Cloning and sequence of the cDNA corresponding to the variable region of immunoglobulin heavy chain MPC11.

Poly(A)-containing mRNA from mouse myeloma MPC11 was transcribed into cDNA which was cloned in the PstI site of the plasmid pBR322. The transformants were screened by hybridization with a cDNA fragment, derived from plasmid p gamma(11)7, corresponding to the 5' portion of the constant region of MPC11 heavy chain. Several positive transformants were found to contain various lengths of the variable region of the heavy chain. We describe the structure and sequence of one of these clones, pV(11)2, which contains cDNA corresponding to the entire variable region of MPC11 heavy chain and extends to codon 248 in the constant region. The protein sequence deduced from the DNA sequence indicates that the variable region of MPC11 heavy chain contains 121 amino acids and belongs to subgroup II of mouse heavy chains. Comparison of this sequence with other heavy chain sequences suggests a J (joining) segment of 16 residues which overlaps five residues of the third hypervariable region. The cDNA sequence shows that there is no discontinuity between the end of the variable region and the beginning of the constant region.     

Cloned MPC 11 myeloma cells express two kappa genes: a gene for a complete light chain and a gene for a constant region polypeptide.

Cloned MPC 11 mouse plasmacytoma cells synthesize a complete kappa light chain and also a kappa light chain constant region fragment. Partial amino terminal sequences of the in vitro forms of these two proteins have been determined. Both in vitro products contain typical light chain leaders; leaders are defined as the amino terminal sequences present on in vitro products but absent from the in vivo products found in living cells. The in vitro form of the MPC 11 complete light chain contains a leader sequence plus variable and constant region sequences. The in vitro form of the MPC 11 light chain constant region fragment contains a different leader sequence attached directly to a complete constant ragion sequence and has no variable region sequences. Thus the MPC 11 light chain fragment is not a degradation product of the MPC 11 complete light chain (or of any other complete light chain) and must be coded by a separate gene. The results reveal two unusual features of MPC 11 cells: first, expression of a unique variant light chain gene coding the light chain constant region fragment, and second, expression of two different kappa light chain genes (coding the complete light chain and the variant constant region fragment) in a single cell. In addition, evidence is provided that the in vitro forms of kappa light chains, three of which are presented here for the first time, include a minimum of three partially homologous but quite different leader sequences.     

Formation of intermolecular disulfide bonds on nascent immunoglobulin polypeptides.

The initial step of intermolecular covalent assembly of immunoglobulins molecules involves formation of heavy chain-light chain or heavy chain-heavy chain disulfide bonds. Using QAE-Sephadex chromatography to isolate microsomal nascent polypeptides, we have shown that this initial step of intermolecular covalent assembly occurs, to a substantial extent, on nascent heavy chains, as well as on completed heavy chains as previously demonstrated by others. In MPC 11 mouse myeloma cells, completed light chains are assembled covalently to nascent heavy chains, whereas in MOPC 21 mouse myeloma cells, completed heavy chains are assembled covalently to nascent heavy chains. These results are consisted with the heavy-light half-molecule being the major initial intermediate in the assembly of MPC 11 IgG2b and heavy-heavy dimer being the major initial intermediate formed in assembly of MOPC 21 IgG1. The nascent MPC 11 heavy chain must be at least 38,000 daltons in size before assembly with the light chain occurs, even though the heavy chain cysteine involved in this disulfide bond is 131 residues (approximately 15,000 daltons) from the NH2 terminus. In addition, pulse-chase labeling studies of MPC 11 cells have shown that the assembly of completed light chains with the nascent heavy chain must occur within a few minutes of the synthesis of the light chain even though a large excess of unassembled MPC 11 light chains remain inside the cell for an average time of 2 h before being secreted.     

A conditional secretory mutant in an Ig L chain is caused by replacement of tyrosine/phenylalanine 87 with histidine.

The kappa-chain of the myeloma MOPC 21 is an unusual L chain, in that it is not secreted unless complexed with a H chain. This nonsecreted kappa-chain seems to be retained in the endoplasmic reticulum in association with the protein BiP/GRP78, both in myeloma cells and when expressed in COS-1 fibroblasts. By assaying the fate of the MOPC 21 kappa-chain and its mutated derivatives in COS-1 cells, we show that the cause of the nonsecreted phenotype is the presence of histidine in position 87 of the variable domain. When this amino acid is changed back to the tyrosine that usually occupies position 87, secretion of the unassembled kappa-chain is restored. As in B lymphoid cells, co-expression of gamma H chains in COS-1 cells complements the mutation in the L chain, and rescues secretion of the arrested kappa. Thus, the presence of histidine at position 87 creates a conditional L chain secretory mutant: it is not compatible with normal transport of free L chain, but can be rescued in the presence of H chain.     

Mouse myeloma cells that make short immunoglobulin heavy chains: pleiotropic effects on glycosylation and chain assembly

Two variants in immunoglobulin heavy chain production, derived from the MPC 11 mouse myeloma cell line, make short heavy (H) chains with identical precise deletions of the CH3 domain. The CH3 domain is expressed in the H chain mRNA from both variants. Although in vitro translation of this mRNA produces one H chain species, deleted heavy chains are secreted as heavy-light (HL) and H2L2 moieties in contrast to MPC 11, which secretes only H2L2 . The heavy chains of HL apparently contain more carbohydrate (CHO+) than do the H chains of H2L2 , and inhibition of N-linked glycosylation results in the secretion of relatively more H2L2 . Here we present evidence suggesting that (a) the absence of the CH3 domain has led to conformational changes in these molecules, (b) these changes permit posttranslational glycosylation, and (c) unrestrained glycosylation can frequently yield unusual CHO+ structures that make complete assembly unlikely.     

The specificity of chain interactions among immunoglobulins. Combinations of γ chains with κ chains of the same subgroup as in the parent immunoglobulin G

1. The specificity of combination of heavy and light chains from selected human immunoglobulins was examined in the light of greater structural information than in previous studies. Heavy (gamma) chains from immunoglobulin G (kappa) myeloma proteins were allowed to combine with their homologous light (kappa) chains or with other kappa chains of the same variable-region subgroup. The affinity of each such pairing was assessed by having the test kappa chain compete with a standard population of normal light chains. 2. There was a spread of affinities among the heavy-light pairings with the homologous pairings having an average affinity significantly higher than the heterologous pairings. 3. It follows that (a) the preference shown for homologous heavy-light pairings is not explicable simply in terms of the known subdivisions of the variable and constant regions of the chains, and (b) it is unlikely that those residues specifying the subgroups of kappa-chain variable regions have a predominant role in the formation of interchain bonds with the gamma-chain variable regions.     

Synthesis of a carboxyl-terminal (constant region) fragment of the immunoglobulin light chain by a mouse myeloma cell line

The MPC11 mouse myeloma cell line synthesizes not only heavy chains and light chains but also an 11,600 molecular weight light chain fragment. The fragment comprises 1% of the newly synthesized protein, compared to 8% for the complete light chain. Similar amounts of fragment are produced by a number of heavy plus light chain producing subclones, 18 independently generated light chain producing variant clones, and five independent non-producing variant clones. For both the heavy plus light chain producing and the non-producing cell types, less than 20% of the fragment appears to be secreted, while the remainder is metabolized with a half-life of 30 minutes. Radiochemical peptide analyses and radiochemical amino -terminal sequence analyses are consistent with the fragment containing most of the peptide sequences present in the carboxyl-terminal half (constant region) of the parent kappa light chain, but none of the variable region peptides. The fragments produced by a heavy plus light chain producing clone and a non-producing variant clone were identical by radiochemical peptide analysis. The results suggest that the constant region fragment may be a primary gene product, and in addition, they raise the possibility that the fragment may be specified by a gene discrete from the gene specifying a light chain.     

Nucleotide sequence of an unequal sister chromatid exchange site in a mouse myeloma cell line.

The mouse myeloma cell line MPC 11 carries two C gamma 2a immunoglobulin heavy-chain genes on the expressed chromosome, a duplication shown to have occurred through unequal sister chromatid exchange (USCE). In the present report, we present the nucleotide sequence of the USCE joint and show that both breaks occurred within tracts of repeated TC dinucleotides. Additional TC dinucleotide tracts and two oligonucleotide segments (N sequences) were inserted at the USCE site.     

The disulphide bridges of a mouse immunoglobulin G1 protein

[(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.     

The complete amino acid sequence of a mouse κ light chain

The complete amino acid sequence of the kappa-chain of the mouse myeloma protein MOPC 21 was established. The protein was reduced and alkylated with iodo[2-(14)C]acetic acid, and 21 tryptic peptides were isolated, mainly by paper electrophoresis and paper chromatography. Three large tryptic peptides (of 35, 36 and 42 residues), which were difficult to isolate in this manner, were obtained pure and in excellent yields by a combination of Sephadex G-50 gel filtration in 1% (w/v) NH(4)HCO(3) and chromatography on a DEAE-cellulose column in ammonium acetate buffer, pH8.1. Peptides overlapping the tryptic peptides were isolated from a chymotryptic digest. The chain is 214 residues long. Microheterogeneity of two peptides was observed and is believed to be due to deamidation. It was not excluded that such deamidation could occur in serum from which the protein was isolated. The sequence is compared with the sequences of two other mouse kappa-chains, and with the human kappa-chain basic sequences.     

Abnormalities in the glycosylation of immunoglobulin heavy chain and an h-2 transplantation antigen in a mouse myeloma mutant.

Two mutant cell lines derived from the MPC-11 mouse myeloma synthesize immunoglobulin with abnormal heavy chains and normal light chains. The defective heavy chains have molecular weights of 38,000-42,000 (M3.11) and 50,000 daltons (ICR 11.19) as compared to 55,000 daltons of the wild-type. The glycosylation of the defective heavy chains demostrated several unusual features: first, 30-50% of the M3.11 heavy chain contained no carbonydrate, while 100% of the wildtype and ICR 11.19 heavy chains were glycosylated; second, the glycopeptides of the M3.11 heavy chains revealed an altered gel filtration pattern when compared with the wild-type; and third, digestion with an endoglycosidase indicated that the heterogeneity of the wild-type and M3.11 glycopeptides involved structural changes in the core region of the oligosaccharide. Examination of two other glycoproteins (the major histocompatibility complex antigens) in these cell lines showed that in M3.11, the H-2D but not the H-2K product was abnormally glycosylated and contained a smaller glycopeptide. However, in a subclone of M3.11 that had lost the ability to produce immunoglobulin heavy chains, the H-2D glycopeptide had returned to wild-type size. We concluded from these studies that the defective M3.11 immunoglobulin heavy chain interfered both with its own glycosylation and the glycosylation of another protein, H-2D.     

Cloning and complete nucleotide sequence of mouse immunoglobulin gamma 1 chain gene.

The 6.6 kb DNA fragment coding for the immunoglobulin γ1 chain was cloned from newborn mouse DNA using λgtWES·λB as the EK2 vector. The complete nucleotide sequence (1823 bases) of the γ1 chain gene was determined. The cloned gene contained the entire constant region gene sequence as well as the poly(A) addition site, but not the variable region gene. The results indicate that the variable and constant region genes of immunoglobulin heavy chain are separated in newborn mouse DNA. The constant region genes of other gamma chains (that is, γ2a, γ2b and γ3) are not present in the cloned DNA fragment. The sequence demonstrates that the γ1 chain gene is interrupted by three intervening sequences at the junction of the domains and the hinge region, as previously shown in the γ2b and α chain genes and in the γ1 chain gene cloned from myeloma. The results suggest that the intervening sequence was introduced into the heavy chain gene before divergence of the heavy chain classes, and also support the hypothesis that the splicing mechanism has facilitated the evolution of eucaryotic genes by linking duplicated domains or prototype peptides not directly adjacent to one another. Comparison of the nucleotide sequence of the γ1 chain gene around the boundaries of the coding and intervening sequences with those of other mouse genes revealed extensive divergence, although short prevalent sequences of AG-GTCAG at the 5′ border of the intervening sequence and TCTGCAG-GC at the 3′ border were deduced. A limited homology of nucleotide sequences was found among domains and between the hinge region and the 5′ portion of the CH2 domain. Comparison of 3′ untranslated sequences from the γ1 and γ2b chain genes and the mouse major β-globin gene shows significant homology and a palindrome sequence surrounding the poly(A) addition site.     

Novel human light chain V kappa segment: serologic and structural analyses of the kappa III-like Bence Jones protein and IgG kappa light chain REE

Immunochemical and sequence analyses of kappa light chain REE (Bence Jones protein REE and the light chain isolated from IgG kappa myeloma protein REE) revealed antigenic and structural features not previously described for human kappa-chains. Although closely related to proteins of the V kappa III subgroup, light chain REE is readily distinguished from light chains classified serologically as members of the kappa IIIa or kappa IIIb sub-subgroups. Light chains REE (Bence Jones protein REE and light chain REE) are identical in sequence and differ from kappa III proteins by at least 10 uncommon amino acid substitutions in the first three framework regions. Further, kappa-chain REE is unique by virtue of a four-residue deletion in the third complementarity-determining region. The deletion encompasses the three carboxyl-terminal residues in the V kappa-encoded segment and the first residue at the site of V-J recombination. Urine specimens from patient REE also contained a light chain fragment that lacked the first (amino-terminal) 85 residues of the native light chain but otherwise was identical in sequence to the light chain REE. The extensive amino acid differences and unique length of the V kappa segment in light chain REE indicate that this kappa-chain is the product of an unusual V kappa III gene or, alternatively, represents a rarely expressed and novel human V kappa gene.     

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.     

Mapping of immunoglobulin variable region genes: relationship to the ''deletion'' model of immunoglobulin gene rearrangement

Five families of variable region genes of mouse kappa chains were analyzed by Southern blot hybridization to determine their relative chromosomal map positions. Map positions were deduced by Vk gene deletion from antibody-producing cells expressing upstream Vk genes and retention in cells expressing downstream genes. The Vk regions expressed in the myelomas M0PC167, MPC11, M0PC21 and ABPC20 are members of Vk families exhibiting one, three, six and six major germline hybridization bands respectively. The gene order of the five families in germline DNA was found to be VM167-VM11-(VM21, VA20)-VABE8-Jk-Ck. As expected in a deletion model of immunoglobulin gene rearrangement, a sequence located just 5' of J1 in germline DNA was found to be absent from some antibody producing cells which had not retained any germline Ck genes. However, other cell lines contained this sequence in rearranged contexts, suggesting that any deletion model of immunoglobulin V-J joining, as well as V gene mapping, must take into account the possibilities of stepwise rearrangements and reintegration of "deleted" DNA.     

Isolation, sequence analysis and characterization of cDNA clones coding for the C chain of mouse C1q. Sequence similarity of complement subcomponent C1q, collagen type VIII and type X and precerebellin.

A mouse macrophage lambda gt11 cDNA library was screened using a genomic DNA clone coding for the C-chain gene of human C1q. Approximately 600,000 recombinant phage plaques were hybridized with peroxidase-labeled human C-chain probe and detected by enhanced chemiluminescence. Five positive clones were obtained. The size of the full-length cDNA is 1019 bp. The sequence identity of the nucleotide sequence with human C1q C chain is 79%, the identity of the deduced amino acid sequences is 73%. The mouse C1q C chain exhibits the same structural features as the human C chain, e.g. conservation of the cysteine residues. Like the mouse A chain, the mouse C chain has an RGD sequence that may be recognized by receptors of the integrin family. No RGD sequences have been found in any of the human C1q chains. The size of the C-chain mRNA (1.2 kb) and its tissue distribution (macrophages being the cell type with the highest mRNA concentration) are identical to the mRNA of the mouse A and B chains. Alignment of human and mouse C1q A, B and C chains exhibits two blocks of highly conserved residues within the C-terminal globular regions. Three other proteins, collagen type VIII and type X and precerebellin share this similarity with C1q, indicating the structural and probably functional importance of these regions within the non-collagenous domains of the molecules.