The immunoglobulin heavy chain class switch

Conclusions While much has been learned concerning the molecular structural basis for the heavy chain class switch, many questions relating to the regulation of the switch remain unanswered, or at least controversial. Identification of the enzyme system which mediates the class switch, as well as other regulatory, possibly X-linked, genes should provide the necessary key to our understanding of this unique process.AbbreviationsB cell lymphocyte derived from the bone marrow in adult mammals or the bursa of Fabricius in chickens - bp base pair - C immunoglobulin constant region - CDR complementarity-determining region of the immunoglobulin variable region - D diversity gene segment of the immunoglobulin heavy chain variable region gene - H immunoglobulin heavy chain - Ig immunoglobulin - J joining region gene segment of the immunoglobulin variable region gene - kb kilobase - L immunoglobulin light chain - LPS lipopolysaccharide - Pyr pyrimidine - S-, s-site, s-region switch rearrangement site - SCE sister chromatid exchange - sIg surface immunoglobulin - T cell lymphocyte derived from the thymus - USCE unequal sister chromatid exchange - V immunoglobulin variable region     

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.     

Origins of immunoglobulin heavy chain domains

Summary Using computer programs that analyze the evolutionary history and probability of relationship of protein sequences, we have investigated the gene duplication events that led to the present configuration of immunoglobulin C regions, with particular attention to the origins of the homology regions (domains) of the heavy chains. We conclude that all of the sequenced heavy chains share a common ancestor consisting of four domains and that the two shorter heavy chains, alpha and gamma, have independently lost most of the second domain. These conclusions allow us to align corresponding regions of these sequences for the purpose of deriving evolutionary trees. Three independent internal gene duplications are postulated to explain the observed pattern of relationships among the four domains: first a duplication of the ancestral single domain C region, followed by independent duplications of the resulting first and last domains. In these studies there was no evidence of crossing-over and recombination between ancestral chains of different classes; however, certain types of recombinations would not be detectable from the available sequence data.     

Nucleotide sequences of immunoglobulin mu heavy chain deletion mutants.

Mutants of an IgM producing hybridoma cell line were isolated which produce mu heavy chain fragments. Two such mutants were found to have internal deletions in the mu gene and the nucleotide sequence of the deletion endpoints was determined. No evidence was found for a role of the heavy chain switch region in the formation of these deletions. The implications of these mutants in defining the requirements of immunoglobulin gene expression are discussed.     

Homogeneous rabbit immunoglobulin lacking group a allotypes: amino acid sequence analysis of the heavy chain.

The partial amino acid sequence of rabbit a-negative heavy chain has been determined for residues 1--43 as: less than EEQLEESGGGLVQPGGSLKLSCKGSGFDFSVYGVTWVRQAPGK; and for residues 64--120 as: MNGRFTISSDNAQNRLYLQLNSLTAADTATYFCARSMVVVAGVHSYFDVWGPGTLVTV. Comparison of this sequence with the human heavy chain subgroup III shows homology of 78% suggesting that a common ancestral variable region gene existed in mammals prior to speciation. The constant region of the a-negative chain is structurally identical with that of a-positive chains, whereas the variable region differs substantially between a-positive and a-negative molecules. These findings support the concept that two genes encode one immunoglobulin polypeptide chain and demonstrate the existence in the rabbit of variable region subgroups similar to those reported for humans and other species. A novel approach to the initial fragmentation of the heavy chain was developed in this study. This method, which involved digestion of the H chain with the protease V8, produced a free N terminus and should have wide application in future studies on heavy chains with blocked amino terminals.     

Allelic variants of rearranged immunoglobulin heavy and light chain genes in hybridoma PTF-02 and parent myeloma

The hybridoma PTF-02 secretes an antibody against pig transferrin. Rearranged genes for heavy and light immunoglobulin chains have been studied in the genomes of this hybridoma and in the parent myeloma P3-X63.Ag8.653. The hybridoma was shown to contain three rearranged allelic variants of the heavy chain gene's locus. The gene H2, responsible for synthesis of the heavy chain of the antibody to transferrin, was transmitted in the hybridoma cell from a lymphocyte. Two other genes (H1 and H3) were found both in the hybridoma and parent myeloma genomes. The gene H1 was identified in MOPC21 myeloma, which is a precursor of the X63.Ag8 descendent line. Rearranged k genes were also identified both in the hybridoma and parent myeloma. A functional (K2) gene and a fetal (F) gene appeared in the hybridoma genome from an antigen-stimulated normal lymphocyte. The fetal gene was lost in the course of continuous cultivation of the hybridoma PTF-02 cell line. The gene K1 was transmitted from the myeloma used for fusion. In such a way, the pedigree of rearranged heavy and light chain genes in the hybridoma PTF-02 was established. The results obtained in this work may be relevant to many hybridomas whose immortalizing fusion partner is a MOPC21 derivative, and allow one to identify and isolate functional variable genes to create recombinant constructions.     

An evolutionary conserved motif is responsible for immunoglobulin heavy chain packing in the B cell membrane

All species of vertebrates synthesize immunoglobulin molecules, which differ in an number of aspects but also share a few common features responsible for their function, such as the presence of a transmembrane domain in the membrane bound form of the immunoglobulin heavy chain (IgTMD) that ensures communication with the signal transducing Igα-Igβ peptides. We have analyzed the gene sequence encoding the IgTMD of different heavy chain isotypes of very distant species, from shark to mammals. The IgTMD sequences show a high degree of sequence identity and their encoding nucleotide sequences were shown to be subject to purifying selection at most sites. We have built molecular models of seven IgTMDs from different vertebrate species and have investigated the formation of homodimer in a palmitoyl oleoyl phosphatidylcholine (POPC) lipid bilayer by molecular dynamics simulations. We found that the conserved FXXXFXXS/TXXXS motif, never observed to date in protein transmembrane chains, is responsible for the two heavy chains association through two pairs of Phe-Phe hydrophobic interactions and two pairs of Ser/Thr-Ser/Ser hydrogen bonds. This interaction pattern, which stabilizes the dimer conformation in the lipid bilayer, was unique, being different from any other pattern identified in transmembrane helices to date.     

Physicochemical and immunochemical properties of heavy chains of immunoglobulin G typical of malignant growth

Molecular weight of heavy chains of immunoglobulin G typical of cancer is studied immunoglobulin and may be responsible for manifestation of certain anomalous acid and peptide composition of this protein heavy chains as compared with immunoglobulin G in blood serum of healthy people. Immunochemical methods helped detecting an antigenic determinant (or determinants) which is arranged in the heavy chains of the studied immunoglobulin and may be responsible for manifestation of certain anomalous properties of cancer-typical immunoglobulin G molecules. A set of bromo-cyanogenic fragments differing from the spectrum of these fragments in the heavy chains of normal immunoglobulin G is formed following a specific chemical effect of bromo-cyanogen on the heavy chains of immunoglobulin G typical of cancer. Essential differences are found in dancyl-fingerprints of the heavy chains of the compared proteins. Everything mentioned is a result of changes in the primary structure of the heavy chains of immunoglobulin G typical of cancer.     

Comparative studies of two types of bovine immunoglobulin G heavy chains

;Fingerprints' of bovine colostrum and serum immunoglobulin G1 heavy chains were extremely similar, but different from serum immunoglobin G2 heavy chains. Serum immunoglobulin G1 and immunoglobulin G2 heavy chains were treated with cyanogen bromide. The fractions from the C-terminal end of the heavy chains were isolated and the amino acid sequence of this fraction from immunoglobulin G2 was:His-Glx-Ala-Leu-His-Asx-His-Tyr-Met-Gln-Lys-Ser-Thr-Ser-Lys-Ser-Ala-GlyThe amino acid composition of this fraction from immunoglobulin G1 was the same except for the methionine, which in immunoglobulin G1 was replaced by threonine.     

Retention and loss of immunoglobulin heavy chain alleles in helper T cell hybridoma clones

Retention or loss of immunoglobulin heavy chain genes was studied in 20 functional T cell hybridoma clones. DNA probes representing C mu, C alpha and JH genes, as well as VH subgroups II and III were hybridized with restriction enzyme fragments of hybridoma DNA by the Southern filter hybridization technique. Parental alleles of the hybridoma cells were distinguished on the basis of polymorphism of the lengths of restriction enzyme fragments. All clones retained the alleles of the lymphoma parent cell BW-5147 at all four loci. Thirteen clones lost both CH and VH alleles of the immune partner cell, whereas seven retained both VH alleles, and at least C alpha of the antigen-specific partner. Hence, T cell function in these cells is compatible with the loss of most immunoglobulin heavy chain alleles. This is interpreted to indicate either gene rearrangement and deletion, or chromosome loss. Accordingly, the T cell receptor is either controlled by two split gene loci in chromosome 12, at the two respective (5' and 3') ends of the mouse heavy chain gene family, or by a gene(s) outside chromosome 12.     

An immunoglobulin heavy chain variable region gene is generated from three segments of DNA: VH, D and JH

We have determined the sequences of separate germline genetic elements which encode two parts of a mouse immunglobulin heavy chain variable region. These elements, termed gene segments, are heavy chain counterparts of the variable (V) and joining (J) gene segments of immunoglobulin light chains. The VH gene segment encodes amino acids 1-101 and the JH gene segment encodes amino acids 107-123 of the S107 phosphorylcholine-binding VH region. This JH gene segment and two other JH gene segments are located 5' to the mu constant region gene (Cmu) in germline DNA. We have also determined the sequence of a rearranged VH gene encoding a complete VH region, M603, which is closely related to S107. In addition, we have partially determined the VH coding sequences of the S107 and M167 heavy chain mRNAs. By comparing these sequences to the germline gene segments, we conclude that the germline VH and JH gene segments do not contain at least 13 nucleotides which are present in the rearranged VH genes. In S107, these nucleotides encode amino acids 102-106, which form part of the third hypervariable region and consequently influence the antigen-binding specificity of the immunoglobulin molecule. This portion of the variable region may be encoded by a separate germline gene segment which can be joined to the VH and JH gene segments. We term this postulated genetic element the D gene segment, referring to its role in the generation of heavy chain diversity. Essentially the same noncoding sequences are found 3' to the VH gene segment and as inverse complements 5' to two JH gene segments. These are the same conserved nucleotides previously found adjacent to light chain V and J gene segments. Each conserved sequence consists of blocks of seven and ten conserved nucleotides which are separated by a spacer of either 11 or 22 nonconserved nucleotides. The highly conserved spacing, corresponding to one or two turns of the DNA helix, maintains precise spatial orientations between blocks of conserved nucleotides. Gene segments which can join to one another (VK and JK, for example) always have spacers of different lengths. Based on these observations, we propose a model for variable region gene rearrangement mediated by proteins which recognize the same conserved sequences adjacent to both light and heavy chain immunoglobulin gene segments.     

Biallelic heavy chain immunoglobulin gene rearrangement in acute nonlymphocytic leukemia

Acute nonlymphocytic leukemia (ANLL) was diagnosed in 18 patients based on morphological, cytochemical and immunological criteria. Leukemic cells of these cases were subjected to Southern blot analysis and subsequent hybridization to heavy chain immunoglobulin and T-cell-receptor gene probes. A rearrangement within the immunoglobulin joining region was detected in 2 cases, while the T-cell-receptor beta-chain gene was in germline configuration in all samples investigated. These data confirm recent reports indicating that immunoglobulin heavy chain gene rearrangements are not restricted to B-lineage neoplasms.     

Gene transfer of immunoglobulin light chain restores heavy chain secretion

Several lines of evidence suggest that immunoglobulin (Ig) light (L) chain plays a role in the secretion of heavy (H) chain. For example, myeloma variant lines, which synthesize the Ig H chain but not the L chain, fail to secrete H chain protein. Here we have tested directly the role of chain assembly in the control of Ig secretion by the transfer of functional L chain genes into two such L chain-defective myeloma mutants. A lambda 2 or kappa L chain gene was introduced into variant lines of the mouse myelomas MOPC 315 (IgA, lambda 2) or PC7 (IgM, kappa), respectively. Although the two mutant lines are unable to secrete the H chain they produce, rescue of secretion of complete Ig protein molecules (IgA or IgM) was observed after transfection. These results imply that the secretory apparatus of these cells is intact and that the failure to secrete free H chain reflects a structural feature intrinsic to that protein. The implications of these results with respect to control of secretion of multi-subunit proteins are discussed.     

Co-translational modification of nascent immunoglobulin heavy and light chains

We have investigated the in vivo co-translational covalent modification of nascent immunoglobulin heavy and light chains. Nascent polypeptides were separated from completed polypeptides by ion-exchange chromatography of solubilized ribosomes on QAE-Sephadex. First, we have demonstrated that MPC 11 nascent heavy chains are quantitatively glycosylated very soon after the asparaginyl acceptor site passes through the membrane into the cisterna of the rough endoplasmic reticulum. Nonglycosylated completed heavy chains of various classes cannot be glycosylated after release from the ribosome, due either to rapid intramolecular folding and/or intermolecular assembly, which cause the acceptor site to become unavailable for the glycosylation enzyme. Second, we have shown that the formation of the correct intrachain disulfide loop within the first light chain domain occurs rapidly and quantitatively as soon as the appropriate cysteine residues of the nascent light chain pass through the membrane into the cisterna of the endoplasmic reticulum. The intrachain disulfide loop in the second or constant region domain of the light chain is not formed on nascent chains, because one of the cysteine residues involved in this disulfide bond does not pass through the endoplasmic reticulum membrane prior to chain completion and release from the ribosome. Third, we have demonstrated that some of the initial covalent assembly (formation of interchain disulfide bonds) occurs on nascent heavy chains prior to their release from the ribosome. The results are consistent with the pathway of covalent assembly of the cell line, in that completed light chains are assembled onto nascent heavy chains in MPC 11 cells (IgG₂b), where a heavy-light half molecule is the major initial covalent intermediate; and completed heavy chains are assembled onto nascent heavy chains in MOPC 21 cells (IgG₁), where a heavy chain dimer is the major initial disulfide linked intermediate.     

Posttranslational association of immunoglobulin heavy chain binding protein with nascent heavy chains in nonsecreting and secreting hybridomas

A rat monoclonal antibody specific for immunoglobulin (Ig) heavy chain binding protein (BiP) has allowed the examination of the association of BiP with assembling Ig precursors in mouse B lymphocyte-derived cell lines. The anti-BiP monoclonal antibody immunoprecipitates BiP along with noncovalently associated Ig heavy chains. BiP is a component of the endoplasmic reticulum and binds free intracellular heavy chains in nonsecreting pre-B (mu+, L-) cell lines or incompletely assembled Ig precursors in (H+, L+) secreting hybridomas and myelomas. In the absence of light chain synthesis, heavy chains remain associated with BiP and are not secreted. The association of BiP with assembling Ig molecules in secreting hybridomas is transient and is restricted to the incompletely assembled molecules which are found in the endoplasmic reticulum. BiP loses affinity and disassociates with Ig molecules when polymerization with light chain is complete. We propose that the association of BiP with Ig heavy chain precursors is a novel posttranslational processing event occurring in the endoplasmic reticulum. The Ig heavy chains associated with BiP are not efficiently transported from the endoplasmic reticulum to the Golgi apparatus. Therefore, BiP may prevent the premature escape and eventual secretion of incompletely assembled Ig molecules.     

Using a heavy chain-loss hybridoma 26.4.1LL for studying the structural basis of immunoglobulin chain association

One of the mechanisms contributing to antibody diversity is created by the association of different heavy and light chains. The combinability of heavy and light chains has been studied previously in two systems: in vitro chain recombination and hybrid hybridoma. Here, a novel in vivo chain combination assay system involving a heavy chain-loss variant, 26.4.1LL, producing two kappa light chains (L(DEX) and L(MPC)) different in size is described. In conjunction with DNA transfection, immunoprecipitation and SDS-PAGE, the structural basis of noncovalent interaction between heavy and light chains can be elucidated systematically by examining the relative association tendency of a heavy chain with two light chains. To demonstrate the usefulness of this system, three stably transfected 26.4.1LL cell lines expressing gamma2b heavy chains, designated as H(DEX), H(CHI) and H(ARS), respectively, with structural interrelated variable regions were generated: H(DEX) differs from H(CHI) only in framework regions whereas H(CHI) differs from H(ARS) in complementarity-determining regions. The relative amounts (R values) of L(DEX) and L(MPC) associated with the heavy chains H(DEX), H(CHI) and H(ARS) in the assembled immunoglobulin molecules were found to be 1.02, 0.64 and 0.05, respectively, suggesting that the complementarity-determining regions and framework regions contribute equally to the V(L)-V(H) interaction. This conclusion is consistent with previous observations based on calculation of the buried area in the V(L)-V(H) interface, thus demonstrating the usefulness of this system.     

Partially functional outer-arm dynein in a novel Chlamydomonas mutant expressing a truncated gamma heavy chain

The outer dynein arm of Chlamydomonas flagella contains three heavy chains (alpha, beta, and gamma), each of which exhibits motor activity. How they assemble and cooperate is of considerable interest. Here we report the isolation of a novel mutant, oda2-t, whose gamma heavy chain is truncated at about 30% of the sequence. While the previously isolated gamma chain mutant oda2 lacks the entire outer arm, oda2-t retains outer arms that contain alpha and beta heavy chains, suggesting that the N-terminal sequence (corresponding to the tail region) is necessary and sufficient for stable outer-arm assembly. Thin-section electron microscopy and image analysis localize the gamma heavy chain to a basal region of the outer-arm image in the axonemal cross section. The motility of oda2-t is lower than that of the wild type and oda11 (lacking the alpha heavy chain) but higher than that of oda2 and oda4-s7 (lacking the motor domain of the beta heavy chain). Thus, the outer-arm dynein lacking the gamma heavy-chain motor domain is partially functional. The availability of mutants lacking individual heavy chains should greatly facilitate studies on the structure and function of the outer-arm dynein.     

The N-terminal sequence of the heavy chain of rabbit immunoglobulin IgG

The absence of an N-terminal amino acid with a free alpha-amino group from the heavy chain of rabbit immunoglobulin IgG has been confirmed and no evidence could be found of a blocking formyl, acetyl or propionyl group. The N-terminal amino acid appears to be pyrrolid-2-one-5-carboxylic acid (PCA) in all molecules. A mixed amino acid sequence follows in the approximate proportions: PCA-Ser-Val-Glu-Glu-Ser-Gly-Gly-Arg, 50%; PCA-Ser-Leu-Glu, 20%; PCA-Glu(NH(2)), 20%. The heavy chains of a purified antibody, namely anti-(human serum albumin), and of immunoglobulin IgG from a rabbit homozygous at the allotypic loci both showed a similar mixed N-terminal sequence.     

Ordered rearrangement of immunoglobulin heavy chain variable region segments

The immunoglobulin heavy chain variable region is encoded as three separate libraries of elements in germ-line DNA: VH, D and JH. To examine the order and regulation of their joining, we have developed assays that distinguish their various combinations and have used the assays to study tumor cell analogs of B-lymphoid cells as well as normal B-lymphoid cells. Abelson murine leukemia virus (A-MuLV) transformed fetal liver cells - the most primitive B-lymphoid cell analog available for analysis - generally had DJH rearrangements at both JH loci. These lines continued DNA rearrangement in culture, in most cases by joining a VH gene segment to an existing DJH complex with the concomitant deletion of intervening DNA sequences. None of these lines or their progeny showed evidence of VHD or DD rearrangements. Heavy chain-producing tumor lines, representing more mature stages of the B-cell pathway, and normal B-lymphocytes had either two VHDJH rearrangements or a VHDJH plus a DJH rearrangement at their two heavy chain loci; they also showed no evidence of VHD or DD rearrangements. These results support an ordered mechanism of variable gene assembly during B-cell differentiation in which D-to-JH rearrangements generally occur first and on both chromosomes followed by VH-to-DJH rearrangements, with both types of joining processes occurring by intrachromosomal deletion. The high percentage of JH alleles remaining in the DJH configuration in heavy chain-producing lines and, especially, in normal B-lymphocytes supports a regulated mechanism of heavy chain allelic exclusion in which a VHDJH rearrangement, if productive, prevents an additional VH-to-DJH rearrangement.