No full-text available
To read the full-text of this research,
you can request a copy directly from the author.
Evidence for and the Significance of ‘Two Genes, One Polypeptide Chain’
Abstract
All immunoglobulins (Ig’s) consist of one or more basic units composed of identical pairs of heavy (H) and light (L) polypeptide chains (Figure 1). Each chain folds into several compact globular domains, connected by relatively narrower but more exposed areas. Each domain is approximately 110 amino acids in length and characterized by an intrachain disulfide bond connecting two cysteine residues approximately 60 amino acid residues apart (Figure 1). All Ig polypeptide chains can be divided into an amino-terminal portion, the variable (V) region, and a carboxylterminal portion, the constant (C) region. The V regions of both H and L chains are equivalent in size to a domain. The C regions of L chains are of similar size, whereas those of H chains are two to four times longer. Based on the degree of amino acid sequence homology, the V regions have been divided into three main groups, Vk
, Vλ, and VH. The C regions have been divided into k and λ types (L chains) as well as γ, α, µ, ∈, and δ classes (H chains) according to their antigenic and serological properties. Some of the L-chain types and H-chain classes are further divided into subtypes and subclasses, respectively. Details of Ig structure are given in earlier Chapters. A general review on this subject was given by Gally (1973). This chapter is devoted to the genetic aspects of the synthesis of Ig polypeptide chains.
The immune system consists of two major components, cellular immunity and humoral immunity. The latter is expressed through the synthesis of soluble antibodies. Under normal conditions, antibodies are formed by bone- marrow-derived lymphocytes (B cells) in reaction to the stimulation of antigens which enter the body by inhalation, ingestion, and/or penetration of the skin. In the laboratory, antibodies are usually produced by deliberate immunization by injection of antigens intravenously, intradermally, subcutaneously, etc., in the presence of adjuvants.
We report 10 Japanese patients with Sjgren''s syndrome (SS) who developed monoclonal gammopathies (MG). One was of the IgG class, five of IgA, three of IgM, and one of IgG/IgM. The monoclonality of 7 of 10 M proteins was studied using antiidiotypic (Id) antibodies against M proteins. Four (three IgA and one IgM) of 10 M proteins had rheumatoid factor (RF) activity. Hemagglutination inhibition tests and enzyme-linked immunosorbent assays (ELISA) showed that the RF activity was inhibited by anti-Id antibodies in all four monoclonal RFs. In two patients examined, many cells infiltrating into the salivary glands were stained with anti-Id antibodies. Our review of 19 Japanese SS patients with MG revealed that the non-IgM class predominated (13/19). This contrasts with 19 reported non-Japanese SS patients, among whom 14 were IgM. In both Japanese and non-Japanese patients there was a higher incidence of MG in primary than in secondary SS. The difference in the dominant heavy-chain class may reflect a difference in the genetic factors affecting B cell differentiation in immunologically disordered states.
Structural studies were performed on five monoclonal immunoglobulins isolated from patients with carcinoma of the colon. Serologic analysis revealed that two of the five proteins shared idiotypic antigenic determinants; these two but none of the three others had VHIII heavy chains. The results demonstrated a close structural similarity between the heavy chains of these two proteins both in their antigen-binding sites (the hypervariable region) and in their framework regions. Determination of the NH2-terminal amino acid sequences indicated that the light chain variable regions of all five proteins were either VxII or VxIII. These data suggest that monoclonal immunoglobulins in patients with carcinoma of the colon have restricted heterogeneity and that, in some cases the production of monoclonal immunoglobulin(s) and the development of a solid tumor in a given patient may be related events.
An analysis was made of the immunoglobulin surface markers of the cells of patients with chronic lymphatic leukemia (CLL) in view of previous evidence of their monoclonal B-cell character. The simultaneous presence of IgM and IgD on the surface of the majority of lymphocytes was demonstrated by both immunofluorescence and hemagglutination inhibition in most cases. However, cases were observed with surface IgM without IgD as well as cases with IgD without IgM. IgG and IgA were absent. Studies of the light chains indicated only a single class in a given case. In addition to bound light chains, free light chains were readily demonstrated in most cases through the use of antisera specific for "free chain" determinants. It thus appeared that there are three major types of surface Ig on CLL lymphocytes, IgM, IgD, and free light chains.
An Ig molecule containing L chains and H chains similar to human delta-chains has been detected on the surface of radioiodinated murine lymphoid cells. Newborn mice have only IgM on their splenocytes. Between 10 and 15 days, the IgD-like molecule appears and increases in amount until 3 mo of age, when it is the predominant cell surface Ig in terms of radioactivity. IgD is found only in peripheral lymphoid tissues and is present in larger amounts on peripheral lymph node cells (approximately 85% of surface Ig) than on splenocytes (approximately 50%). IgD is also present in comparable amounts on cells from both nu/nu and germfree mice, indicating that its expression may be independent of both thymic influence and antigenic stimulation. These studies suggest that there is a switch from cell surface IgM to IgD that occurs during differentiation of virgin B lymphocytes in the spleen.
To determine the amino acid sequence of the human k Bence-Jones Protein Ni, the protein was completely reduced and aminoethylated and then digested with trypsin [EC 3.4.4.4]. Twenty-five major tryptic peptides were isolated by chromatography on Dowex 1-x2. The complete sequences of 18 of these peptides were determined. These comprised the entire variable NH2-terminal half and 7 peptides from the COOH-terminal half of the protein. Only the amino acid compositions and partial sequences of the other peptides were determined. Only one of the tryptic peptides in the NH2-terminal half of the molecule had the same sequence as that reported for the corresponding region of other human k chains, whereas all the tryptic peptides covering the sequence from residue 108 to the COOH-terminus corresponded exactly to the sequences reported for the constant region of human k chains.
This chapter describes human allotypes and genetic dogma. The γ-chains of human IgG occur in four immunological subclasses that have been shown to differ in their primary sequences. It has been also demonstrated that except for Gm (8) and Gm (9), a Gm antigen is confined to a single γ chain subtype. These observations led to the postulate that there is a gene for each subtype (multiple loci) and that the Gm antigens associated with a subtype are determined by the gene for the subtype. This attractive hypothesis follows from the widely accepted aphorism of current genetic dogma, one gene-one polypeptide chain. At the time of its proposal in 1964 and until very recently, the association between the immunological subtype and some Gm antigens was the sole evidence in its support
Immunoglobulins comprise a diverse family of phylogenetically related proteins. This family includes antibodies, as well as other proteins that are evolutionary homologous as evident by their physicochemical, physiological or antigenic properties. Immunoglobulins occur in the body fluids of almost all vertebrate species. They are biologically characterized by their ability to bind specifically to foreign substances, thereby stimulating some demonstrable physiological response that enhances the survival of the organism. This response might result in the death of the cell bearing the antigenic substances, the engulfment and enzymatic digestion of the antigen, sneezing, coughing, or a number of other physiological reactions. Much of immunology and most of immunopathology concerns itself with elucidating the events that are subsequent to the binding of antigen to antibody. This chapter describes the structure of immunoglobulins and the molecular genetics underlying that structure.
We shall focus attention today on two types of rellular differentiation in which highly specific cells are produced. The first example is the development of the brain.
The heterogeneity of immunoglobulins of a single vertebrate species is because of the fact that there are a number of genes in the germ line that dictate the structure, or control the expression, of different immunoglobulin polypeptide chains. Genetic polymorphisms of many of these genes have been discovered by serologic and chemical methods. The genetic markers on different portions of various polypeptide chains of the immunoglobulins have provided powerful tools for the study of the genetic basis of antibody structure. The allotypes of human immunoglobulins were discovered during an investigation into the anti-immunoglobulins that were present in sera of patients with hypogammaglobulinemia. The modern era of investigation of rabbit allotypes began with the discovery of isoprecipitins in the sera of rabbits injected with specific precipitates formed between antiserum of a second rabbit and corresponding antigen. Since these initial discoveries, investigations of allotypes have contributed to the understanding of immunoglobulin structure and genetics and to the concepts of the complexities of cellular interactions in development, immunity, and specific immune responses. This chapter reviews the knowledge of the genetically different forms of immunoglobulins in man, mouse, and rabbit.
The idiotype present on the Fab of a phosphorylcholine-binding IgA myeloma protein TEPC 15 (T15) of BALB/c origin was found in normal serum of BALB/c mice. Molecules carrying the T15 idiotype in normal serum could be adsorbed with Sepharose phosphorylcholine beads and R36A pneumococci. The T15 idiotype is absent in germ-free BALB/c but appears when the mice are conventionalized. A survey of normal sera of inbred strains for the T15 idiotype showed it to be present in BALB/c, 129, C57L, C58, and ST and absent or in low levels in CBA, C3H, C57BL/6, C57BL/Ka, C57BL/10, SJL, B10.D2, DBA/2, RIII, A, AL, AKR, NZB, and NH inbred strains of mice. The T15 idiotype is associated with some but not all strains carrying the IgCH allotypes found in BALB/c. Linkage of genes controlling the T15 idiotype in normal serum to the IgCH locus of BALB/c was demonstrated in F2 progeny of a BALB/c and C57BL cross, Bailey's recombinant inbred strains, C x BD, C x BE, C x BG, C x BH, C x BI, C x BJ, C x BK, and CB20 congenic strains. Among these strains, only those possessing the IgCH locus of BALB/c including the F2 progeny consisting of BALB/c homozygotes and BALB/c/C57BL heterozygotes and C x BG and C x BJ recombinants showed the T15 idiotype.
A large proportion of the human peripheral blood lymphocytes of adults and newborns having IgD were found also to have IgM on their membranes and vice versa. A few lymphocytes had one of these classes only. IgD and IgM could be capped independently on the same cell. The possibility that IgD was acquired by a cytophilic process was excluded by the finding that IgD-bearing cells were of one light chain type only, and by the direct demonstration of reappearance of IgD on the lymphocyte membrane during incubation in an IgD-free culture medium. On the basis of these findings, it is proposed that IgD functions as a lymphocyte antigen receptor.
Excerpt
“The dogmas of the past are inadequate to the stormy present ... as our case is new, so must we think anew and act anew. We must disenthrall ourselves.” Abraham Lincoln: Message to Congress, December 1, 1862.
In our first presentation in this volume (Hood, Gray, Sanders, and Dreyer) we dealt rather extensively with specific protein structural studies and the significance of such work to the subject of this symposium. As a consequence, we have elected to take advantage of the high degree of freedom permitted participants and devote this paper to the discussion of speculations prompted by various aspects of the structural studies. We do, however, review some of the conclusions of the earlier paper and present a few more hard facts in the form of sequence data. A specific mechanism (“copy-splice”) for light chain synthesis is then elaborated, based on the assumption that the germ-line theory of specificity...
Zusammenfassung: Bence-Jones-Protein Rei. wurde aus dem Harn eines Plasmazytom-Patienten durch Ammoniumsulfat-Fällung isoliert und durch Ionenaustauschchromatographie gereinigt. Wie eine Molekulargewichtsbestimmung zeigt, handelt es sich um die monomere Form des Proteins. Die Präparation enthielt eine Nebenfraktion, die aus dem Dimeren der variablen Teile bestand und dadurch gekennzeichnet war, daß sie kristallisiert werden konnte. Röntgenkristallographische Daten erlauben eine atomare Auflösung und mit Hilfe der Aminosäuresequenz die Konstruktion eines dreidimensionalen Modells, an dem eine Aussage über die antigenbindenden Abschnitte gemacht werden kann. Es handelt sich dabei um die hypervariablen Abschnitte, die am einen Ende des Moleküls im Dimeren die Wände einer etwa 15 õ breiten Tasche bilden. Die Sequenzbestimmung erfolgte an den tryptischen Peptiden, die durch chromatographische Verfahren gereinigt und in Homologie zu anderen K-Ketten angeordnet wurden. Das Protein ist aufgrund seiner charakteristischen Aminosäuresequenz der Subgruppe I der K-Ketten zuzuordnen und zeigt wie diese ein streng geordnetes Muster von Aminosäureaustauschen. Seine Sequenz ist eine weitere Bestätigung für den evolutionären Ursprung der Antikörpervariabilität.
Two antibodies to Group C streptococcal carbohydrate isolated from an individual rabbit had similar relative binding affinities for a Group C immuno-adsorbent column. Their light chains were similar, if not identical, as were the constant regions of their heavy chains. Differences in the variable regions of the H chains of the two antibodies were detected by chemical analysis. The two antibodies had serologically identical idiotypic determinants although one antibody possessed the a3 allotype and the other had no detectable group a marker. The occurrence of such antibodies indicates the absence of obligatory associations between group a allotypes and idiotypic specificities, despite the fact that both determinants have antigenic components in the VH region of the H chain.
Es wird über Untersuchungen zur Aufklärung der Primärstruktur des Bence-Jones-Proteins Bau. berichtet. Das Protein hat 211 Aminosäuren. Auf Grund der Aminosäuresequenz gehört es zur Subgruppe IV der λ-ketten. Es hat die subgruppenspezifischen Deletionen in den Positionen 1, 27a-c, 95/96. Ein Vergleich der individualspezifischen Austausche des Proteins Bau. mit anderen Proteinen der Subgruppe IV zeigt noch weitergehende Koppelungsgruppen innerhalb dieser Subgruppe. Die vorliegenden Sequenzergebnisse stehen in Einklang mit einer in der Evolution erfolgten Entstehung der Antikörpervariabilität.
Excerpt
The current concept of protein biosynthesis holds that for each type of polypeptide chain there is a corresponding structural gene. When this concept is applied to immunoglobulins, several basic problems develop, chiefly because there are so many immunoglobulins. First, the immunoglobulins within a species or an individual are an extremely heterogeneous group of molecules. The heterogeneity is in a small part due to the several generic types of immunoglobulin chains that serve as subunits of the 4-chain immunoglobulin molecular unit. By far the greatest source of heterogeneity are the primary structural variations among seemingly closely related types of chains (Potter, Dreyer, Kuff, and McIntire, 1964). For example, kappa chains resemble each other in roughly 70 or more per cent of the amino acid sequence as exemplified by the comparisons of the sequences in the mouse (Gray, Dreyer, and Hood, 1967) and man (Putnam, Titani, and Whitley, 1966). In the mouse,...
THE Cygnus OB2 stellar association contains the most heavily reddened early type star known1,2. If all interstellar grains were to be removed from the line of sight to this eleventh magnitude star, it would rival Sirius as the brightest in the sky, its B-V colour excess being 3.7 magnitudes3.
Protein NEI, which has a molecular weight of 23 500 and contains 216 amino acids, was isolated from the urine of a patient with multiple myeloma by salt-precipitation and purified by ion-exchange chromatography, block electrophoresis and gel filtration. 23 tryptic peptides have been isolated from the aminoethylated protein and 13 chymotryptic peptides from the carboxymethylated protein by ion-exchange chromatography. Sequence studies have mainly been carried out with the tryptic peptides; the chymotryptic peptides have been used for overlaps in the variable region, since the alignment of the tryptic peptides by homology was not unambiguous.
Protein NEI belongs to subgroup II of the λ-chains. It has a series of subgroup specific exchanges and a subgroup specific deletion at position 96. Protein NEI differs from all other λ-chains of subgroup II by possessing a sixth cysteine residue. This additional cysteine residue at position 88 is so far unique, as it is adjacent to an invariant cysteine at position 87. Protein NEI also contains a carbohydrate moiety attached to an aspartic acid or asparagine (Asx) residue at position 93, one of the hypermutable areas of the variable part.
Furthermore we conclude by comparison with other λ-chains that all completely elucidated λ-chains can be ordered into four subgroups. The subgroups and linkage groups within subgroups are in complete agreement with an evolutionary origin of antibody variability and are incompatible with somatic hypermutation models.
Amino acid sequence analysis has been done on a κ Bence Jones protein (Tew) from a case of primary amyloidosis with the objective of determining the sequence of the variable region. Twenty-two tryptic peptides accounting for 182 residues were isolated and were completely or partially sequenced. Chymotryptic digestion yielded 32 peptides which supplied many overlaps. Sequenator analysis was performed for the first 42 residues of the amino terminus. From the combined data the sequence of the variable region (residues 1-108) was deduced. The composition, partial sequence data, and alignment of the peptides of the constant region (residues 109-214) correspond exactly to the sequence established for human κ light chains of the same allotype. The V region includes five extra residues (30a-30e) and is characteristic of the κII subgroup. A computer analysis of the sequence of the Tew Bence Jones protein in comparison with other human κ light chains was undertaken to establish quantitative criteria for subgroup classification. In terms of minimum nucleotide mutations the Tew protein differs from other subgroup κII proteins by an average of only 0.2 base/amino acid residue position, whereas other human κ chains differ from Tew by an average of 0.4-0.6 base/position. The Bence Jones protein and the tissue amyloid protein from this patient appear to be identical in primary structure as indicated by identity in the amino-terminal sequence for 27 residues and similarity in peptide maps, amino acid composition, and other properties.
A total of six human λ-chain proteins have been found which possess three hitherto unreported constant region amino acid substitutions. The three variant residues are always found in association with the previously described Oz (−) and Kern (+) substitutions. λ-Chains containing the three variant replacements have also been demonstrated in the light chain fractions of IgG derived from pooled normal human serum and from the sera of each of four individuals. In addition, studies of monoclonal λ-chains have demonstrated that the variant constant region sequence is found in association with a specific variable region substitution. The results indicate that this isotypic λ-chain constant region is the product of a specific gene.
Anti-p-azophenylarsonate (anti-Ar) antibodies elicited in all strain A/J mice tested share one or more idiotypic specificities. These specificities are also found in the anti-Ar antibodies of mice of the closely related strain, AL/N, but not in those of BALB/c mice.
Anti-Ar antibodies were elicited in congenic mice in which the IgCH locus of AL/N mice, which controls allotypic markers in the constant regions of heavy chains, had been introgressively backcrossed for nine generations onto a BALB/c background; the mice were then rendered homozygous for the AL/N allotypic determinant. On the average, these antibodies were quantitatively equivalent, with respect to content of the cross-reactive idiotype, to those of AL/N mice. This indicates that the gene controlling the idiotype is closely linked to the IgCH locus. Since idiotype must be a function of V region sequences, the results suggest close linkage of VH and CH genes. The cross-reactive idiotype was found in nearly all F1 mice (C57/BL x A/J or BALB/c x A/J) tested.
A human IgA1 λ monomeric myeloma protein (Vo) was found to be antigenically deficient to other IgA proteins. The molecular weight of the heavy chains was 42,000 daltons, compared with a figure of 58,000 daltons found for normal α chains. In contrast, both the light (L) chains and the Fd′ fragment had molecular weights identical to those of other IgAl proteins. Two cyanogen bromide fragments were isolated from the heavy (H) chains: one fragment consisted of the first 85 amino acid residues of the variable region, whereas the second one contained the remainder of the foreshortened α chain. Neither the carboxy-terminal octapeptide of normal α chains nor the cyanogen bromide fragment containing 30 amino acid residues and located in normal α chains in a position preceding that of the octapeptide were found. The structural moiety missing in Vo and responsible for its antigenic deficiency was found to be a peptide consisting of more than 100 amino acid residues from the C-terminal end of the α chain, probably involving the entire CH3 domain.
The role of the thymus and bursa in the ontogeny of IgA, IgG, and IgM was studied in fowl by using isolated or combined bursectomy (Bx) and thymectomy (Tx) at hatching. Bx birds had markedly elevated serum IgM levels together with moderately decreased IgG and IgA. Tx birds had normal IgM levels but moderately decreased IgG and IgA. BxTx birds had normal IgM levels but markedly depressed IgG and lacked IgA totally even when followed to 5 months of age. The predominant IgA-containing cells in the duodenal lamina propria of normal birds were completely replaced by IgM-containing cells in BxTx birds. These experimental models document the requirement for normal thymus-derived and bursa-derived cell interactions in the ontogeny of both IgG and the IgA system and should provide useful approaches to studies of the post bursal differentiation of the plasma cell lines.
The separate genetic control of the variable and constant parts and the importance of subgroups and their subdivisions in the evolutionary origin of antibody specificity has been discussed13-19. Single amino-acid changes in the constant part could be due either to allotypic differences (as in the Inv-factors in ?-chains1,20-24, the Gm-factors in ?-chains25 or the Am-factors in ?-chains26·27), which are inherited codominantly in a Mendelian manner, or to gene duplication, as is the case in the Oz-factor, an arginine-lysine exchange in position 191 of the constant part of ?-chains28·29. This exchange therefore represents a beginning isotypic variation in this chain type.£ 0.5-? = 0,005 ?
The H chain of the IgG protein Mcg has a deletion of 15 amino acids which starts at a position 216 residues from the amino terminal end of the molecule. This protein is thus complementary to those seen in H chain disease where the deletion occurs within the first 215 amino acid residues and a normal structure is resumed at position 216.
A Bence Jones protein with phenotype Inv (1, –2) was isolated from the urine of a patient with multiple myeloma. Inv typing of the patient's relatives established the presence of anInv
1 allele in the kindred, and that the patient's genotype wasInv
1/Inv
3. Hence, the absence of Inv (2) in the Bence Jones protein was shown to be genetic and not an artifact caused by the disease. The tryptic peptide-containing residues 191 through 194 were isolated and shown to be composed of Leu, Tyr, Ala, Cys, with Leu at the amino end. Hence, the residue at 191 is the same as that present in Inv (1, 2) Bence Jones proteins. More detailed study of the tryptic peptides established that residue 153 is Val rather than Ala as in all other K chains thus far studied. The primary sequence: Ala153, Leu191 determines Inv (1, 2); Ala153, Val191 determines Inv (3); and Val153, Leu191 determines Inv (1). The Val153, Val191 sequence has not been observed. It may correspond to Inv (–). These data are strikingly similar to the data for the Kern and Oz isotypes (changes at 154 and 191, respectively) in the chain. As in the case of theK chain, only three of the four possible combinations have been observed. The implications of this parallelism and of crystallographic findings on chains, reported by others, are discussed.
Genetic markers related to the antigen binding specificity of mouse immunoglobulins (idiotypes and fine specificity characteristics) are used to investigate the polymorphism of genes encoding heavy chain variable regions (V
H
genes). The distribution of eight such specificity related markers in inbred strains and their segregation together with allotype indicate linkage to genes that specify the heavy chain constant regions (C
H
genes). A number of recombinant mice allowed the construction of a chromosomal map consisting of threeV
H subloci, oneC
h sublocus, and a region which contains nonimmunoglobulin coding genes. All data are consistent with a linear arrangement of genes in this linkage group so thatV
H
andC
H
genes are discontinuous. Each of theV
H
subloci is correlated to a particular set of antigen binding specificities which include protein antigens, polysaccharides, and haptens. This suggests that specific complementarity determining sequence portions are conserved in the germ line of the mouse.
In an attempt to account for antibody specificity and complementarity in terms of structure, human κ-, human λ-, and mouse κ-Bence Jones proteins and light chains are considered as a single population and the variable and constant regions are compared using the sequence data available. Statistical criteria are used in evaluating each position in the sequence as to whether it is essentially invariant or group-specific, subgroup-specific, species-specific, etc.
Examination of the invariant residues of the variable and constant regions confirms the existence of a large number of invariant glycines, no invariant valine, lysine, and histidine, and only one invariant leucine and alanine in the variable region, as compared with the absence of invariant glycines and presence of three each of invariant alanine, leucine, and valine and two each of invariant lysine and histidine in the constant region. The unique role of glycine in the variable region is emphasized. Hydrophobicity of the invariant residues of the two regions is also evaluated. A parameter termed variability is defined and plotted against the position for the 107 residues of the variable region. Three stretches of unusually high variability are noted at residues 24–34, 50–56, and 89–97; variations in length have been found in the first and third of these. It is hypothesized that positions 24–34 and 89–97 contain the complementarity-determining residues of the light chain—those which make contact with the antigenic determinant. The heavy chain also has been reported to have a similar region of very high variability which would also participate in forming the antibody-combining site. It is postulated that the information for site complementarity is contained in some extrachromosomal DNA such as an episome and is incorporated by insertion into the DNA of the structural genes for the variable region of short linear sequences of nucleotides. The advantages and disadvantages of this hypothesis are discussed.
Two immunoglobulins, IgA(K) and IgG(K), were isolated from the serum of a single patient with two monoclonal components (biclonal proteins). After chain separation, the light chains from each molecule were found to be identical by the following criteria: electrophoretic mobilities under various pH and dissociating conditions, amino acid compositon, fingerprint analysis of tryptic peptides and of 14C-succinylated chymotryptic peptides, and amino acid sequence of the N-terminal 40 residues. The heavy chains were indistinguishable for the N-terminal 45 amino acid residues. These data are consistent with the hypothesis that a single heavy chain variable (VH) region may be associated with two different heavy chain constant (CH) genes.
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.
ALLOTYPIC genetic variations in the variable (V) part of immunoglobulin (Ig) chains have been clearly identified only in the domestic rabbit. Three allotypic specificities, a1, a2 and a3, controlled by allelic genes, a1, a2 and a3, at the a locus, are present on the variable region of heavy chains of all classes (IgM, IgG, IgA and IgE)1-3. These allotypic specificities reflect multiple amino acid differences in the V region of Ig heavy chains4.
This chapter discusses the structure and role of the variable regions of the immunoglobulins (Ig) molecules. Based on amino acid sequence data, hypervariable regions make up a significant part of the variable regions, occupy relatively constant locations in a variety of Ig molecules even from different species, and appear to be intimately associated with the antibody-combining site. In addition, the idiotypic determinants that mark the antigenic individuality of particular Ig molecules are based on the properties of some or all of the hypervariable regions. In contrast, outside the hypervariable regions there is a considerable invariance of sequence, and these invariant sections occur in approximately the same position in Igs of different V region subgroups. The polymeric Ig molecules are intriguing, because one of them, IgM, is structurally the most complex, phylogenetically the most primitive, and ontogenetically the earliest of the Ig molecules. The other polymeric Ig molecule, IgA, evolved relatively recently but retained that portion of the IgM Fc sequences responsible for polymerization and, in addition, developed a mechanism for transport across epithelial cells. The most detailed view of the molecular orientation of the immunoglobulin V region, which is consistent with the known serological and immunochemical data is obtained by X-ray diffraction analyses.
This chapter presents the allotypic studies in the rabbit and offers more detailed information on several specific topics to which allotypic studies have contributed. The chapter focuses on the rabbit allotypes and gives an overview of the developments. There is a paucity of structural information on rabbit immunoglobulins and an optimal amount of genetic information has been obtained in studies on this species by the use of the immunoglobulin antigenic markers. The chapter explains allotypic phenomena adequately by involving regulator genes in the linkage, somatic recombination, and selective gene expression that have been observed in genetic studies employing allotypes. It is difficult to visualize these complex genetic relationships in terms of the repressors and corepressors that have been described for nonmammalian systems involving regulation. The possibility has been raised that rabbit immunoglobulin allotypes are not products of allelic structural genes, however are products of regulator genes. The studies on allotypes and idiotypes have made significant contributions to the knowledge of genetic control of immunoglobulin synthesis, knowledge that is, in many instances, relevant to more general biological topics.