Structural studies on induced antibodies with defined idiotypic specificities. V. The complete amino acid sequence of the light chain variable regions of anti-p-azophenylarsonate antibodies from A/J mice bearing a cross-reactive idiotype.

The complete amino acid sequence of the variable regions of light chains derived from anti-p-azophenylarsonate antibodies from A/J mice bearing a cross-reactive idiotype is reported. At least two and probably more than three distinct light chains are associated with this idiotypically characterized antibody. The antibodies have several differences in their "framework" structures but evidence is presented indicating that all three light chain hypervariable regions have a homogeneous sequence. The data are discussed in relation to the various theories of antibody diversity. In addition, the findings support the view that hypervariable regions, idiotypic determinants, and the antibody-combining site involve, to a large extent, the same molecular structures.     

Comparison of the amino acid sequences of the variable domains of two homogeneous rabbit antibodies to type III pneumococcal polysaccharide.

The amino acid sequences of the V (variable) regions of the H (heavy) and L (light) chains derived from rabbit antibody K-25, specific for type III pneumococci, were determined; this is the second homogeneous rabbit antibody besides antibody BS-5 whose complete sequence of the V domain has been established (Jaton, 1974d). The V regions of L chains BS-5 and K-25 (both of allotype b4) differ from each other by 19 amino acid residues; 11 of these 19 substitutions are located within the three hypervariable sections of the V region. On the basis of seven amino acid differences within the N-terminal 28 positions, it is suggested that L chain K-25 belongs to a different subgroup of rabbit K chains and L chain BS-5. H chain K-25 (allotype a2) differs from another H chain of the same allotype by one amino acid substitution within the N-terminal 70 positions in addition to interchanges occurring in the first two hypervariable sections. H chain K-25 was compared with H chain BS-5 (allotype a1) and with the known V-region rabbit sequences. Allotype-related differences between a1, a2 and a3 chains appear to occur within the N-terminal 16 positions and possibly in scattered positions throughout the V-region. In the hypervariable positions, variability between the two antibodies is remarkably more pronounced within the third hypervariable section of both H and L chains than within the first two.     

Structural studies on induced antibodies with defined idiotypic specificities. II. The light chains of anti-p-azophenylarsonate antibodies from A/J mice bearing a cross-reactive idiotype.

N-terminal amino acid sequence analyses have been performed on three preparations of light chains of A/J mice. Light chains derived from the IgG of unimmunized animals were compared to light chains of anti-p-azo-phenylarsonate (anti-Ar) antibodies possessing a cross-reacting idiotype (CRI); the latter were derived from the ascites fluid of a single A/J mouse, or from the pooled ascites fluids of 18 A/J mice. The heavy chains of these same two antibody preparations had previously been shown to comprise a single, homogeneous sequence to position 40. With few exceptions, the first 26 positions of light chains derived from unimmunized animals were extremely heterogeneous; the heterogeneity is comparable to that observed in a composite of sequence data on light chains of BALB/c myeloma proteins. Although the light chains obtained from anti-Ar antibodies possessing the CRI (whether from the pool of 18 A/J mice or from a single mouse) were more restricted in their sequence, at several positions as many as four alternative amino acids were detected. These studies indicate that an antibody population with defined idiotypic specificity, and very possibly identical heavy chain sequences, may contain at least four distinct light chains. The feasibility of structural studies on antibodies induced in individual mice is further demonstrated.     

k Chain variable regions from three galactan binding myeloma proteins.

A series of seven BALB/c myeloma proteins has been identified with binding specificity for antigens containing beta(1 leads to 6)-D-galactopyranosyl moieties. We have determined the primary amino acid sequence of the first 108 residues from the light chains of three of these proteins. The framework portions of the variable regions of these three light chains are identical with residue 100 at which position three different amino acids are found in the three chains. An additional interchange was found at position 106 in one of the proteins. Based on recent DNA sequence studies suggesting that the variable region ends at residue 97, these substitutions indicate the possible existance of multiple genes coding for the region beginning at residue 98 and continuing toward the carboxy terminus. A single amino acid interchange was observed in complementarity determining regions occurring in L3. This substitution (Ile-Trp) would require changes in all three codon bases to produce the respective amino acids if one were derived from the other. Two of these chains are thus indistinguishable for their first 100 amino acids and are the first pair of k chains to exhibit complete identity over their variable regions.     

The structure of the variable gene for the kappa chains of antibodies produced by hybridoma PTF-02

The genome of hybridoma PTF-02 has two genes for the kappa chains, and only one of these codes for the synthesis of the antibody light chains. The nucleotide sequences corresponding to the leader peptide and to the variable region of this gene were determined. An amino acid sequence corresponding to exons has been proposed on the basis of the nucleotide sequence. A nucleotide sequence adjacent to the gene at the 5'-end has also been determined, in particular, the precise localization of TATA- and CAT-boxes as well as those of the conservative deca (dc) and pentadeca (pd) sequences. The structure of the regulatory region in the gene is similar to that in the myeloma genomes. However, the 5'-region differs in its nucleotide composition and in the frequency of dc sequences from the DNA sequences adjacent to the 5'-end of eukaryotic genes which do not belong to the immunoglobulin family.     

Analysis of the diversity of murine antibodies to dextran B1355: N-terminal amino acid sequences of heavy chains from serum antibody.

The N-terminal amino acid sequences of two gamma and two mu chains from normally induced serum antibodies to dextran in BALB/c mice are presented. These heavy chains are derived from antibodies with three distinguishable idiotypes. These variable region (VH) sequences are all identical as far as they have been analyzed (27 to 53 residues). The light chains from these antibodies are all of the lambda type and are identical by isoelectric focusing analysis. Accordingly, the diversity of dextran antibodies appears to reside primarily in the heavy chains. The implications of these observations for antibody diversity are discussed.     

Structural characterization of H chain-associated idiotopes of anti-p-azophenylarsonate monoclonal antibodies

The majority of antibodies directed against p-azophenylarsonate (Ars) protein conjugates elicited during secondary immune responses of A/J mice bear a heritable cross-reactive Id (CRIa or IdCR) which corresponds to the utilization of a unique combination of variable region gene segments that can differ by somatic mutations. One such monoclonal anti-Ars antibody, 44-10, bears IdCR as defined by rabbit antisera but does not react with two anti-idiotypic mAb, 5Ci and AD8, which react with all primary (unmutated) IdCR+ antibodies and some secondary response IdCR+ antibodies. We therefore determined the complete sequence of antibody 44-10, which differs from the germline encoded (unmutated) IdCR+ antibody 36-65 at four positions in the H chain V region (VH): position 55 in the second complementarity determining region, 100 and 107 (D-gene junctions) and 110 (in JH2). The 44-10 L chain is unmutated. Sequence analyses of five other secondary immune response anti-Ars IdCR+ antibodies chosen on the basis of sharing one or more of the amino acid substitutions found in 44-10, were correlated with idiotypic expression of this set of antibodies. The results suggest that the mutation at VH position 55 (Asn----Lys) is responsible for loss of the 5Ci idiotope. To substantiate this hypothesis, oligonucleotide-directed mutagenesis of the germline encoded (unmutated) IdCR+ antibody was used to produce two mutants, one with VH Lys 55 and the other containing residues at positions 100, 107 and 110 identical to those found in 44-10. Id binding studies on these mutants confirm that 5Ci idiotope loss is due to conformational changes resulting from a mutation at VH position 55. This mutation also results in loss of the AD8 idiotope in the structural context of antibody 44-10.     

Shared N-terminal sequences in monclonal IgMkappa and IgGkappa proteins from a patient with a complex multiple paraprotein disorder.

The N-terminal sequence analyses were performed on the heavy (H) and light (L) chains of the idiotypically identical IgM kappa and IgG kappa paraproteins isolated from the serum of patient, Cam. The N-terminal 39 residues of the kappa chains of the IgM and IgG were identical and belonged to the human V kappa III subgroup. This sequenced stretch included the first L chain hypervariable region. The N-terminal 27 residues of the variable regions (VH) of the respective mu and gamma 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 IgM kappa and IgG kappa 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 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, IgC, and IgA proteins.     

Isolation and characterization of human VkIII germ-line genes. Implications for the molecular basis of human VkIII light chain diversity

To understand the relative importance of germ-line genes in the generation of the functional human antibody repertoire, it is first necessary to define the number of variable region genes and to determine their fine structure. We have focused on the human VkIII variable region gene family because of its association with autoantibodies. A human genomic library was screened with a VkIII cDNA probe and subsequently with a VkIII germ-line gene probe. Seven different VkIII clones were isolated and characterized by restriction mapping and sequence analyses. Three clones have identical restriction enzyme sites over a 12-kilobase (kb) region, contain identical sequences over an 895-base pair (bp) region, and thus are likely to be different isolates of the same human VkIII gene. Another two clones have identical restriction enzyme sites over a 5-kb region, are identical over a stretch of 905 bp sequenced, and likely represent independent isolates of another human VkIII gene. The remaining two VkIII clones consist of two additional VkIII genes which are homologous to each other, but are quite different from the first two VkIII genes. Thus, four new human VkIII genes were defined. Together with four other VkIII genes previously isolated by other investigators, a total of eight human VkIII germ-like genes have now been described. A comparison of the deduced amino acid sequences of these genes with the reported amino acid sequences of all human VkIII light chains suggests that at least one additional VkIII gene exists in the germ line. Among the eight identified human germ-line VkIII genes, three are pseudogenes. Of the remaining five potential functional genes, one gene seems to encode a majority of the VkIII light chains which have been sequenced. Possible explanations for this phenomenon are discussed.     

Primary structure of the variable regions of two canine immunoglobulin heavy chains.

The complete amino acid sequences of the variable regions of two canine immunoglobulin heavy chains have been determined by automated Edman degradation and found to be strongly homologous to the human VHIII subgroup. The canine sequences were identical with each other at 76 of 113 residue positions. Twenty-three of the 37 differences are located within the four hypervariable regions previously defined by the sequences of several human VHIII proteins. Forty-five of 77 framework residue positions are invariant in the seven human and two canine VHIII proteins which have been completely sequences. The canine proteins are 78% homologous to the framework of the human prototype. Phylogenetically associated residues before the first hypervariable region were confirmed and several potential phylogenetically associated residues were identified between the first and third hypervariable regions. This study represents the first complete amino acid sequences of VH regions of spontaneously occurring, nonhuman homogeneous immunoglobulins. The date demonstrate a high degree of preservation of VHIII structure in another species.     

Rabbit variable kappa light chain regions: subgroups contain polypeptides encoded by multiple genes.

Two identical light chain variable regions were identified in anti-streptococcal Group A-variant antibodies elicited in litter-mate rabbits by hyperimmunization with vaccine. In addition, one rabbit produced two additional clonally restricted antibodies to this polysaccharide antigen. The partial amino acid sequence of the light chain of one of these antibodies was identical with the dominant antibody light chain sequence, while the light chain of the other antibody, also partially established, showed significant variations in the framework-associated regions with identical CDRI and II. Since all of these light chains were from a small subset of rabbit kappa light chain pools (b4 allotype) the data suggest, together with other light chains reported in the literature, that more than one copy of variable region genes are present in the germ-line per subgroup. Furthermore, framework associated amino acid substitutions are not random; this suggests the existence of some "ordered" mechanism for linked amino acid substitutions (presumably recombination). Furthermore one light chain can pair with more than one heavy chain to yield functional antibodies.     

Sequence of a rabbit anti-Micrococcus lysodeikticus antibody light chain.

The complete sequence of rabbit antibody light-chain L 120 has been elucidated. The antibody was raised against Micrococcus lysodeikticus bacteria and is specific for the external part of the cell wall. All protein used in this work was obtained from a single 50-mL bleeding. The variable region of L 120 is compared to 13 other sequences of chains of different specificities. The constant region of this b4 k chain is identical to that of two other constant regions published earlier. The general structure of the rabbit light chain is compatible with the three-dimensional folding proposed for human myeloma chains.     

Sequence similarities and cross-idiotypic specificity of L chains among human monoclonal IgM kappa with anti-gamma-globulin activity

The complete amino acid sequence of five light chain variable (V) regions of human monoclonal IgM kappa rheumatoid factors (RF) was determined, and their cross-reactive idiotypes (CRI) were characterized with antibodies induced by immunization with synthetic peptides PSL2 and PSL3, corresponding to the second and third complementarity-determining regions (CDR) of the SIE light chain. Together with two additional RF studied previously, all seven RF belong to the V kappa IIIb sub-subgroup. The region encoded by the V kappa gene segment (positions 1 to 95) in all seven proteins was virtually identical in primary structure, whereas the sequence from positions 96 to 108 defined the usage of the J kappa 1 gene in three proteins and the J kappa 2 gene in four of them. Position 96 contributed by the recombination of the V kappa and J kappa gene segments showed the presence of four different amino acid residues. Both anti-PSL2 and anti-PSL3 bind efficiently to all separated L chains when analyzed by the Western blot technique, and the binding was inhibited specifically by the corresponding peptides. The results reveal that the majority of human IgM-RF light chains are derived from a single germ line V kappa gene or a family of closely related V kappa III germ line genes, and express two "primary structure-dependent" CRI, which are largely dependent on the amino acid sequence of the second and third light chain CDR.     

Primary structure of a human IgA1 immunoglobulin. V. Amino acid sequence of a human IgA lambda light chain (Bur).

The sequence of the lambda light chain of the Bur IgA1 molecule has been determined. It comprises 214 amino acid residues with a blocked NH2 terminus and lacks carbohydrate. The V-region sequence is of the VlambdaII subgroup and contains the coupled interchanges Arg-7 and Cys-87. The Lv3 region is comparatively short and hydrophobic in nature and lends support for the designation of this area as a hypervariable deletion region. The C-region exhibits the Mcg+ Kren+ Oz- isotypes. These appear coupled with substitution at position 100 (in the V-region). The pattern of nonrandom association of V- and C-regions and H and L chains is discussed in terms of the generation of antibody diversity. With the companion papers in this series, the complete primary structure of a human IgA1 molecule is established.     

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.     

Comparative study on the structure of the light chains of human immunoglobulins. II. Assignment of a new subgroup.

The primary structure of the variable region of the human lambda type Bence Jones protein NIG-48 was determined by analysis of the N-terminal sequence of the completely reduced and aminoethylated protein, as well as of five cyanogen bromide fragments. The variable region of NIG-48 contains 112 amino acid residues. The protein NIG-48, having a unique sequence of the variable region, a low degree of homology (about 50%) with lambda chains of the five other subgroups and the addition of two residues around 65, may represent a new subgroup, namely V lambda VI.     

The V-region sequence of the H chain from a third rabbit anti-pneumococcal antibody.

The amino acid sequence of the V (variable) region of the heavy (H) chain of rabbit antibody BS-1, raised against type III pneumococcal vaccine, is reported. Together with the sequence data of the V region of the light (L) chain previously determined [Jaton (1974a) Biochem. J. 141, 1-13], the present work completes the analysis of the V domain of the homogeneous antibody BS-1. The V domains (VL + VH regions) of this antibody are compared with those of two other anti-(type III) pneumococcal antibodies BS-5 and K-25 [Jaton (1975) Biochem. J. 147, 235-247]. Except for the second hypervariable section of the L chains, these antibodies have very different sequences in the hypervariable segments of the V domains. Within the third hypervariable region of the H chain, each antibody has a different length: BS-1 is three amino acids shorter than K-25 and two amino acids shorter than BS-5. When the sequences in that section are aligned for maximal homology, only two residues, glycine-97 and leucine-101, are common to the three antibodies. On the basis of the amino acid sequences of these three anti-pneumococcal antibodies, the results do not support the concept of a simple correlation between primary structure in the hypervariable sections (known to determine the shape of the combining site) and antigen-binding specificity.     

Complete heavy and light chain variable region sequence of anti-arsonate monoclonal antibodies from BALB/c and A/J mice sharing the 36-60 idiotype are highly homologous

Structural and serologic studies on murine A/J monoclonal anti-arsonate antibodies resulted in the identification of a second idiotype family (Id36-60) in addition to the predominant idiotype family (IdCR). Id36-60, unlike IdCR, is a dominant idiotype in the BALB/c strain but is a "minor" idiotype in the A/J strain. The complete heavy and light chain variable region (VH and VL) amino acid sequences of a representative Id36-60 hybridoma protein from both the A/J and BALB/c strains have been determined. There are only four amino acid sequence differences between the VH of antibody 36-60 (A/J) and antibody 1210.7 (BALB/c). Two of these differences arise from single nucleotide changes in which the A/J and BALB/c Id36-60 VH germline gene sequences differ. The two other differences are the result of somatic mutation in hybridoma protein 36-60. In addition, Id36-60 heavy chains employ the same D and JH3 segments in both strains. The entire Vk2 VL of 36-60 and 1210.7 differ by only two amino acids, suggesting that like the heavy chains, they are derived from highly homologous VL genes. The same Jk segment is used in both antibodies. A comparison of the amino acid sequence data from Id36-60-bearing hybridomas suggests that a heavy chain amino acid difference accounts for the diminished arsonate binding by the 1210.7 hybridoma protein. Because the 1210.7 heavy chain is the unmutated product of the BALB/c VH gene, somatic mutation in VH may be required to enhance Ars affinity in this system.     

Recombinant anti-polyamine antibodies: identification of a conserved binding site motif.

Polyamines are small linear polycations found ubiquitously in eukaryotic cells. They are involved in nucleic acid and protein synthesis and rises in cellular polyamine levels have been correlated with cell proliferation. Antibodies to these molecules have potential as prognostic indicators of disease conditions and indicators of treatment efficacy. Antipolyamine monoclonal antibodies of differing but defined specificities have been generated in our laboratory using polyamine ovalbumin conjugates as immunogens. These antibodies show small but significant cross reactivities with other polyamine species; IAG-1 cross reacts with spermidine (8%), JAC-1 with spermine (6%) and JSJ-1 with both putrescine (11%) and spermine (6%). We have rescued and sequenced the heavy and light chain variable regions of all three of these antibodies. While the light chains of two antibodies, IAG-1 and JSJ-1, were 93% homologous at the amino acid level, none of the heavy chains displayed any significant sequence homology. However, computer-generated models of all three antibody binding sites revealed a three-dimensionally conserved polyamine binding site motif. The polyamine appears to bind into a negatively charged cleft lined with acidic and polar residues. The cleft is partially or completely closed at one end and the specificity of the interaction is determined by placement of acidic residues in the cleft. Aromatic residues contribute to polyamine binding interacting with the carbon backbone. The polyamine-binding motif we have identified is very similar to that observed in the crystal structure of PotD, the primary receptor of the polyamine transport system in Escherichia coli.