Amino terminal sequence of heavy and light chains from ratfish immunoglobulin

The ratfish,Callorhinchus callorhinchus, a representative of the Holocephali, has a natural serum hemagglutinin (M _r 960 000), composed of heavy (M _r 71000), light (M _r 22 500), and J (M _r 16 000) chains. To approach the mechanisms that generate diversity at this level of evolution, the amino terminal sequence of the heavy and light chains was determined by automated microsequencing. The chains are unblocked and have modest internal sequence heterogeneity. The heavy chains show sequence similarity with the terminal region of the heavy chain from the horned shark,Heterodontus francisci, and other species. In contrast to the heavy chain, the ratfish light chains display low sequence similarity with their shark kappa counterparts. However, their similarity with the variable region of the chicken lambda light chains is about 75%.     

Conservation among vertebrate immunoglobulin chains detected by antibodies to a synthetic joining segment peptide

We used affinity purified antibodies produced against a synthetic peptide sequence corresponding to the entire J beta of a human T cell receptor gene to screen sera of man, mouse and other vertebrates to determine the presence of cross-reactive molecules. Little evidence for free alpha/beta heterodimers was found, but the antibody reacted with light chains of many vertebrate species, including characterized myeloma proteins of man and mouse. Some vertebrate orders, notably Aves, lacked polypeptide chains cross-reactive with J beta, but detectable determinants occurred in primitive vertebrates such as the galapagos shark (Carcharhinus galapagensis). In addition to the strong cross-reaction with purified light chains, human heavy chains reacted weakly with the antibody. The cross-reaction correlated with the sequence of the denatured immunoglobulins and was inhibitable with free peptide. These results establish the similarity of T cell receptor beta chains to immunoglobulin chains and support the conclusion that J region sequences were conserved, not only within mammalian immunoglobulins and T cell receptors, but in vertebrate evolution.     

Conservatism in the Evolution of Immunoglobulin

IMMUNOGLOBULINS of all vertebrate species studied to date show two basic structural features¹: (1) they are composed of two types of polypeptide subunits termed light chains and heavy chains and (2) they possess various amino-acid sequences. The differences in primary sequence observed in single immunoglobulin classes reside chiefly in the amino-terminal portions of the polypeptide chains which have been classed as variable or V regions^2,3. This heterogeneity has provided an obstacle to the determination of the primary sequence of antibodies. Most knowledge of immunoglobulin structure therefore derives from analysis of homogeneous immunoglobulins produced by human and murine plasmacytomas^2,3. Definite assignments of homology among vertebrate species are difficult to make because of the dearth of amino-acid sequence data for lower species. The relatively small quantities of immunoglobulin, the difficulty of purification and the innate heterogeneity prevent the attainment of complete sequence information from critical species such as the lamprey^4,5.     

Amino acid sequence of the N-terminal sixty-nine residues of heavy chain derived from a homogeneous rabbit antibody

The sequence of the N-terminal 69 residues of heavy chain from a homogeneous rabbit antibody to type III pneumococcal polysaccharide was determined. The sequence is similar to that found in heavy chains of normal pooled rabbit immunoglobulins of the same allotype Aa1. Two regions of the homogeneous heavy chain (residues 35-46 and 62-69) are very similar to corresponding regions of heavy chains from rabbit Aa2 immunoglobulin, as well as from mouse, guinea-pig and human immunoglobulins. In contrast, residues 47-62 appear to be variable. Comparison in this section with another homogeneous anti-pneumococcal antibody (Strosberg et al., 1972) of related specificity and of the same allotype indicates sequence variation in at least three positions. An antibody to group C streptococcal carbohydrate of allotype Aa2 (Fleischman, 1971) differs by five amino acids in the same region of the heavy chain. Sequence variability between these three antibodies does not occur in homologous positions within this variable section. Allotype-related sequences could not be identified in section 34-65.     

Recombinant shark natural antibodies to thyroglobulin

As cartilaginous fish are the vertebrates most distal from man to produce antibodies, fundamental information regarding conservation and variation of the antigen binding site should be gained by comparing the properties of antibodies directed against the same antigen from the two species. Since monoclonal cell lines cannot be generated using shark B cells, we isolated antigen binding recombinant single chain Fv antibodies (scFv) comprising of the complete variable regions from shark light and heavy chains. Thyroglobulin was used as the selecting antigen as both sharks and humans express natural antibodies to mammalian thyroglobulin in the absence of purposeful immunization. We report that recombinant sandbar shark (Carcharhinus plumbeus) scFvs that bind bovine thyroglobulin consist of heavy chain variable regions (VH) homologous to those of the human VHIII subset and light chain variable regions (VL) homologous to those of the human Vlambda6 subgroup. The homology within the frameworks is sufficient to enable the building of three-dimensional models of the shark VH/VL structure using established human structures as templates. In natural antibodies of both species, the major variability lies in the third complementarity determining region (CDR3) of both VH and VL.     

Structure and significance of beta2-microglobulin.

beta2-Microglobulin shares many structural features with the homology regions of the immunoglobulins. Particularly significant is the fact that its amino acid sequence is homologous to the sequences of the constant regions of both classes of light chains (kappa and lambda) and to the constant homology regions of at least three classes (gamma, mu and epsilon) of heavy chains, especially the carboxyl-terminal regions Cgamma3 Cmu4 and Cepsilon4. Molecules similar to human beta2-microglobulin have been found in other vertebrate species. The properties of beta2-microglobulin suggest that the gene for this protein may have evolved from a precursor gene that by duplication gave rise to immunoglobulin light and heavy chains. Furthermore, the observation that beta2-microglobulin is synthesized by and appears on the surfaces of a variety of cell types, including nonlymphoid cells, suggests that the concepts derived from analysis of the immune system may be applicable to other areas of cell biology. In particular, the close association of this immunoglobulin-like molecule with the histocompatibility antigens has a number of implications for the origin, structure, and function of these as well as other cell surface glycoproteins.     

Genomic organization and sequences of immunoglobulin light chain genes in a primitive vertebrate suggest coevolution of immunoglobulin gene organization.

The genomic organization and sequence of immunoglobulin light chain genes in Heterodontus francisci (horned shark), a phylogenetically primitive vertebrate, have been characterized. Light chain variable (VL) and joining (JI) segments are separated by 380 nucleotides and together with the single constant region exon (CI), occupy less than 2.7 kb, the closest linkage described thus far for a rearranging gene system. The VL segment is flanked by a characteristic recombination signal sequence possessing a 12 nucleotide spacer; the recombination signal sequence flanking the JL segment is 23 nucleotides. The VL genes, unlike heavy chain genes, possess a typical upstream regulatory octamer as well as conserved enhancer core sequences in the intervening sequence separating JL and CL. Restriction mapping and genomic Southern blotting are consistent with the presence of multiple light chain gene clusters. There appear to be considerably fewer light than heavy chain genes. Heavy and light chain clusters show no evidence of genomic linkage using field inversion gel electrophoresis. The findings of major differences in the organization and functional rearrangement properties of immunoglobulin genes in species representing different levels of vertebrate evolution, but consistent similarity in the organization of heavy and light chain genes within a species, suggests that these systems may be coevolving.     

Immunoglobulin heavy chain gene organization and complexity in the skate, Raja erinacea

Immunoglobulin heavy chain genes from Raja erinacea have been isolated by cross hybridization with probes derived from the immunoglobulin genes of Heterodontus francisci (horned shark), a representative of a different elasmobranch order. Heavy chain variable (VH), diversity (DH) and joining (JH) segments are linked closely to constant region (CH) exons, as has been described in another elasmobranch. The nucleotide sequence homology of VH gene segments within Raja and between different elasmobranch species is high, suggesting that members of this phylogenetic subclass may share one VH family. The organization of immunoglobulin genes segments is diverse; both VD-J and VD-DJ joined genes have been detected in the genome of non-lymphoid cells. JH segment sequence diversity is high, in contrast to that seen in a related elasmobranch. These data suggest that the clustered V-D-J-C form of immunoglobulin heavy chain organization, including germline joined components, may occur in all subclasses of elasmobranchs. While variation in VH gene structure is limited, gene organization appears to be diverse.     

Early Evolution of Immunoglobulin Genes

Considerable progress has been made in the characterizationof immunoglobulin genes from several lower vertebrate taxa.Isolation and identification of immunoglobulin genes in phylogeneticallyprimitive species is based predominantly on heterologous crosshybridization.The unit, clustered organization of heavy chain segmental elementsobserved in the germline of the horned shark (Hinds and Litman,1986) has also been found in another elasmobranch. Studies todetermine whether the clustered organization is universal throughoutthe entire cartilaginous fish assemblage are ongoing. In contrast,the ray-finned (bony)fishes appear to possess a mammalian-typeheavy chain gene organization. Additionally, immunoglobulingenes are being characterized in two relict fish species whoseexact systematic relationships are unknown. Isolation of putativeimmunoglobulin genes from the phylogenetically- ancient hagfishis being attempted using a PCR-based approach. Other ongoingor future research efforts involve characterization of lowervertebrate light chain genes, heavy chain isotype evolution,and the divergence of the immunoglobulins and T-cell antigenreceptors     

Nucleotide sequence of a cDNA clone encoding a rabbit immunoglobulin-lambda light chain: the V lambda region differs markedly from that of other species

A cDNA clone (pDH7) has been isolated which encodes the entire leader peptide and variable (V) region and most of the constant (C) region of a rabbit lambda-light chain. Although similar to amino acid sequences derived from fragments of isolated lambda-chains from several Basilea rabbits, differences in the first framework region (FR1) suggest that at least two germ-line V lambda genes are expressed. There are major differences between rabbit V lambda sequences and light chains of other species: in particular, rabbit lambda-chains have an additional four amino acids in the vicinity of the FR2-CDR2 junction. The same region also has significant homology with the human D2 germ-line mini-gene sequence, especially with a 14-nucleotide sequence previously shown to be homologous to human and rabbit heavy chain CDR2 sequences. Similar homologies in other heavy and light chain sequences suggest that D-gene segments may be derived from VH genes, perhaps by transposition. The framework regions of the rabbit lambda-chain encoded by clone pDH7 show the greatest homologies with those of human kappa- and lambda-sequences (46 to 54% homology), with that of chicken sequence (55%), and least with murine V lambda sequences (40%).     

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.     

Light chain variable region sequence of rabbit antipneumococcal type III polysaccharide antibody 3368.

The amino acid sequence of the amino-terminal 111 residues (variable region) for the light chain of the homogeneous rabbit antipneumococcal type III polysaccharide antibody 3368 was determined. This sequence was obtained principally through automated Edmann degradations of the intact light chain and of peptides generated by tryptic digestion of the citraconylated light chain. With these methods only 2 mumol of purified light chain was required to determine the reported sequence. When compared with the light chains of four other antipneumococcal type III polysaccharide antibodies, the 3368 light chain exhibits a unique sequence in those segments of the variable region that contribute to formation of the antigen binding site (complementarity-determining regions) (10 or 11 residue differences in 12 positions). The 3368 light chain also demonstrates an insertion of three residues relative to the other four light chains in the complementarity-determining region at positions 89 to 98. These five light chains have greater than 80% sequence homology for the portion of the variable region which is not involved in antigen binding (framework).     

Antibody response inHeterodontus

Summary Appropriately selected phylogenetic models are capable of providing insight into genetic mechanisms which may have become obscured during the passage of evolutionary time. In higher vertebrates a complex multigenic family encodes immunoglobulin-variable regions. The mechanisms involved in the expansion of the gene family and the stable maintenance of large numbers of individual genes presently are not understood. By defining the nature of antibody diversity in lower vertebrate species, it may be possible to approach such issues at a more fundamental level. Analyses of the immunoglobulins inHeterodontus francisci (horned shark), a representative phylogenetically primitive elasmobranch, indicate that this species may represent a useful developmental model.     

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.     

The primary structure of the constant region of Basilea-rabbit immunoglobulin lambda-chains.

The amino acid sequence of the constant region of the immunoglobulin lambda-chain of the rabbit (C lambda) has been determined. This chain was obtained from a homogeneous anti-(type II pneumococcal polysaccharide) antibody fraction raised in Basilea rabbit 4548. The C lambda-region sequence was established by the analysis of tryptic and some overlapping chymotryptic peptides and partly by homology with known C lambda-region sequences of lambda-chains from other species. Peptides were isolated by a combination of methods including high-voltage paper electrophoresis, gel filtration and high-pressure liquid chromatography. The peptides were subjected to automated Edman degradation in the presence of Polybrene. The sequence showed no evidence of heterogeneity. Large interspecies similarities, as well as amino acid interchanges, are apparent among various C lambda regions; the degrees of homology between rabbit C lambda region and the C lambda region from human, porcine and murine chains are 72.6, 71 and 72% respectively. The similarity of lambda-chains of different species is much greater than that found between rabbit kappa- and lambda-chains (about 34% homology). Genetic implications of these results are discussed.     

Characterization of a recombinant monoclonal antibody by mass spectrometry combined with liquid chromatography

In this report, we present the characterization of a humanized monoclonal antibody specific for the human epidermal growth factor receptor (hEGFR). Direct analysis by matrix assisted laser desorption ionization mass spectrometry (MALDI-MS) of peptide mixtures and chromatographically isolated fractions allowed identification of 94.0% and 85.4% of the amino acid sequence of light and heavy chains, respectively. Microheterogeneity sources were identified in light and heavy chains and a previously unreported posttranslational modification for immunoglobulins was found. One N-glycosylation site was identified in the heavy chain with non-sialylated bianntenary fucosylated structures. This study is one of the first to assess the potential of MALDI-MS in combination with more conventional protein chemistry techniques for the characterization of monoclonal antibodies.     

Relationship of human variable region heavy chain germ-line genes to genes encoding anti-DNA autoantibodies

In order to identify the V region genes encoding systemic lupus erythematosus (SLE)-derived anti-DNA autoantibodies, we have determined the nucleotide sequence of heavy chain mRNA from several DNA-binding immunoglobulins secreted by human hybridomas. We used the technique of cDNA primer extension for determining sequences of the VH, D, and JH gene segments of anti-DNA autoantibodies from three different primary hybridoma growths from an SLE patient and one hybridoma from a leprosy patient. Immunoglobulins from two of the SLE hybridomas expressed the same idiotype, Id-16/6, which is also expressed on immunoglobulins in sera of patients with active SLE. Their mRNA sequences showed complete homology to each other in the V, D, and J genes and more than 99% homology to the VH26 germ-line gene sequence, a member of the human VHIII gene family. The VH mRNA sequence of the third SLE hybridoma, 21/28, which was idiotypically unrelated to the other two, was 93% homologous to a different VH germ-line gene sequence, HA2, a member of the human VHI gene family. The fourth anti-DNA-producing hybridoma, 8E10, was derived from a leprosy patient of different ethnic origin than the SLE patient. It was idiotypically related to 21/28 and expressed a VH segment gene identical to that of 21/28. Hybridomas 21/28 and 8E10 shared sequence homology with the VH26 anti-DNA antibodies in the first complementarity-determining region. In addition, 21/28 shared sequence homology with the Id-16/6+ group in the region encoded by the D and J gene segments. Our findings indicate that some SLE autoantibodies are encoded by unmodified or scarcely modified VH germ-line genes that are conserved in the human population and identify two distinct VH germ-line genes that can encode segments of anti-DNA immunoglobulins.     

Qualitative and quantitative analysis of serum immunoglobulins of four Antarctic fish species

Immunoglobulins from the Antarctic fish species Trematomus bernacchii, Trematomus hansoni, Trematomus newnesi, and Chionodraco hamatus were analysed in whole serum and after purification by affinity chromatography on protein A-sepharose. Using SDS-PAGE, the apparent masses of the heavy and light chains were, respectively, 83.5 kDa and 27.5 kDa for T. bernacchii, 83.5 kDa and 27 kDa for T. hansoni, 81 kDa and 27.5 kDa for T. newnesi, and 74.5 kDa and 30 kDa for C. hamatus. It was not possible to purify immunoglobulins from T. newnesi due to their low concentration in serum. Heterogeneity in mass of both heavy and light chains was observed in all species. By using a polyclonal antibody raised against sea bass immunoglobulins, cross-reactivity was observed with heavy and light chains of all species. With this antibody, an indirect enzyme-linked immunosorbent assay (ELISA) was developed and results showed the relative immunoglobulin concentration in sera of the Antarctic fish species considered, using as standard sea bass immunoglobulins. Received: 25 November 1996 / Accepted: 3 February 1997     

A double monoclonal IgG1 kappa and IgG2 kappa in a single myeloma patient. Variation in clonal products and therapeutic responses

Two electrophoretically homogeneous immunoglobulins were detected in the serum of a patient with multiple myeloma. The heavy chains were of different classes (gamma 1 and gamma 2). The light chains of both were kappa, but had different electrophoretic mobilities on polyacrylamide gels. Amino acid sequence analysis, carbohydrate determinations, and biosynthetic experiments indicated that the difference seen in their electrophoretic mobility was due to the glycosylation of one but not the other kappa-chain. The primary structure of both chains demonstrated that they both used a V kappa 1 and a J kappa 2 gene segment and the same C kappa allele, Km(1,2), and that both contained the same junctional three amino acid deletion. However, they varied by 19 amino acids in the first 94 amino terminal residues encoded for by the V kappa gene, with some of the substitutions requiring two base changes in the appropriate codons. Moreover, the malignant "clones" producing the two proteins differed in their responses to chemotherapy. These data indicate that, although the two clones producing the serum proteins were different at the time of study, they may have arisen from the same precursor clone.