VH Sequences Research Articles

Amino acid sequence of the heavy chain variable region from the A/J mouse anti-arsonate monoclonal antibody 36-60 bearing a minor idiotype.

In addition to the predominant idiotype family (IdCR) associated with the murine A/J anti-azophenylarsonate response, a second idiotype family (Id36-60) was defined on the basis of serologic cross-reactivity and amino acid sequence homology among monoclonal antibodies [Marshak-Rothstein, A., Margolies, M.N., Benedetto, J.D., & Gefter, M.L. (1981) Eur. J. Immunol. 11, 565-572]. The complete variable region amino acid sequence of the A/J IgG2a hybridoma protein 36-60 heavy chain was obtained by automated Edman degradation of the intact chain and fragments generated by cleavage with CNBr and by tryptic digestion of a succinylated CNBr peptide. A comparison of the Id36-60 heavy chain sequence to that of the IdCR heavy chain variable region reveals only 45% homology between them. The structural data indicate that different genes encode the VH, D, and JH gene-encoded sequences in the two idiotypes.

The limited diversity of the mouse gamma-chains anti-GAT repertoire does not seem to be noticeably amplified upon class switch.

NH2-terminal sections of H and L chains isolated from five monoclonal anti-GAT antibodies derived from BALB/c mice have been sequenced upon to residue 43. Four among these five antibodies, sharing similar public idiotypic determinants, possess extremely conserved sequences, both for the H, which is apparented to the VH II type, and the L chains, which belong to the V kappa I subgroup. VH sequences are identical up to residue 43 and contain the common sequences (residues 1 to 32) defined for the H chains derived from the DBA/2 IgM anti-GAT monoclonal antibodies. Light chains are also remarkably conserved, a rather unusual situation for kappa-chains. The fifth antibody that expresses only part of the public idiotypic determinants contains very distinctive H and L chains. Its heavy chains are close to the VH I subgroup, whereas its kappa-chains permit definition of a new V kappa subgroup. The repertoire appears to be highly conserved between BALB/c and DBA/2 mice, and does not seem larger in IgG than in IgM antibodies. This latter observation does not speak in favor of a switch-linked amplification of diversity.

Immunoglobulin idiotopes expressed by T cells. I. Expression of distinct idiotopes detected by monoclonal antibodies on antigen-specific suppressor T cells.

The idiotopic repertoire expressed by antigen-specific suppressor T cells (Ts) generated by Streptococcus pneumoniae strain R36a (Pn) in BALB/c strain mice was investigated using a panel of five monoclonal anti-idiotopic antibodies against TEPC-15/HOPC-8 myeloma proteins. Previous studies suggested that the anti-idiotopic antibodies recognize distinct idiotopic determinants within the T15 idiotype, and that Pn-reactive B cells express all of those idiotopes as shown by a specific inhibitory effect of the anti-idiotopic antibodies on induction of anti-Pn response in vitro as well as on the mature antibody plaque-forming cells. In this study we asked the question of whether anti-idiotopic (Id) can block the inductive and/or effector phases of generation of Ts which act on the Pn-reactive B cells. The presence of anti-Id during the activation of T cells with Pn did not prevent the generation of Ts. However, suppression mediated by Ts on responder lymphocytes (cultures of spleen cells or B cels) was inhibited (reversed) by four out of five anti-Id. Some of the antibodies recognize hapten (phosphorylcholine)-inhibitable Id in the paratope of Ig whereas others are directed against nonparatopic Id. These data indicate that the antigen receptor on Ts includes VH sequences both within and without the immunoglobulin in paratope, and that the Id repertoir of Ts overlaps with that of B cells.

Polymorphism in immunoglobulin heavy chains suggesting gene conversion.

Complete heavy (H) chain variable region (V region; amino acids 1-118) sequences have been determined for three phosphocholine (PCho)-binding monoclonal antibodies of CBA mouse strain origin. Two of these were found to differ from the sequence of the BALB/c T15 germline VH segment (segment of the V region that includes amino acids 1-95) at four positions but were identical to the allelic form of T15 (C3) found in C57BL. The third VH segment, HP101.6G6 (6G6), was clearly the product of a second, related VH gene, probably the allele of the BALB/c V11 gene, a second member of the P-Cho VH gene family. Thus, more than one VH gene is capable of encoding heavy chains of PCho-binding antibodies. The 6G6 VH segment differs from VII at seven positions; four of these distinguishing amino acids are encoded in other membranes of the PCho VH gene family. We postulate that the origin of the 6G6 VH sequence can most easily be explained by a process of gene conversion occurring between the least three members of the PCho VH family.

Identification of D segments of immunoglobulin heavy-chain genes and their rearrangement in T lymphocytes.

The finding that the diversity (D) and joining (JH) but not the variable (VH) DNA segments of mouse immunoglobulin heavy-chain genes are joined in the DNA of some cloned cytolytic T cells, led to identification and sequencing of three different D DNA segments. Two segments identified on the embryo DNA carry on both the 5' and 3' sides two sets of characteristic sequences separated by a 12-base pair spacer, which have been implicated as recognition signals for a recombinase. The third segment, identified in a form joined with a JHDNA segment in a T cell, carries the recognition signal on the 5' side. These results support the 12/23-base pair model for somatic generation of immunoglobulin V genes, and rule out the possibility that the cytolytic T cells use assembled VH, D and JH sequences to encode their antigen receptors.

Structure and multiplicity of genes for the human immunoglobulin heavy chain variable region.

Several genes for the variable region of immunoglobulin heavy chains (VH genes) have been isolated from human fetal liver DNA by using a cDNA plasmid probe containing a mouse VH sequence. The detectable VH genes are separated by 12-16 kilobases of DNA, and hybridization experiments show about 23 hybridizing VH genes in DNA of three different individuals. The complete nucleotide sequence of one of these human VH genes shows that it belongs to the human VHIII subgroup. The VH gene appears to contain an intervening sequence (104 bases in length) within a precursor sequence, between residues -4 and -5. The precursor sequence is itself 19 codons in length. The 3' end of the V gene seems to be at codon 93 or 94, and this is followed by the conserved sequences C-A-C-A-G-T-G and G-A-C-A-C-A-A-A-C-C. The presence of these sequences suggests that similar enzymatic mechanisms are involved in the integration of V genes in both heavy and light chains.

Shared N-Terminal Sequences in Monoclonal IgMκ and IgGκ Proteins from a Patient with a Complex Multiple Paraprotein Disorder

Abstract The N-terminal sequence analyses were performed on the heavy (H) and light (L) chains of the idiotypically identical IgMκ and IgGκ paraproteins isolated from the serum of patient, Cam. The N-terminal 39 residues of the κ chains of the IgM and IgG were identical and belonged to the human VκIII subgroup. This sequenced stretch included the first L chain hypervariable region. The N-terminal 27 residues of the variable regions (VH) of the respective µ and γ heavy chains were also identical and belonged to the human VHIII subgroup. These identical VH sequences were unique with lysine residues at positions 13 and 19. This dual lysine substitution has not been seen in 37 other human VHIII sequences reported in the literature. This N-terminal sequence homology in the V-regions of Cam IgMκ and IgGκ paraproteins and the shared idiotypy expressed by Cam IgM, IgG, and IgA proteins strongly suggest the existence of complete structural homology in the variable regions of the H and L chains of these Ig molecules of three separate Ig classes. At the cellular and genetic level, these results point toward a common clonal origin for the idiotypically related Ig molecules and suggest that identical V-region (VH and VL) genes were utilized by the Cam lymphoid clone in the biosynthesis of the respective IgM, IgG, and IgA proteins.

Structural Evidence for an Unblocked VHI Sub-Subgroup of Human Heavy Chains

The N-terminal amino acid sequence of an unblocked human heavy chain of an IgGK paraprotein from patient Tho is reported. This unblocked VH sequence belongs to the VHI subgroup and has striking structural homology to an unblocked VHI sequence previously reported for the IgGK paraprotein isolated from the serum of patient Bro. Direct sequence comparison of the Tho and Bro proteins reveals complete structural identity in 25 of the N-terminal 27 residues, with unique amino acid substitutions shared at the N-terminus and positions 16, 18, and 24. This remarkable sequence homology suggests that the two VH sequences represent examples of an unblocked VHI sub-subgroup. The Tho and Bro sequences possess an ala-glu-val basic triplet at positions 9 to 11 in common with the other previously reported VHI proteins which help to further establish this sequence triplet as a recognizable VHI subgroup-specific region. Moreover, in this regard, the unblocked sequences of Tho and Bro emphasize the importance of extending an unblocked VHI sequence beyond positions 9 to 11 before a subgroup assignment can be determined.

Comparative studies on monotypic IgM lambda and IgG kappa from an individual patient. IV. Immunofluorescent evidence for a common clonal synthesis

Previous studies have presented evidence of shared idiotypic antigenic determinants located within the variable (VH) region of the heavy chains of monotypic IgMlambda and IgGkappa isolated from the serum of an individual patient, Bro, with Waldenström macroglobulinemia. Comparative N-terminal VH sequence analyses have demonstrated that the respective micron and gamma chains belong to separate VH subgroups. The entire VH sequence of the Bro micron chain has been reported, but the VH sequence of the Bro gamma chain still awaits completion. We report the results of an immunofluorescent analysis of cytoplasmic Ig of lymphoid cells isolated from the patient's peripheral blood and bone marrow. Between 6% and 9% of the cytoplasmic Ig-positive lymphoid cells exhibited fluorescent evidence for the dual presence of kappa and lambda chains are well as micron and gamma chains. These results strongly suggest that the idiotypically related Bro IgMlambda and IgGkappa paraproteins are derived from a common clonal origin. Moreover, these findings extend the results of a previous study that has demonstrated the dual presence of IgGkappa and IgGlambda paraproteins within individual myeloma plasma cells. Collectively, these studies suggest that a single neoplastic lymphoid clone may not necessarily be restricted to the synthesis of Ig proteins of the identical light chain class. These findings may have a broad implication for the understanding of surface and cytoplasmic Ig markers of neoplastic lymphoid cells in certain other lymphoproliferative disorders.

Comparative Studies on Monotypic IgM λ and IgGκ From an Individual Patient. IV. Immunofluorescent Evidence for a Common Clonal Synthesis

Abstract Previous studies have presented evidence of shared idiotypic antigenic determinants located within the variable (VH) region of the heavy chains of monotypic IgMlambda and IgGkappa isolated from the serum of an individual patient, Bro, with Waldenstrom macroglobulinemia. Comparative N-terminal VH sequence analyses have demonstrated that the respective micron and gamma chains belong to separate VH subgroups. The entire VH sequence of the Bro micron chain has been reported, but the VH sequence of the Bro gamma chain still awaits completion. We report the results of an immunofluorescent analysis of cytoplasmic Ig of lymphoid cells isolated from the patient's peripheral blood and bone marrow. Between 6% and 9% of the cytoplasmic Ig-positive lymphoid cells exhibited fluorescent evidence for the dual presence of kappa and lambda chains are well as micron and gamma chains. These results strongly suggest that the idiotypically related Bro IgMlambda and IgGkappa paraproteins are derived from a common clonal origin. Moreover, these findings extend the results of a previous study that has demonstrated the dual presence of IgGkappa and IgGlambda paraproteins within individual myeloma plasma cells. Collectively, these studies suggest that a single neoplastic lymphoid clone may not necessarily be restricted to the synthesis of Ig proteins of the identical light chain class. These findings may have a broad implication for the understanding of surface and cytoplasmic Ig markers of neoplastic lymphoid cells in certain other lymphoproliferative disorders.

Amino acid sequence of the VH region of a human myeloma immunoglobulin (IgG New)

The amino acid sequence of the heavy-chain variable region of the human immunoglobulin. New has been determined. Since the amino terminus of the heavy chain was blocked, the sequence of residues 1-69 was established by digesting the appropriate CNBr fragment separately with trypsin, chymotrypsin, and thermolysin and sequencing the resulting peptides. The region from residues 70 to 120 was present in another CNBr fragment which was submitted directly to automatic Edman degradation. The result of this experiment extended the sequence to residue 100. The primary structure of the remaining portion of the VH region was determined by automatic Edman degradation of a lysine-blocked tryptic peptide derived from this region which included residues 98-214. The sequence of the VH region of New corresponds most closely to VH sequences of proteins in the VH II subgroup. This primary structure makes it possible to construct a model from the high-resolution electron-density map of protein New.

Evidence for the induction of two antibodies with identical combining sites in outbred animals.

THE mechanism by which the immune system produces an apparently limitless array of antibodies in response to a variety of antigenic stimuli remains an unresolved biological question. Primary structural analysis has shown that immunoglobulin heavy (H) and light (L) chains have both variable (V) and constant (C) regions of amino acid sequence. Within the VH and VL region sequences there are areas of hypervariability which are thought to be associated with the antibody combining site. It is the amino acid substitutions within certain of these hypervariable areas that result in the multiplicity of antibody specificities (sites) expressed in nature. It seems clear that separate genes code for the V and C regions of H and L chains and that integration of V- and C-region genes occurs at the DNA level1. One can account for antibody isotypic diversity by postulating a limited number of C-region genes which are transmitted from generation to generation in the germ line. The difficulty arises in the attempt to account for the apparent large numbers of V-region genes required to explain antibody combining site diversity. V-gene counting has been accomplished by RNA–DNA hybridisation2–4. Evidence from these studies has not definitely demonstrated whether the capacity to generate large numbers of combining sites exclusively arises by either the inheritance of a complete set of VH and VL genes in the germ line or by somatic mutation. The repeated occurrence of V-gene products has, however, been indicated by sequence analysis of myeloma proteins5–7, isoelectric focusing8–10, and idiotypic analysis11–13.

Genetics of the immunoglobulin variable region

On March 25 and 26, 1974, the Tumor Immunology Program of the National Cancer Institute sponsored a workshop on recent progress in the genetic analysis of antibody structural genes. The intent of the meeting was: 1. To define the genetics of markers currently used. In addition to serological studies of defined V-region aUotype markers, investigators have studied other phenotypes, such as idiotype, the magnitude of response to certain antigens, distinctive crossreactivities elicited by certain antigens, unique isoelectric spectra, and others. It is still not known whether these phenotypes are coded for by V-region structural genes. 2. To accumulate linkage data from a variety of systems. For the variety of phenotypes studied, close linkage to CH-region genes could be demonstrated. Combined data from diverse sources yielded valuable information on the linkage relationship of VH and CH. Furthermore, there are indications that genes of the H-chain loci are arranged in an organized fashion. 3. To exchange information on the use of appropriate strains. Because of the complexities of mouse genetics, further attempts to identify additional V-region markers should focus on those strains of mice for which appropriate congenic partners exist and which have been used to derive established recombinant-inbred lines. Detailed linkage analysis is required for strains known to differ by other V-region phenotypes. Abstracts of papers presented at this workshop are appended. However because of the large amount of data from divergent sources, certain topics have beefi summarized and interpreted, hopefully in a unifying way. Until recently, V-region genetics has centered mainly on studies in the rabbit. The particular advantage offered by this species has been serologically defined VH markers associated with VH sequence differences. In combination with CH markers,