and their use in calculating structures.     

Symmetry of binding sites of a mouse IgA myeloma protein (MOPC 315)

We have investigated the mechanism of monovalency of the 7S subunit of a mouse IgA myeloma protein (MOPC 315) against a large antigen. This subunit, although it clearly can bind two molecules of a small hapten, fails to precipitate or hemagglutinate the relevant multivalent antigen. In an equilibrium Farr assay, we have shown that the subunit has only one valence for a univalent 40,000 molecular weight antigen (dinitrophenyl-dextran). We have investigated how various levels of affinity labeling quantitatively affect (a) the valence observed in the equilibrium Farr assay against a large antigen, and (b) the binding of the MOPC 315 to an insoluble antigenic matrix. Our results indicate that the Fab regions of the 7S subunit are arranged symmetrically and that the inactivity of one of them toward a large antigen is probably due to steric hindrance caused by the antigen bound to the adjacent site.     

The role of nitro groups in the binding of nitroaromatics to protein MOPC 315.

Two series of dinitrophenyl haptens, in which chlorine replaces one or both nitro groups, were used to investigate, by a combination of high-resolution 1H n.m.r. and fluorescence quenching, the presence of groups in the combining site of protein MOPC 315, which form hydrogen bonds to the aromatic-ring substituents of the hapten. The large differences in binding constants on successive replacement of nitro groups were shown to be due to specific hapten-substituent-protein interactions by (a) showing that there was little difference in the interaction between these haptens and 3-methylindole (a model for the residue tryptophan-93L with which the hapten stacks in protein MOPC 315), (b) proving by 1H n.m.r. that the mode of hapten binding is constant and (c) showing that the differences in Kd were consistent with the relative hydrogen-bonding capacities of chlorine and the nitro moiety. In this way it was established that each nitro group forms a hydrogen bond. Furthermore, from consideration of the 1H n.m.r. chemical shifts of several dinitrophenyl haptens and their trinitrophenyl analogues, it was shown that there is no distortion of the o-nitro group on binding to the variable fragment of protein MOPC 315.     

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.     

Comparison of the dimensions of the combining sites of the dinitrophenyl-binding immunoglobulin A myeloma proteins MOPC 315, MOPC 460 and XRPC 25 by spin-label mapping

The mouse immunoglobulin A myeloma proteins MOPC 315, MOPC 460 and XRPC 25 all possess dinitrophenyl (Dnp)-binding activity. Differences in specificities were shown by measuring the affinities of a variety of haptens. By using a series of Dnp-spin-labelled haptens, the dimensions of the binding sites of the three myeloma proteins were compared by the method described for protein MOPC 315 [Sutton, Gettins, Givol, Marsh, Wain-Hobson, Willan & Dwek (1977) Biochem. J.165, 177-197]. The dinitrophenyl ring is rigidly held in all three sites. The depths of the sites are all 1.1-1.2nm, but there are differences in the lateral dimensions at the entrance to the sites. For protein XRPC 25 these dimensions are 0.75nmx0.8nm, which may be compared with 0.85nmx1.1nm for protein MOPC 315 and >/=1.0nmx1.1nm for protein MOPC 460. The site in protein MOPC 460 is more symmetrical with respect to the plane of the dinitrophenyl ring than in either of the other two myeloma proteins and also allows greater penetration of solvent. In protein XRPC 25 a positively charged residue was located at the entrance to the site, similarly positioned to that reported for protein MOPC 315 [Sutton, Gettins, Givol, Marsh, Wain-Hobson, Willan & Dwek (1977) Biochem.J.165, 177-197]. All three proteins possess lanthanide-binding sites, but only in protein MOPC 315 is there antagonism between lanthanide and hapten binding. However, the effects of the diamagnetic La(III) on the electron-spin-resonance spectra of bound Dnp spin labels in both proteins MOPC 460 and XRPC 25 suggest an interaction between the two sites. Comparison of this effect with that caused by the addition of the paramagnetic Gd(III) enables the distance between the lanthanide- and hapten-binding sites to be calculated. In both proteins MOPC 460 and MOPC 315 the metal site is approx. 1.0nm from the nitroxide moiety of the spin-labelled hapten, but in protein XRPC 25 this distance is at least 2.0nm.     

Arrangement of lambda light chain genes in mutant clones of the MOPC 315 mouse myeloma cells

The synthesis of lambda light chains and the arrangement of the lambda-chain genes was examined in cells of the mouse myeloma MOPC 315, which is an alpha lambda 2 producer, and in several mutants derived from it. The mutants produce lambda 2 chains only (MOPC 315.26, MOPC 315.34, and MOPC 315.37) or fail to produce alpha and lambda 2 chains (MOPC 315.25 and MOPC 315.36). Messenger RNA from the lambda 2 chain-producing cells directed the synthesis of a lambda 2 chain precursor and a fragment of the lambda 1 chain (lambda 1 F) in a wheat embryo cellfree system, whereas mRNA from the cells that do not produce lambda 2 chains directed the synthesis of lambda 1 F only. DNA from the parental MOPC 315 cells and from the lambda 2 chain-producing cells contained discrete EcoRI restriction fragments coding for rearranged lambda 1 and lambda 23 chain genes and their respective germ-line V and J-C regions. DNA from the no-Ig-producing cells contained fragments coding for the rearranged lambda 1 chain gene and the germ-line V lambda 2 region, but it lacked the sequences coding for the rearranged lambda 2 chain gene and the germ-line V lambda 1 and J-C lambda 1 regions. These results suggest that rearrangements of the lambda 1 and lambda 2 chain genes occur on different chromosomes in MOPC 315 cells and imply that rearrangements of the lambda 1 and lambda 2 chain genes on the same chromosome may be mutually exclusive.     

Binding of antigen-bearing fluorescent liposomes to the murine myeloma tumor MOPC 315.

Small unilamellar lipid vesicles bearing the DNP-hapten on their surfaces and containing the water-soluble fluorescent dye carboxyfluorescein were formed by sonication. These vesicles were incubated with cells from the murine myeloma tumor MOPC 315, which secrete and also bear on the cell surface an immunoglobulin with affinity for the nitrophenyl hapten. At 0 degrees C the cells bound an average of several thousand vesicles at saturation. This binding was specific for the nitrophenyl hapten on the vesicle since it was abolished by an excess of soluble nitrophenyl derivative, by omission of the hapten from the vesicle, or by substitution for MOPC 315 of a tumor lacking receptors for the nitrophenyl hapten. Specific binding of vesicles was greater when cells were incubated at 37 degrees C. The study suggests that ligand-bearing vesicles can be a useful marker for cell surface immunoglobulin. However, in spite of the ability to "target" vesicles to cell surface determinants, binding did not result in increased delivery of vesicle contents to the cytoplasm.     

Post-translational fate of variant MOPC 315 lambda chains in Xenopus oocytes and mouse myeloma cells

The post-translational fates of three immunoglobulin lambda chain variants of MOPC 315 were investigated in mouse plasmacytoma cell lines and in mRNA-microinjected Xenopus oocytes. Quite unexpectedly we found that one non-secretory variant chain (lambda-43) underwent extensive post-translational N-glycosylation: however the presence of the oligosaccharide moiety did not account for the nonsecretory phenotype nor did it affect the rate of degradation of this lambda chain. Another variant chain (lambda-47) at first believed to be non-secretory, was found to be secreted from oocytes at a very low level, but mostly as a lambda-lambda dimer. In myeloma cells a low level of lambda-47 chain was secreted and again lambda-lambda dimers were the favoured secretory form. The secretory lambda-48 chain also formed lambda-lambda dimers, whereas lambda-43, which was never secreted, was only found as a monomeric lambda chain in both oocytes and myeloma cells. A similar relationship between assembly and secretion was found when oocytes were coinjected with MOPC 21 heavy (gamma 1) chain mRNA and MOPC 315 lambda chain mRNAs. The wild type lambda chain (lambda-48) was able to assemble with the gamma chain in a covalently bound tetramer (gamma gamma lambda lambda). The variant lambda-47 chain was also able to form gamma gamma lambda lambda tetramers, whereas the lambda-43 was not, even when glycosylation was prevented by tunicamycin. Both types of tetramer were secreted. These data reinforce the idea that conformational changes play a major role in the routing of secretory proteins and that the cellular mechanisms by which these changes are recognized are not cell-type specific.     

Regulation of immunoglobulin biosynthesis in the murine plasmacytoma MOPC 315.

We have examined certain aspects of IgG biosynthesis by constructing hybrids between MPC11 (gamma2b, kappa) and MOPC 315 (alpha,lambda2) that have lost the ability to synthesize one or the other heavy chain. Cells express the three chains in a stable fashion, and both autologous (parental) and heterologous (nonparental) H and L chain pairs form and are secreted. The alpha H chain was found in polymeric form when associated with the heterologous kappa L chain. The lambda2 L chain covalently assembled to the heterologous gamma2b H chain. Surprisingly, autologous pairing was always favored over heterologous pairing in vivo by 5 to 10:1 in terms of rate of assembly. Similar ratios were maintained in the secreted protein. These results suggest that co-expression of particular H and L chain pairs is predetermined. Evolution presumably operates to improve antigen recognition as well as rate of assembly of active molecules.     

Cloning of immunoglobulin kappa light chain genes from mouse liver and myeloma MOPC 173

The organization of the kappa chain constant region gene was compared in DNA from an immunoglobulin-producing mouse myeloma (MOPC 173) and from liver. In situ hybridization using the Southern blotting technique revealed constant region gene-containing EcoRI-DNA fragments of 14 and 20 kb in the myeloma tissue whereas one EcoRI-DNA fragment with a length of 15 kb was found in liver DNA. After enrichment by RPC-5 chromatography and preparative electrophoresis the 14 kb fragment from MOPC 173 DNA and the 15 kb fragment from liver DNA were cloned in the bacteriophage lambda vector Charon 4A using in vitro packaging. Extensive characterization of the two fragments by restriction endonuclease mapping, in situ hybridization, and electron microscopy (R-loop and heteroduplex) showed that both fragments contain the constant region but no MOPC 173 variable region gene. Both fragments are homologous over a length of 12.5 kb including the constant region but differ from one another starting about 2.7 kb from the 5' end of the constant region gene. This indicates that the 14 kb EcoRI-DNA fragment from the myeloma tissue clearly resulted from somatic DNA rearrangement although it does not seem to carry the MOPC 173 variable region gene. These observations suggest that somatic DNA rearrangement of immunoglobulin light chain genes can involve both homologous chromosomes.Images     

Characterization of the combining site of mouse myeloma protein M315

The interaction of M315 with 2,4-dinitrophenyl haptens was studied. 2,4-Dinitroaniline (DNP-NH2) showed maximum affinity to M315 at about pH 4. The pH dependence of the association constant of DNP-NH2 to M315 showed three transitions at pH 4.7, at pH 7.2, and below pH 9, respectively. Since the DNP-NH2 molecule has no charged group in this pH range, the transitions were explained in terms of amino acid residues with ionizable side chains in M315. Judging from the pK values and the effect of succinylation, these transitions were concluded to be related to ionizations of carboxyl, imidazole, and phenol groups, respectively. Measurement of the fluorescence of affinity-labeled M315 suggested that the transition at pH 4.7 reflected an equilibrium between two forms of M315 with different conformations of the combining site. The contribution of the amino acid sequence on the light (L) chain to the interaction with haptens was studied by use of antibodies (Abs) reconstituted from the heavy chain of M315 (H315) and either a homologous or a heterologous L chain. The reconstituted heterologous Ab (H315L952) showed similar pH dependence of binding to DNP-NH2 to that of the homologous Ab (H315L315). Moreover, the two Abs showed no appreciable difference in binding to DNP-haptens of different sizes. These results suggested that the difference in the amino acid sequences of L315 and L952, which originated by a somatic hypermutation, has little effect on the ligand binding.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interactions of the lanthanide- and hapten-binding sites in the Fv fragment from the myeloma protein MOPC 315.

1. The interactions of lanthanide metals and dinitrophenyl spin-label haptens with the Fv fragment of the mouse myeloma protein MOPC 315 were investigated by the techniques of fluorescence, e.s.r. (electron spin resonance) and high-resolution n.m.r. (nuclear magnetic resonance). 2. The protein fluorescence of Fv fragment at 340nm is quenched by the haptens (fluorescence enhancement, epsilon=0.15) and enhanced by Gd(III) (epsilon=1.14) and other lanthanides. The binding of the haptens studied here is insensitive to pH in the range 5.5-7.0 (dissociation constant KH=0.3-1.0 muM) and shows 1:1 stoicheiometry. The binding of Gd(III) also shows 1:1 stoicheiometry, but is pH-dependent; the binding constant (KM) varies from 10 muM at pH7.0 to 700 muM at pH4.8. La(III) binding is less sensitive to pH. The pH-dependences of the metal-binding constants imply that a group in the protein with pKa greater than or equal to 6.2 is involved in the binding, and probably also other groups with lower pKa values. 3. The apparent binding of the haptens is weakened about 20-fold by Gd(III), and vice versa. An equilibrium scheme involving a ternary complex with an interaction between the two binding sites is derived in Appendix I to explain the experimental results at two pH values. 4. Time-dependent fluorescence changes are observed in the presence of Gd(III) at pH5.5. A two-state kinetic scheme involving a 'slow' conformational change in the Fv fragment is derived in Appendix II to explain this time-dependence. This scheme is consistent with the antagonistic equilibrium behaviour. 5. The e.s.r. changes in the spin-label haptens on binding to Fv fragment and on the subsequent addition of lanthanides are consistent with the binding scheme for haptens and lanthanides proposed from the fluorescence studies. A difference between the limiting quenching of the e.s.r. signal from the bound haptens in the presence of saturating concentrations of Gd(III) and La(III) is attributed to dipolar interactions between bound Gd(III) and the nitroxide moiety of the bound hapten. The residual quenching with Gd(III) allows an estimate of 1.2nm to be made for the distance between the two paramagnetic centres. 6. The 270 MHz proton difference spectrum of the Fv fragment resulting from the addition of La(III) suggests that any metal-induced conformational changes are small and involve relatively few amino acid residues on the Fv fragment...     

Specificity of interactions of hapten side chains with the combining site of the myeloma protein MOPC 315.

The pKa values of the three histidine residues in the Fv fragment (variable region of the heavy and light chains) of the mouse myeloma protein MOPC 315, measured by high resolution n.m.r. (nuclear magnetic resonance), are 5.9, 6.9 and 8.2. The perturbation of the pKa of one of the histidines (pKa 6.9) on the addition of hapten and the narrow linewidth of its proton resonances suggests that it is at the edge of the combining site. References to the model of the Fv fragment [Padlan, Davies, Pecht, Givol & Wright (1976) Cold Spring Harbor Symp. Quant. Biol. 41, in the press] allows assignment of the three histidine residues, histidine-102H, histidine-97L and histidine-44L. The determination of the pKa of the phosphorus group, by 31P n.m.r., of a homologous series of Dnp- and Tnp- (di- and tri-nitrophenyl) haptens has located a positively charged residue. Molecular-model studies on the conformations of these haptens show that the residue is at the edge of the site. The model suggests that the positively charged residue is either arginine-95L or lysine-52H.     

A protein related to immunoglobulin light chain detected in mouse myeloma cells

A protein (denoted L′) which is similar in structure to immunoglobulin light chain has been isolated from the mouse plasma cell tumor, RPC-20. L′ has a molecular weight which is about 6000 daltons less than light chain. The exact nature of the relationship between L′ and light chain has not been established.