Activity of multiple light chain genes in murine myeloma cells producing a single, functional light chain

Two cloned lambda 1-producing myelomas (HOPC-1, MOPC-104E) contain rearranged kappa genes and levels of mature-sized kappa RNA comparable to those found in kappa-producing myeloma cells. Another lambda 1-producing myeloma tumor line (HOPC-2020) and a lambda 1-containing B cell leukemia line (BCL1) also contain significant levels of kappa RNA. One lambda 11-producing line (MOPC-315) contains no detectable kappa RNA, but it also has no kappa genes in the embryonic configuration. kappa-related proteins are not detectable in the lambda 1-producing lines by standard procedures, but by sensitive methods at least two lines contain kappa protein fragments. The MOPC-104E line produces both a 14.5K kappa fragment that is not readily detectable because of its low rate of synthesis and short half-life (T 1/2 less than 5 min), and a major 16.5K protein that lacks kappa cross reactivity but is demonstrable by translation of purified MOPC-104E kappa RNA. The HOPC-1 kappa RNA also encodes a short-lived 14K kappa fragment. The MPC-11 line, which produces a mature kappa RNA and protein as well as an 800 base kappa fragment RNA and kappa protein fragment, has both kappa alleles rearranged, one apparently aberrantly between J and C kappa. Two different kappa RNA species, one the same size as the MPC-11 kappa fragment RNA, frequently are present in kappa RNA-containing Abelson murine leukemia virus-transformed lymphoid cells as well as in 18 and 19 day murine fetal liver. For light chains, neither allelic nor isotype exclusion is generally evident in myeloma and lymphoma cells; rather both produce only a single functional light chain. Models of light chain activation must explain restriction by considering the functional properties of the light chain rather than light chain gene expression.     

Untranslated immunoglobulin kappa light chain mRNA in a lambda light chain-producing mouse myeloma, MOPC104E

Fourteen clones were isolated in culture from a mouse myeloma, MOPC104E. All clones had kappa and lambda types of light chain mRNAs in approximately equimolar quantity as assayed by hybridization with specific complementary DNA (cDNA). However, the myeloma produces and secretes only lambda-type light chain protein. Both kappa- and lambda-type mRNAs in these clones were indistinguishable from kappa- and lambda-type mRNAs of other myelomas with respect to (a) adsorption to oligo-(dT) cellulose, (b) molecular size (12.6 S), and (c) thermal stability of the hybrids formed with corresponding cDNA. The kappa chain mRNA of MOPC104E cells, however, was translated very inefficiently both in vivo and in vitro, whereas the lambda chain mRNA was translated efficiently. These results indicate that each cell of MOPC104E myeloma synthesizes a crippled kappa chain mRNA in addition to a normal lambda chain mRNA.     

Messenger RNA for immunoglobulin light and heavy chains in MOPC-315 plasmacytoma and variants

Mouse plasmacytoma MOPC-315 produces light and heavy immunoglobulin chains. The variants, MOPC-315 NP-1 and MOPC-315 NR, synthesize only heavy or light chains, respectively. To eludicate the inability of MOPC-315 variants to produce intact light or heavy chains, complementary DNAs (cDNAs) to the mRNAs were prepared. From the kinetics of DNA-RNA hybridization, the RNA of the MOPC-315 NP-1 variant was shown to contain few or no sequences homologous with MOPC-315 light chain mRNA. Thus, the inability of this variant to produce light chain results from the absence of light chain mRNA. In contrast, the polyadenylated mRNA fraction of the MOPC-315 NR variant, which does not synthesize heavy chain, contains about two-thirds of the sequences present in heavy chain mRNA. Thus, this variant contains a fragment of heavy chain mRNA.     

Glutamine as a precursor to N-terminal pyrrolid-2-one-5-carboxylic acid in mouse immunoglobulin λ-type light chains. Amino acid-sequence variability at the N-terminal extra piece of λ-type light-chain precursors

The mRNA molecules coding for three mouse immunoglobulin lambda-type light (L) chains (MOPC-104E lambda(1), RPC-20 lambda(1), MOPC-315 lambda(2)) programme the cell-free synthesis of precursors larger than the mature proteins. Radioactive amino acid-sequence analyses of each of the three precursors labelled with [(3)H]alanine, [(3)H]serine, [(3)H]glutamine, [(3)H]glutamic acid and [(3)H]threonine showed that an extra piece, at least 18 residues long, is linked to the N-terminus of the mature L-chains. The N-terminal extra-peptide segment may be 19 residues long, since analyses of precursors labelled with [(35)S]methionine indicated an additional N-terminal methionine residue which was recovered in low yields. Presumably this is the initiator methionine, which is known to be short lived in eukaryotes. The mature forms of MOPC-104E, RPC-20 and MOPC-315 lambda L-chains are blocked at the N-termini by pyrrolid-2-one-5-carboxylic acid (pyroglutamic acid). Sequence analyses of precursors labelled with [(3)H]glutamine and [(3)H]glutamic acid showed incorporation only of glutamine in a position that matches with the position of pyrrolid-2-one-5-carboxylic acid in the mature forms of all three precursors, and incorporation of glutamic acid in other positions. The data showed the absence of glutamine-glutamic acid interconversion, since the radioactive peaks obtained from either (3)H-labelled amino acid were discrete, and free from cross-contamination. These results prove that glutamine is the precursor amino acid of pyrrolid-2-one-5-carboxylic acid at the N-termini of the mature MOPC-104E lambda(1), RPC-20 lambda(1) and MOPC-315 lambda(2) L-chains. Thus the formation of pyrrolid-2-one-5-carboxylic acid by cyclization of glutamine is a post-translational event which occurs after, or concomitant with, cleavage of the extra piece from the precursor to yield the mature L-chain. The variable (V) regions (110 amino acid residues) of mouse lambda L-chains are quite similar: when compared with that of MOPC-104E lambda(1) chain, the V-region of RPC-20 lambda(1) chain differs in one residue, and the V-region of MOPC-315 lambda(2) chain differs in 11 residues. The partial sequence data show that the N-terminal extra pieces of the two lambda(1) L-chain precursors have, so far, identical partial sequences; the extra piece of the lambda(2) L-chain precursor differs from these in at least three out of 19 positions.     

Purification of immunoglobulin heavy chain messenger RNA by immunoprecipitation from the mouse myeloma tumor, MOPC-31C.

Immunoglobulin heavy chain mRNA was purified from immunoprecipitated polysomes derived from the mouse myeloma tumor, MOPC-31C. The purified mRNA migrated predominantly as a single band upon polyacrylamide gel electrophoresis in 98% formamide and the molecular weight of this mRNA was calculated to be 700,000. This mRNA was as active as the purified light chain mRNA when it was employed as a template in a cell-free protein synthesizing system from wheat germ. The translation product had a molecular weight of 55,000 daltons, and migrated slightly faster than mature heavy chain upon polyacrylamide gel electrophoresis in sodium dodecylsulfate. The protein synthesized by the direction of this mRNA was shown to yield tryptic peptides corresponding to those derived from the mature heavy chain protein except that one missing peptide was replaced by another additional peptide. DNA complementary to the mRNA was synthesized by RNA-dependent DNA polymerase from avian myeloblastosis virus. Hybridization kinetic analysis between the heavy chain mRNA and its complementary DNA indicated that the RNA was essentially homogenous with rabbit globin mRNA as a standard.     

Synthesis and glycosylation of the MOPC-46B immunoglobulin in kappa chain in Xenopus laevis oocytes.

Polyadenylated mRNA isolated from MOPC-46B plasmacytoma, which secretes a glycosylated kappa chain, was injected into $\text{Xenopus laevis}$ oocytes. Analysis of the resulting product showed that [1-¹⁴C]mannose was incorporated into the MOPC-46B kappa chain. Light chains synthesized in oocytes injected with mRNA from MOPC-321 plasmacytoma, which secretes a nonglycosylated kappa chain, failed to incorporate label from [1-¹⁴C]mannose. Thus, protein glycosylation in the oocyte is apparently specific in that carbohydrate is incorporated only into the kappa chain synthesized as a glycoprotein by myeloma cells. It is thus evident that the general signals for glycosylation have remained stable during independent evolution of the amphibia and mammalia.     

Cotranslational cleavage of immunoglobulin light chain precursors by plasmacytoma microsomes.

Murine plasmacytoma endoplasmic reticulum which has been freed of ribosomes by EDTA treatment is capable of the cotranslational proteolytic processing of representative λ₁,λ₂, and k immunoglobulin light chain precursors. Messenger RNA fractions from the MOPC-104E, MOPC-315, and MOPC-46B tumor lines were used to direct the synthesis of the light chain precursors in a cell-free system derived from Krebs II ascites cells. The precursor cleavage activity of the plasmacytoma membranes is comparable in activity and in characteristics to that of two well-defined membrane preparations: Krebs II ascites intracellular membranes (E. Szczesna and I. Boime, 1976, Proc. Nat. Acad. Sci. USA73, 1179–1183) and EDTA-treated rough endoplasmic reticulum from canine pancreas (34., 35., J. Cell Biol.67, 852–862). The efficiency of the cleavage reaction appears to be dependent upon the precursor being utilized as a substrate. An assay suitable for a preliminary characterization of the plasmacytoma membrane preparations is described.     

Amino acid-sequence variability at the N-terminal extra piece of mouse immunoglobulin light-chain precursors of the same and different subgroups.

The proteins programmed in the wheat-germ cell-free system by the mRNA coding for the MOPC-63 mouse myeloma L (light) chain were labelled with six radioactive amino acids: [35S]methionine, [4,5-3H]leucine, [3,4-3H]proline, [3-3H]serine, [4,5-3H]isoleucine or [2,3-3H]alanine. Amino acid-sequence analyses showed that over 90% of the total cell-free product was one homogeneous protein, which corresponds to the MOPC-63 L-chain precursor. In this precursor an extra piece, 20 amino acid residues in length, precedes the N-terminus of the mature L chain. The extra piece contains one methionine residue at the N-terminus, six leucine residues, which are clustered in two triplets at positions 6, 7, 8 and 11, 12, 13, one proline residue at position 16, and one serine residue at position 18. The closely gathered leucine residues, as well as their abundance (30%), suggest that the extra-piece moiety is hydrophobic. In the precursors, the extra piece is coupled to the variable region of the L chain. Partial sequences of precursors of L chains of the same and different subgroups that were labelled with the above six radioactive amino acids indicate that the extra piece is part of the variable region. Thus the precursors of MOPC-63 and MOPC-321 L chains, which are of the same subgroup, have extra pieces of identical size (20 residues), and so far their partial sequences are also identical (see above). On the other hand, in the precursor of MOPC-41 L chain, which is of a different subgroup, the extra piece is 22 residues in length. Further, the sequence of the MOPC-41 extra piece differs in at least ten positions from sequences of the extra pieces of the precursors of MOPC-63 and MOPC-321 L chains.     

Comparing domain interactions within antibody Fabs with kappa and lambda light chains

IgG antibodies are multi-domain proteins with complex inter-domain interactions. Human IgG heavy chains (HCs) associate with light chains (LCs) of the κ or λ isotype to form mature antibodies capable of binding antigen. The HC/LC interaction involves 4 domains: VH and CH1 from the HC and VL and CL from the LC. Human Fabs with κ LCs have been well characterized for their unfolding behaviors and demonstrate a significant level of cooperativity and stabilization when all 4 domains are intact. Very little is known regarding the thermodynamic properties of human Fabs with λ LCs. Here, we dissect the domain contributions to Fab stability for both κ and λ LC-containing Fabs. We find the cooperativity of unfolding between the constant domains, CH1/Cλ, and variable domains, VH/Vλ, within λ LC-containing Fabs is significantly weaker than that of κ LC-containing Fabs. The data suggests there may not be an evolutionary necessity for strong variable/constant domain cooperativity within λ LC-containing Fabs. After investigating the biophysical properties of Fabs with mismatched variable and constant domain subunits (e.g., VH/Vκ paired with CH1/Cλ or T cell receptor Cα/Cβ), the major role of the constant domains for both κ- and λ-containing Fabs may be to reduce the hydrophobic exposure at the VH/VL interface. Even though Fabs with these non-native pairings were thermodynamically less stable, they secreted well from mammalian cells as well behaved monodisperse proteins, which was in contrast to what was observed with the VH/Vκ and VH/Vλ scFvs that secreted as a mixture of monomer and aggregates.     

RNA associated with murine intracisternal type A particles codes for the main particle protein.

Intracisternal type A particles were isolated from MOPC-104E myeloma grown subcutaneously and from N 4 neuroblastoma cells in culture. Polyadenylated RNA was prepared from the particles and tested in a cell-free translation system derived from rabbit reticulocytes. RNA from the two sources directed the synthesis of multiple polypeptides with similar distributions of electrophoretic mobilities in sodium dodecyl sulfate-containing polyacrylamide gels, including one conponent of the same size as the major A-particle structural protein (73,000 daltons). Analysis of the RNAs by electrophoresis in methyl mercury-containing agarose gels revealed a 35S component common to A-particles from both cell types. This was a major component of the N4 preparations, whereas a 28S species predominated in the case of MOPC-104E. These two RNAs (35S from N4 cells and 28S from MOPC-104E), when isolated on isokinetic sucrose gradients, each directed the synthesis of a 73,000-molecular-weight polypeptide that comigrated on gels with authentic A-particle structural protein. Idnetity of the cell-free product was confirmed by two-dimensional analysis of the [35S]methionine-labeled tryptic peptides. The N4 RNA preparations also contained a major32S component which did not code effectively for the A-particle structural protein.     

Some characteristics of the in vivo antitumor immunity exhibited by mice cured of a large MOPC-315 tumor by a low dose of melphalan

Summary BALB/c mice cured of a large MOPC-315 or MOPC-104E plasmacytoma following treatment with a low dose (2.5 mg/kg) of melphalan (L-PAM) were resistant to challenge with the other plasmacytoma but to a much lesser extent than to challenge with the autochthonous plasmacytoma. The resistance of the L-PAM-cured MOPC-315-tumor bearers to challenge with MOPC-104E tumor cells was increased when the MOPC-104E tumor cells were admixed with MOPC-315 tumor cells prior to their inoculation. This enhanced resistance to MOPC-104E cells was due to elimination of the MOPC-104E tumor cells through an innocent bystander killing effect since it did not render the mice more resistant to a subsequent challenge with MOPC-104E tumor cells alone. Administration of carrageenan to L-PAM-cured MOPC-315-tumor bearers 1 day after the challenge with the mixture of MOPC-104E and MOPC-315 tumor cells drastically reduced the ability of the mice to resist the tumor challenge. All of the tumors that developed in such mice were of MOPC-104E origin only (as judged by the binding specificity of the myeloma proteins secreted by the tumor cells as well as that present on their surface) even though (a) the tumor inoculum used consisted of up to 10-fold more MOPC-315 than MOPC-104E tumor cells and (b) the MOPC-315 tumor cells divide more rapidly. The same protocol of carrageenan treatment did not reduce the ability of normal BALB/c mice to develop in vivo a primary cell-mediated cytotoxic response nor a primary antibody response indicating that it has no effect on the initiation of an immune response. Therefore, it is conceivable that carrageenan treatment reduced the ability of L-PAM-cured MOPC-315-tumor bearers to reject a challenge with MOPC-315 and MOPC-104E tumor cells by interfering at the effector stage. The ability of the L-PAM-cured MOPC-315-tumor bearers to reject the MOPC-315 cells present in the challenge mixture was reduced when the mice were treated with anti-Thy 1.2 antibody but not with carrageenan, indicating that T-cells independent from carrageenansensitive effector cells are required for the rejection of the MOPC-315 tumor cells. Thus, at least two different effector mechanisms participate in the rejection of a challenge composed of MOPC-315 and MOPC-104E tumor cells by L-PAM-cured MOPC-315-tumor bearers. One of the mechanisms is dependent on the action of carrageenansensitive effector cells while the other does not require carrageenan-sensitive effector cells but is T-cell-dependent.Supported by Research Grant IM-435 from the American Cancer Society and Research Grant CA-35761 from the National Cancer InstituteIn partial fulfillment of the requirements for the Doctor of Philosophy degree     

Suppression of myeloma establishment in adult mice with anti-micron antiserum.

Uniform suppression of MOPC-104E tumor development was observed in adult BALB/c mice to which 0.4 ml of high titered anti-μ antiserum had been administered intraperitoneally or intravenously one day before subcutaneous tumor implantation. In contrast, when MOPC-104E cells were exposed to anti-μ antiserum in vitro for 10 min and subsequently injected into adult BALB/c mice, inhibition of tumor development was observed in only about half of the subject animals. Nude mice treated intraperitoneally with anti-μ antiserum were also uniformly refractory to MOPC-104E challenge. Anti-μ antiserum exhibited virtually no cytotoxicity against MOPC-104E cells in vitro. These observations suggest that neither complement-mediated cytotoxicity nor T cell-mediated immunity is likely to be the primary mechanism for anti-μ-mediated suppression of myeloma development in adult BALB/c mice. More plausible explanations for this type of suppression include macrophage arming, some type of antibody-dependent cell-mediated cytotoxicity, and/or opsonization.     

Mass spectrometry analyses of κ and λ fractions result in increased number of complementarity-determining region identifications

Sera from lung cancer patients contain antibodies against tumor-associated antigens. Specific amino acid sequences of the complementarity-determining regions (CDRs) in the antigen-binding fragment (Fab) of these antibodies have potential as lung cancer biomarkers. Detection and identification of CDRs by mass spectrometry can significantly be improved by reduction of the complexity of the immunoglobulin molecule. Our aim was to molecular dissect IgG into κ and λ fragments to reduce the complexity and thereby identify substantially more CDRs than by just total Fab isolation. We purified Fab, Fab-κ, Fab-λ, κ and λ light chains from serum from 10 stage I lung adenocarcinoma patients and 10 matched controls from the current and former smokers. After purification, the immunoglobulin fragments were enzymatically digested and measured by high-resolution mass spectrometry. Finally, we compared the number of CDRs identified in these immunoglobulin fragments with that in the Fab fragments. Twice as many CDRs were identified when Fab-κ, Fab-λ, κ and λ (3330) were combined than in the Fab fraction (1663) alone. The number of CDRs and κ:λ ratio was statistically similar in both cases and controls. Molecular dissection of IgG identifies significantly more CDRs, which increases the likelihood of finding lung cancer-related CDR sequences.     

Molecular cloning of a bovine immunoglobulin lambda chain cDNA

A cDNA library of the bovine mammary gland constructed in pBR322 was screened by mRNA hybrid-selected translation and by differential hybridization. Several immunoglobulin (Ig) lambda light-chain clones were identified and sequenced. Nucleotide sequence comparison of bovine and human Ig lambda chains showed a high degree of homology for constant regions and for J regions. The amino acid (aa) sequence encoded by the constant region of the bovine Ig lambda chain cDNA contains 107 aa with differences at 24 aa positions from the human Ig lambda chain. Three complementarity-determining regions (CDR1,2,3) characteristic of the variable region of bovine Ig lambda chain cDNA can be distinguished. The bovine and human sequences display good homology in the framework region 3 (FR3) but only patches of homology throughout the FR2 region. The 5' end of the bovine Ig lambda chain cDNA fragment of clone 1-14E contains five stop codons: two in CDR1, one in FR1 and two in the hydrophobic prepeptide region. These data suggest that the Ig lambda mRNA of clone 1-14E is transcribed from the V lambda pseudogene.     

Structural mutations in a mouse immunoglobulin light chain resulting in failure to be secreted

Two nonsecreting clones of MOPC 315 mouse myeloma cells were compared with a clone of the same cell line secreting λ light chains. All three clones synthesized λ chains but no detectable heavy chains. The two mutant clones which failed to secrete λ chains both synthesized an altered λ chain. The structural alterations were demonstrated by two independent methods for each mutant, and in each case were located in one peptide derived by cyanogen bromide cleavage. In one clone, the alteration was located in the N terminal 87 amino acids of the protein, and was thus present in the variable region of the light chain. In the other clone, the alteration was located between residues 88 and 175. Light chain precursors, and not mature light chains, were synthesized in a cell-free translation system from polyribosomes derived from each of the three cell lines. The structural alterations characteristic of the two mutant clones were also present on the λ chains synthesized in vitro. These results suggest that the mutant light chains were synthesized as precursor molecules and cleaved in vivo, and that the block in secretion occurred at some stage after precursor cleavage. For the MOPC 315 light chain, the synthesis and cleavage of a precursor polypeptide sequence may be necessary, but does not appear to be sufficient, for subsequent secretion of the protein.     

Characterization of light chain and light chain constant region fragment mRNAs in MPC 11 mouse myeloma cells and variants

Cultured MPC 11 mouse myeloma cells synthesize not only γ_2b heavy and κ light chains but also a carboxyl terminal (constant region) fragment of κ light chain. In vitro translational analysis of total cytoplasmic and microsomal RNA indicates that these cells contain RNA which directs synthesis of both a light chain precursor and a light chain fragment precursor. Variant clones which do not synthesize either heavy or light chains continue to synthesize the light chain fragment. One such “nonproducing” variant was studied in detail. It does not contain translatable mRNA for the intact light chain but does contain RNA which is translated into the light chain fragment precursor. Nucleic acid hybridization analysis with a cDNA probe specific for the constant region of κ light chains revealed that microsomal RNA from the wild-type cell contains both a 14S and a 10S species of κ specific RNA, whereas the variant contains only the 10S species. Translational analysis of these same RNAs indicates that the 14S species codes for the light chain precursor, while the 10S RNA codes for the light chain fragment precursor.