Partial sequence of the variable region of IgG light chains of b5 allotype from A B4B5 rabbit.

The partial sequence (positions 29–71) of the variable region of light chains of predominately b5 allotype from the IgG of a single allotype-suppressed rabbit was obtained by traditional sequencing methods on isolated tryptic and chymotryptic peptides. The peptides from this region were isolated in relatively high yields and probably represent a dominant sequence. The framework sequence between positions 35 and 49 (FR 2) is identical to an FR 2 sequence commonly found in light chains from antibodies produced by b4 rabbits as well as murine and human myeloma light chains, with the exception of an interchange of threonine for proline at position 43 or 44. This may be b5 allotype-related since, to date, all the b4 light chains have had proline, and a b9 light chain was found with arginine at position 43. The fact that a dominant sequence could also be found for positions corresponding to the second complementarity determining region (CDR 2, 50–56) in other species, confirms previous observations that this portion of the light chain is not extremely variable in the rabbit.     

The complete amino acid sequence of the light chain variable region of two monoclonal anti-p-azobenzene-arsonate antibodies bearing the cross-reactive idiotype

Two monoclonal anti-p-azobenzene-arsonate antibodies produced by cell fusion of A/J spleen lymphocytes were selected. It had previously been shown that they both expressed the cross-reactive idiotype (CRI) and that the amino terminal sequence of their heavy chain variable region differed by only one amino acid substitution within the first 55 residues, at residue 41 in the second framework region. A novel, sensitive peptide-mapping method had indicated that the light chain of these two antibodies differed by at least three amino acid substitutions. Here, the complete amino acid sequence of the light chain variable region is presented. There are only 3 amino acid substitutions between the two antibodies, located in the first and second complementarity determining regions and the third framework region respectively. Although the variable region of the light chains of these two monoclonal antibodies show such a high degree of homology they differ by 26 and 27 substitutions from the reference sequence of the light chain of CRI⁺ anti-ABA serum antibodies. In addition, they are homologous to 4 other such CRI⁺ monoclonal antibody light chain sequences published so far, in which only 2 of the above 3 substitutions are not represented. The contribution of the light chain to the CRI is discussed.     

Variable region correlates of group b allotypes: amino acid sequence studies of b9 L chains from homogeneous antibodies.

The amino acid sequence was determined for residues for residues 1 to 88 and residues 110 to 147 for a rabbit light chain (4153-I) with allotype b9 from a homogeneous anti-streptococcal antibody. The amino acid sequence of the L chain from a second antibody (4153-II) was also determined for residues 1 to 49 and 62 to 77. In spite of the large differences in constant region sequence between b4 and b9 L chains, the variable regions of these antibodies are quite similar to those reported for b4 L chains. Both chains bear a b9-specific substitution (glutamic acid) at position 16. The 4153-I chain also has substitutions at positions 70 and 81 that may be exclusive to variable regions of the L chains with the b9 allotype. These allotype-associated VL structural differences offer support to the notion that structural genes for the CL region are either linked to distinct VL gene complexes or that certain V regions are expressed only in concert with certain CL regions.     

Allotypic variations in the amino acid sequence of rabbit IgG kappa light chains in the region of tryptophan 146

Peptides containing tryptophan 146 from rabbit kappa light chains of b5 and b6 allotypes were Isolated and their sequences determined. Between positions 138 and 161 there is 56% homology between b4 and b9, 64% homology between b5 and b9 and 80% homology between b4 and b5. Between positions 138 and 150 there is 86% homology between b4 and b6 and 93% homology between b5 and b6. This confirms the predictions of serological studies that b9 light chain constant regions would differ most from the other allotypes, while b5 and b6 would be most similar.     

Immunohistochemical study of immunoglobulin light chain amyloidosis with antibodies to the immunoglobulin light chain variable region

To detect immunoglobulin (Ig) light chain amyloidosis (AL amyloidosis) in formalin-fixed, paraffin-embedded tissue sections by immunohistochemistry, polyclonal antibodies were generated against synthetic peptides corresponding to amino acids 1-19 of the Ig lambda light chain V lambda VI subgroup (anti-V lambda VI (1-19)) and the Ig kappa light chain Vkappa I subgroup (anti-Vkappa I (1-19)). Anti-V lambda VI (1-19) antibody reacted with amyloid deposits in 21 of 22 Alambda amyloidosis cases, and anti-Vkappa I (1-19) antibody reacted with amyloid deposits in 10 of 11 Akappa amyloidosis cases. Immunoreactivity varied in intensity by case and within specimens. Surprisingly, amyloid deposits were positive for anti-V kappa I (1-19) staining in one case of Alambda amyloidosis. Analysis of anti-V lambda VI (1-19) and anti-Vkappa I (1-19) antibody reactivity by ELISA showed some cross-reactivity with peptides other than antigen peptides. The antibodies were not reactive in all cases of AL amyloidosis examined but may be useful, together with anti-Ig constant region antibodies, for immunohistochemical diagnosis of AL amyloidosis.     

抗迟缓爱德华菌独特型单克隆抗体轻链可变区基因的克隆及序列分析

迟缓爱德华菌(Edwardsiella tarda)属肠杆菌科,为革兰氏阴性菌,兼性厌氧,周生鞭毛,无荚膜,无芽孢[1].该菌引起的原发感染或继发感染,对人或鱼类的致病性均较严重[2,3].为了构建迟缓爱德华菌抗独特型抗体基因工程疫苗,我们应用RT-PCR的方法,从分泌迟缓爱德华菌抗独特型的杂交瘤细胞株1E11中克隆出抗体轻链可变区(VL)基因,并进行了克隆和序列分析.     

The structural correlates of the rabbit light chain b allotypes: Sequence studies of b5 and b6 chains

Rabbit kappa light chains of allotype b5 and b6 were prepared from antibodies of restricted heterogeneity made by animals hyperimmunized with, respectively, strain III and strain II pneumococcal vaccines. The amino-acid sequence of several tryptic peptides were determined. The variable region fragments of the b5 and b6 chains appear to be quite similar to the corresponding fragments of b4 and b9 chains, albeit some residues seem to be allotype associated. In contrast the chains of different allotypes vary right from the start of the constant region in a number of positions, suggesting that b allotypes correlate with amino-acid substitutions in this region. The number of substitutions between the b5 and b6 and the previously determined b4 and b9 constant regions sequences ranges from 20 to 35%. Serological studies suggest that Leporidae b allotypes diverged no more than 2 × 10⁶ years ago. By this time only 1% of the substitutions could be generated by ‘conventional evolution’. Duplication and mutation of the individual C_K genes could account for the high level of divergence observed. The data reported here support the notion that the structural genes encoding the light chain constant regions of the various b allotypes coexist on the same chromosome and that the allelism is controlled by a regulatory mechanism.     

The amino acid sequence of the light chain of Acanthamoeba myosin IC

The amino acid sequence of the light chain of Acanthamoebamyosin IC deduced from the cDNA sequence comprises 149 aminoacids with a calculated molecular weight of 16739. All but the 3N-terminal residues were also determined by amino acid sequencingof the purified protein, which also showed the N-terminus to beblocked. Phylogenetic analysis shows Acanthamoeba myosin IC lightchain to be more similar to the calmodulin subfamily of EF-handcalcium-modulated proteins than to the myosin II essential lightchain or regulatory light chain subfamilies. In pairwisecomparisons, the myosin IC light chain sequence is most similarto sequences of calmodulins (50% identical) and a squidcalcium-binding protein (43% identical); the sequence is37% identical to the calcium-binding essential light chainof Physarum myosin II and 30% identical to the essentiallight chain of Acanthamoeba myosin II, and the essential lightchain and regulatory light chain of Dictyostelium myosin II. Thesequence predicts four helix-loop-helix domains with possiblecalcium-binding sites in domains I and III, suggesting thatcalcium may affect the activity of this unconventional myosin.This is the first report of the sequence of an unconventionalmyosin light chain other than calmodulin     

Amino acid sequence of the constant region of immunoglobulin light chains from Rana catesbeiana

Little is known about the detailed structure of immunoglobulins in non-mammalian vertebrates. We have determined the amino acid sequence of the constant region of immunoglobulin light chains from the bullfrog, Rana catesbeiana. There appears to be one major type of light chain in this species. However, at position 153, about half of the chains have lysine, the remainder having arginine. Variation at this same or at an adjacent position is responsible for allotypic or isotypic variation in human kappa and lambda chains. At three positions (118, 119, and 181), residues that occur in all known kappa and lambda chains in other species are replaced in the Rana catesbeiana sequence. One of these unusual substitutions--the replacement of proline by cysteine at position 119--allows the formation of an extra intrachain disulfide bond within the constant domain, between positions 119 and 214. This bond appears to replace the usual disulfide bridge between heavy and light chains so that, in the immunoglobulins of this species, the light chains are not covalently bonded to heavy chains. The sequence of the Rana catesbeiana constant region is compared to sequences of a variety of mammalian light chain constant regions. We consider the implications of these comparisons for the timing of the divergence of kappa and lambda chains relative to the divergence of the lineages leading to amphibians and to mammals and birds.     

Amino acid sequence of a light chain variable region of a human rheumatoid factor of the Wa idiotypic group, in part predicted by its reactivity with antipeptide antibodies

Antipeptide antisera were raised to the second and third complementarity-determining regions of the light chain derived from a human monoclonal IgM (Sie) which had antigammaglobulin activity and belonged to the Wa cross-reactive idiotypic group of human rheumatoid factors, two of whose members (Sie, Wo1) had been previously sequenced in our laboratory (Andrews and Capra, Biochemistry 20, 5816-5822, 1981). These antisera were found to react with the light chain of another human monoclonal IgM (Go1) that shared the Wa idiotype while antipeptide antisera made to the third CDR of the Sie heavy chain failed to react. The amino acid sequence of the variable region of the Go1 light chain was found to be highly homologous to the light chain of Sie from which the synthetic peptides were derived, particularly in the framework regions and the second and third CDR. This study illustrates that antipeptide antisera are valuable and specific probes for determining the relationship between molecules which exhibit similar antigen binding or idiotypic specificities and, furthermore, such antisera are able to predict amino acid sequences with surprising precision.     

A comparison of the N-terminal amino acid sequences of early and late pools of anti-DNP and anti-DNP-p-aminobenzoylglutamate antibody light chains.

The N-terminal amino acid sequences of early and late pools of anti-DNP and anti-DNP-p-aminobenzoylglutamate (DNP-ABG) antibody light chains were quantitatively determined and compared. The amino acid composition at each locus of the N-terminal 20 amino acid residues of each light chain preparation was determined using automatic sequencing techniques coupled with high-pressure liquid chromatography and mass spectrometry. The sequence data obtained for the light chains corresponding to antibodies isolated early in the immune response (3-4 weeks) were essentially the same as those for light chains from antibodies isolated late in the response (12-14 weeks). In addition, it was observed that the sequence data obtained for the anti-DNP antibody light chain preparation were almost identical to those found for two anti-DNP-ABG antibody light "hain preparations. The sequence data obtained in the present study were compared with those obtained for normal rabbit light chains and with the composite sequence data published for other rabbit anti-hapten light chains.     

Structural studies on induced antibodies with defined idiotypic specificities--VIII. NH2-terminal amino acid sequence analysis of the heavy and light chain variable regions of monoclonal anti-para-azophenylarsonate antibodies from A/J mice differing with respect to a cross-reactive idiotype.

Monoclonal antibodies with specificity for the hapten p-azophenyl arsonate (Ar)^* were generated by somatic cell hybridization of A/J spleen cells and the non-secreting cell line, Sp2/0-Ag14, derived from a Balb/c myeloma. Of four hybridoma products examined, only one bore the previously described A/J anti-Ar cross-reactive idiotype (CRI). The heavy and light polypeptide chains of this CRI positive molecule along with one of of the CRI negative molecules were subjected to amino-terminal sequence analysis and compared with the induced CRI positive antibodies produced upon immunization of A/J mice. The results confirm the extremely restricted expression of V_H and V_L frameworks in the A/J anti-Ar population. Unexpectedly, when the hybridoma proteins were compared to the induced population of antibodies, from one to three amino acid substitutions were detected within the first framework segment (1–30) of each chain of both hybridoma products. The complete covalent structure of two monoclonal antibodies known to differ serologically only with respect to idiotype should prove useful in defining the exact structural basis of an idiotypic determinant.     

The amino acid sequence of a monoclonal gamma 3-heavy chain from a patient with articular gamma-heavy chain deposition disease

Abnormal deposition of proteins, including monoclonal immunoglobulin gamma-heavy chains, may cause tissue damage and organ dysfunction. We here report the amino acid sequence of the free gamma-heavy chains present in serum and urine of the first reported case (patient G. L.) of synovial heavy chain deposition disease. The protein was heavily deleted and consisted of the hinge, in addition to the CH2 and CH3 domains, in a dimeric form, thus lacking its variable domain as well as the CH1 domain. The sequence was consistent with the gamma 3 subclass (gamma 3GL). Gm typing revealed the gamma 3 allotypes G3m(b0) and G3m(b1) in accordance with the residues Pro123, Phe128, Thr171 and Phe268 in gamma 3GL. Furthermore, the gamma 3GL molecule was glycosylated at Asn in position 129. Finally, the gamma 3GL protein was shown to contain a typical binding site for the first complement component, C1q, namely the residues Glu150, Lys152 and Lys154, with the potential of binding and activating complement, causing tissue damage following deposition.     

Evolution of vertebrate IgM: complete amino acid sequence of the constant region of Ambystoma mexicanum mu chain deduced from cDNA sequence.

cDNA clones coding for the constant region of the Mexican axolotl (Ambystoma mexicanum) mu heavy immunoglobulin chain were selected from total spleen RNA, using a cDNA polymerase chain reaction technique. The specific 5'-end primer was an oligonucleotide homologous to the JH segment of Xenopus laevis mu chain. One of the clones, JHA/3, corresponded to the complete constant region of the axolotl mu chain, consisting of a 1362-nucleotide sequence coding for a polypeptide of 454 amino acids followed in 3' direction by a 179-nucleotide untranslated region and a polyA+ tail. The axolotl C mu is divided into four typical domains (C mu 1-C mu 4) and can be aligned with the Xenopus C mu with an overall identity of 56% at the nucleotide level. Percent identities were particularly high between C mu 1 (59%) and C mu 4 (71%). The C-terminal 20-amino acid segment which constitutes the secretory part of the mu chain is strongly homologous to the equivalent sequences of chondrichthyans and of other tetrapods, including a conserved N-linked oligosaccharide, the penultimate cysteine and the C-terminal lysine. The four C mu domains of 13 vertebrate species ranging from chondrichthyans to mammals were aligned and compared at the amino acid level. The significant number of mu-specific residues which are conserved into each of the four C mu domains argues for a continuous line of evolution of the vertebrate mu chain. This notion was confirmed by the ability to reconstitute a consistent vertebrate evolution tree based on the phylogenic parsimony analysis of the C mu 4 sequences.     

Human monoclonal antibodies with different fine specificity for digoxin derivatives: cloning of heavy and light chain variable region sequences.

Human-mouse hybridoma cell lines producing human monoclonal antibodies against the cardiac glycoside digoxin were established after in vitro immunization or direct immortalization of human peripheral blood lymphocytes with digoxin. Three antibodies, designated MO6, LH92 and LH1114, displayed different patterns of fine specificity against digoxin and several digoxin analogues, as elucidated by inhibition ELISA. All three monoclonal antibodies had mu heavy chains, two of them (MO6 and LH114) had kappa light chains and one (LH92) lambda light chains. DNA encoding the variable regions of both heavy and light chains of the three antibodies were amplified from cDNA using the polymerase chain reaction (PCR). The nucleotide sequences of the amplified DNA were determined after subcloning of PCR fragments in M13 vectors. The deduced amino acid sequences revealed considerable sequence differences in the complementarity determining regions between the three antibodies.