Allotype B4 Research Articles

Kappa-chain allotypes and isotypes in the rabbit: cDNA sequences of clones encoding b9 suggest an evolutionary pathway and possible role of the interdomain disulfide bond in quantitative allotype expression.

The constant regions of rabbit kappa light chains are unusual because the sequences of the allotypic forms can differ more from each other than do some variable regions with which they associate. We report the nucleic acid sequence of a full-length cDNA clone of b9 allotype and show comparisons to available sequences of the rabbit kappa allotypes b4, b5, and bas-N4. Our analyses suggest that the primordial rabbit kappa gene encoded a bas-like sequence. They also reveal a surprising difference in the position of the variable region cysteine that forms the interdomain disulfide bond that is unique to most rabbit kappa chains. One b9 cDNA sequence lacks the usual cysteine-80 and instead encodes cysteine-108, which in three-dimensional models appears capable of forming the interdomain disulfide bond with cysteine-171 in the constant region. A partial sequence of a second b9 clone encodes both cysteine-80 and cysteine-108; the translation product of this clone could have a free reactive sulfhydryl group that might lead to an unstable nonfunctional Ig molecule. The fact that pre-B cells with b9 kappa chains do not differentiate and expand into productive Ig-producing cells with frequencies comparable to the other allotypes may be explained if a substantial proportion of the gene products have a free sulfhydryl group. Our sequence results suggest that in cells differentiating to produce kappa light chains of b9 allotype the number and location of the cysteines influence immunoglobulin expression.

Genetics and expression of kappa-type light chains in Basilea rabbits.

In contrast to rabbits of b4, b5, b6, and b9 allotypes whose serum immunoglobulins (Igs) are predominantly composed of kappa-type light chains, rabbits of the mutant Basilea strain have serum Igs that are largely of lambda type. We prepared several antisera that recognized a minor K2 (bas) light chain that is produced by Basilea rabbits. With these antisera we identified the K2 (bas) isotype in the serum of the original b9/b9 male rabbit whose offspring displayed the Basilea mutant phenotype. It was present in one half of his nonmutant offspring which inherited b9 from him and another b allotype from their mothers. Breeding was conducted both in Basel and at the NIH to develop and maintain colonies of mutant Basilea strain rabbits. The data obtained during colony development confirm that the trait of expression of the bas allotype maps to the same genetic region (b locus) that is known to control the allelic b allotypes b4, b5, b6 and b9. Homozygotes or heterozygotes of b4, b5 or b6 allotype (bb/bb) were mated with homozygous bbas / bbas rabbits to produce F1s , and then F2s as well as progeny of backcrosses to both homozygous parental types (bb/bb and bbas / bbas ) were produced. The bas allotype segregates as an allele (or pseudoallele ) at the b locus although there was a deficiency in recovery of homozygous bas offspring in both the F2 and backcross matings to bbas / bbas parental type in the NIH colony. This selective deficiency may reflect a deleterious effect on survival of homozygous bas progeny.

The sequences of rabbit kappa light chains of b4 and b5 allotypes differ more in their constant regions than in their 3' untranslated regions.

We report the sequence of a cDNA clone encoding the entire variable and constant regions of a rabbit kappa light chain of b5 allotype. The deduced amino acid sequence of the variable region (positions 1-95) is 86% homologous to that of a b4 light chain protein [BS-1) (1) but the b4 and b5 constant regions are only 74% homologous. Comparison of this DNA sequence to that of a cDNA clone encoding a b4 constant region shows that the kappa allotypes b4 and b5 have diverged significantly more in their coding region than in the 3' untranslated regions (86% vs 96% nucleotide sequence homologies). This implies either a function for the 3' untranslated region with evolutionary pressures to conserve or an accelerated divergence of the coding regions.

Production of IgG-specific auto-anti-allotypes in b4.2-suppressed rabbits.

Rabbits were completely suppressed for kappa chain allotype b4.2, and autoantibodies against b4.1 or b4.2 could be raised in 2 out of 3 animals. The animal immunized with b4.1 produced anti-b4 and anti-Ms3, two activities which have never as yet been found together in one antiserum. Both autoantisera lacked the capacity to bind b4.2-IgM, whereas they precipitated 4.2-IgG very well. In one animal, anti-b4 IgM activity appeared after the sixth immunization, at the age of 14 months. Allotype suppression was maintained in both rabbits till the end of their lives whereas all control animals recovered within 6 months. The autoantisera which were specific for b4 IgG could not induce suppression in vivo. However, specific inhibition of b4 IgG secretion was observed in vitro.

Serologic and structural characterization of immunoglobulin chains secreted by rabbit-mouse hybridomas.

Serologic and primary structural analyses of Ig chains secreted by several rabbit-mouse hybridomas have shown that these hybrid cells produce heavy (H) or light (L) chains identical to those isolated from rabbit sera. Two of the cell lines (7D2, 7D6) secreted rabbit H chains with a m.w. of 55,000 each of which expressed a full complement of variable and constant region allotypes (a3, d11, e15). These apparently normal rabbit H chains were secreted in a complex with a m.w. about 130,000, and serologic studies indicated that this complex contained a covalently linked mouse kappa L chain. Two other cell lines (4C1, 12F2) produced allotype b4 L chains with m.w. of 23,000 and 25,000, and a third (1D4P5) produced an allotype b5 L chain with a m.w. of 23,000. Serologic analyses indicated that the allotypes on these chains are equivalent to those expressed by normal rabbit Ig molecules. Partial amino acid sequence data obtained for the L chain products showed them to be typical of rabbit L chains, and to be significantly different from mouse L chains.

Isolation and characterization of an active variable domain from a homogeneous rabbit antibody light chain.

The variable domain (VL) of allotype b4 light chains of rabbit IgG was isolated from both nonimmune heterogeneous IgG and a homogeneous antibody directed against type III pneumococcal polysaccharide. Light chains were first isolated and then cleaved under mild acidic conditions between residues 109 and 110. Reduction with dithiothreitol in guanidine hydrochloride cleaved both intradomain disulfide bridges as well as the interdomain disulfide bridge joining the variable and constant domain. The sulfhydryl groups were protected after reduction by p-chloromercuribenzoate. VL was isolated from this mixture of variable and constant domains by affinity chromatography, utilizing sheep antibodies directed against a peptide including residues 110--211 from nonimmune IgG light chain. The isolated VL domain was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and automated Edman degradation. VL from a homogeneous antibody was treated with dithiothreitol to remove p-chloromercuribenzoate, reoxidized, and recombined with homologous heavy chain. The binding of this recombinant to type III pneumococcal polysaccharide was identical with that of the light-chain--heavy-chain recombinant.

Suppression and Enhancement of the Rabbit Primary in Vitro Antibody Response with Anti-Allotype Antibody: Concurrent Effects on Thymidine Incorporation, Blastogenesis, and Cell Surface Ig

Abstract We have adapted an in vitro culture system to study the dose-dependent effects of purified anti-light chain allotype (b4 or b5) antibody on the primary Ab response of rabbit spleen cells to sheep erythrocytes. At high doses, anti-b4 or anti-b5 Ab suppressed the anti-SRBC response and the expression of cell surface immunoglobulins while stimulating blastogenesis and thymidine incorporation. At low doses, anti-allotype Ab markedly enhanced the immune response and, to a lesser extent, thymidine incorporation and blastogenesis. The mitogenic effect of anti-allotype Ab was observed in the absence of antigen only at the higher doses. When tested on heterozygous b4b5 SpC, the high and low doses of Ab selectively modulated the immune response with respect to cells bearing the target allotype. In vivo induced allotype suppression could not be reversed by culturing SpC with high or low doses of purified anti-allotype Ab directed against the expressed allotype. Our data support the concept that anti-Ig Ab reacts directly with B cell surface Ig to trigger a mitogenic response. This trigger, however, is not sufficient to induce differentiation to anti-SRBC secreting cells in the absence of the antigen.

Amino acid sequences of antibody light chain variable regions of pedigreed rabbits: kappa light chain K49-501 (allotype b4 anti-streptococcal group A-variant polysaccharide antibody).

The amino acid sequence of positions 1--150 of a light chain, isolated from another monoclonal rabbit anti-streptococcal group A-variant polysaccharide antibody, was determined. The analysis was performed with 2 mumol of polypeptide chain, using a grossly modified Beckman 890B sequenator. This sequence stretch accounts for the whole variable region and a considerable part of the constant region at a total length of 218 amino acids. This allotype b4 light chain was isolated from a non-precipitating, end-group-specific antibody with a KD = 1.3 X 10(-5)M. This brings the present number of totally known rabbit VL sequences of antigen elicited antibodies to 21. A comparison of these 21 sequences reveals a building plan of ribbit VL homologous to that of human and murine VL regions. The observed variability does follow a pattern of linked amino acid substitutions, indicating that this information must be contained in the germ-line of the rabbit in the form of multiple VL region genes. This conclusion, however, does not rule out the occasional variant being due to somatic rearrangement. Finally, this comparison reveals that the joining peptide between positions 96--110 is also a separate entity in rabbit VL region sequences.

Serologic distinction between the rabbit kappa L chain allotype b4 and an allele b4.

Rabbit kappa light (L) chain allotypes are controlled by the allelic genes b4, b5, b6, b9 and a recently described allele b4v. The product of the b4v gene differs from that of b4 by at least two amino acid substitutions in the constant region. A serologic distinction between the b4 and b4v allotyes has now been detected by a radioimmune assay using one of six anti-b4 sera tested. The distinction was enhanced by fractionation of this antiserum on an immunoadsorbent containing cottontail rabbit IgG. IgG with L chains of the b4v type are deficient in their ability to inhibit binding of b4 IgG to the antibody preparation. L chains isolated from b4 and b4v IgG samples could not be distinguished by this assay indicating that interaction with an H chain is required for expression of the L chain determinants that differentiate b4 from b4v.

Genetic Characterization of a new allele of the rabbit group b Ck allotypes

An inherited variant (b4(v)) of the rabbit kappa-chain allotype b4 is characterized by the presence of serine in place of alanine at position 121 and leucine in place of glutamine at position 124. The variant was traced through eight generations of a pedigreed rabbit family. Genetic analysis of this trait demonstrated that it is an allele of the other group b allotypes, and recent breedings have produced rabbits homozygous for this light-chain type. Two findings, other than the amino acid sequence differences, distinguish b4(v) from b4 in our colony. First, the level of expression of b4(v) in heterozygous rabbits is less than that of b4. For example, in b4b4(v) rabbits, approximately 30% of the preimmune IgG carries b4(v). In b4(v)b5 animals, 46% of the IgG carries the allotype b5, although in animals of allotype b4b5, 38% of the IgG is b5. Second, retrospective analysis of some litters revealed an abnormally low frequency of b4(v) in male heterozygotes. However, male b4(v)b4(v) homozygotes were found at the expected frequency in prospective crosses between b4(v)b5 rabbits.

Allelic exclusion in the B lineage cells of the rabbit.

Allelic exclusion of the K polypeptide chain allotypes b4 and b5 of the rabbit exists for the immunoglobulins of almost all plasma cells and of most mature small B lymphocytes. However, both these alleles are expressed simultaneously in rare plasma cells and in an usually large fraction of the lymphoid cells of the bone marrow. Our experiments support the idea that such bone marrow B lineage cells do not always show allelic exclusion.

Antibody Avidity and Allelic Selection for L Chain Allotypes in Rabbits Hyperimmunized with Streptococcal Vaccine

Abstract An almost absolute selection in favor of allotype b4 occurs in the antibody response of heterozygous b4b9 rabbits immunized with Group C streptococcal vaccine. A possible explanation for this “allelic selection” in favor of b4 is that during the antistreptococcal response the favored allotype (b4) is associated with V region binding sites yielding higher avidity anti-streptococcal antibodies. In order to test this possibility, IgG from heterozygous, hyperimmune rabbits was fractionated by avidity on a Group C immunoadsorbent column and allotypes of the various fractions were quantitatively determined. Contrary to the predicted result, when secondary immunization sera were examined the fractions of highest avidity were enriched in the nonselected allotype. Antibodies from two primary immunization sera lacked the selection for the non-b4 allotype in high avidity fractions observed for the later sera. Thus, the uneven allotype ratios observed in fractions of different avidity from secondary immunization sera can be interpreted as the result of a secondary maturation effect. The observations of allelic selection in heterozygous rabbits were extended to rabbits with allotypes b4b5 and b4b6. Although b4 predominated in immunoglobulin from these rabbits, the allotype ratios in anti-streptococcal antibody were the same as those in total IgG before immunization.

The Homologues of Rabbit b-Allotypes on Hare Immunoglobulin Light Chains: An Evolutionary Puzzle

Abstract Most alloantisera directed against the rabbit allotypes b4, b5, and b6 cross-reacted with hare IgG. The determinants recognized by antisera against the three allotypes were shown to reside on the same hare L chains. Hare immunoglobulins, when injected into b4, b5, or b9 rabbits, elicited the formation of antibodies which reacted with hare IgG and cross-reacted with those rabbit allotypes (except b9) absent from the recipient. The anti-b9 antisera tested lacked reactivity with hare IgG.

Primary structure of the L chain from a rabbit homogeneous antibody to streptococcal carbohydrate. II. Sequence determination of peptides from tryptic and peptic digests.

A complete amino acid sequence containing 210 residues of a rabbit light chain of a homogeneous Group C streptococcal antibody has been determined as: See journal for formula. This light chain is allotype b4 and is of the V-K1 subgroup.

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.

Sequence Variability of Rabbit Antibody Light Chains

Amino acid sequence analyses of the IgG light (L) chains from selected members of a rabbit family with allotypes b4 and b9 showed N-terminal substitutions that segregated with the b4 allotype. The L chains of allotype b4 had tyrosine at step 2, whereas this residue was absent from that step in the b9 L chains. Steps 12 and 13 of b4 L chains yielded a high percentage of glutamic acid, whereas a low percentage was observed for the b9 L chains. These tyrosine and glutamic acid substitutions were observed with the same frequency in L chains from homogeneous antibodies as in the L chains of the IgG samples. The data suggest that, in the family studied, separate and distinct V-region gene complexes are linked to the b4 and b9 allotypic genes.

In Vitro Studies on Allotype Suppression

Abstract Spleen cells from heterozygous rabbits with b locus immunoglobulin allotypes b4 and b5 which had been exposed in utero to maternal antibodies against the paternal allotype (b4) were stimulated in vitro with solubilized T2 phage antigen. The cells of thoroughly suppressed rabbits formed T2 neutralizing antibodies which were identifiable only as having allotype b5. The presence of anti-b5 serum in the cultures inhibited b5 expression and resulted in the formation of antibodies having the previously suppressed allotype, b4. Normal rabbit serum of the same allotypic specificity as the anti-b5 serum (b4) had no effect on the allotype distribution of anti-T2 antibodies. Although partial inhibition of b5 production occurred when the anti-b5 serum was removed from cultures after 5 hr, no reversal of b4 suppression was observed. Possible mechanisms accounting for these results are discussed.

Amino-acid sequence of an allotype b4 light chain from a rabbit antibody to streptococcal carbohydrate.

The complete amino-acid sequence was determined for a rabbit-antibody light chain of allotype b4. The light chain was recovered from approximately 20 g of homogeneous antibody to group C streptococcal carbohydrate, which was obtained from an immunized rabbit. Several enzymic digestions were used to obtain the overlapping peptides necessary for the sequence determination. The L chain has 210 amino-acid residues, as determined by sequence analysis, and a molecular mass of 23,060 daltons. This antibody light chain contains seven half-cystine residues characteristic of the (kappa)B light-chain class. The amino-terminal sequence was determined to be Ala-Asp-Ile-Val-Met, indicating that the chain belongs to the V(kappa)I subgroup.

Allelic Selection of Light Chain Allotypes by Antibodies Raised in Heterozygous Rabbits

Abstract A marked selection for allotype b4 over b9 was observed for anti-streptococcal antibodies produced in rabbits heterozygous for allotypes of groups a and b. Less than 2% of the streptococcal antibodies was of allotype b9 while 20% of the IgG in preimmune sera from the same rabbits was of allotype b9. No such effect was observed with respect to the group a allotypes. In spite of this marked selectivity in b4,b9 heterozygotes for the production of antistreptococcal antibodies of allotype b4, no evidence for linkage between the gene controlling this allotype and immune responsiveness to streptococcal antigens could be detected. Equal amounts of antibody were produced by all immunized offspring (b4,b4; b4,b9; or b9,b9) of heterozygous rabbits.

The aminoterminal sequence of antibody light chains: Evidence for possible inheritance of structural genes

Amino-terminal sequence analyses of 12 rabbit antibody light chains with restricted heterogeneity induced with either streptococcal group or pneumococcal type antigens are described. A comparison of their 27–30 N-terminal residue positions and comparison with the homologous regions of human and mouse κ chains suggest the following conclusions: 1. 1. Rabbit, mouse and human κ chains are homologous. They share the prototype N-terminal sequence Asp-Ile-Val-Met-Thr-Gln and predominantly or exclusively a number of residues further on in the N-terminal 27 position (e.g. Pro 9, Gly 17, Thr 21, Ile 22, Cys 24, Ala 26 and Ser 27). The protoype rabbit light chain sequence appears to start with Ala-Asp-Ile-Val-Met, and is thus longer by one risidue than human and mouse κ chains. Like their human and mouse couinterparts rabbits κ chains can be subgrouped. A minimum number of six subgroups is distinguishable on the basis of the current sequence information. 2. 2. The existence of species specific amino acid residues in the region reported appeats to be doubtful because positions 12 ans 18 are also found to be variable. 3. 3. Rabbit allotype b4 light chains show the greatest variations in the N-terminal 5 amino acid positions with the highest variability index in position 3. 4. 4. The degree of homology of antibody light chain N-termini appears to be a function of the breeding relationship of individual rabbits. For example, antibody light chains of a parent and an offspring rabbit with identical specilities were identical within their N-terminal 22 amino would imply inheritance of structural v-region genes for the synthesis of specific anti-polysaccharide antibodies.