Regions Of Immunoglobulin Light Chains Research Articles

The CDR1 and Other Regions of Immunoglobulin Light Chains are Hot Spots for Amyloid Aggregation

Immunoglobulin light chain-derived (AL) amyloidosis is a debilitating disease without known cure. Almost nothing is known about the structural factors driving the amyloidogenesis of the light chains. This study aimed to identify the fibrillogenic hotspots of the model protein 6aJL2 and in pursuing this goal, two complementary approaches were applied. One of them was based on several web-based computational tools optimized to predict fibrillogenic/aggregation-prone sequences based on different structural and biophysical properties of the polypeptide chain. Then, the predictions were confirmed with an ad-hoc synthetic peptide library. In the second approach, 6aJL2 protein was proteolyzed with trypsin, and the products incubated in aggregation-promoting conditions. Then, the aggregation-prone fragments were identified by combining standard proteomic methods, and the results validated with a set of synthetic peptides with the sequence of the tryptic fragments. Both strategies coincided to identify a fibrillogenic hotspot located at the CDR1 and β-strand C of the protein, which was confirmed by scanning proline mutagenesis analysis. However, only the proteolysis-based strategy revealed additional fibrillogenic hotspots in two other regions of the protein. It was shown that a fibrillogenic hotspot associated to the CDR1 is also encoded by several κ and λ germline variable domain gene segments. Some parts of this study have been included in the chapter “The Structural Determinants of the Immunoglobulin Light Chain Amyloid Aggregation”, published in Physical Biology of Proteins and Peptides, Springer 2015 (ISBN 978-3-319-21687-4).

Mutations in Specific Structural Regions of Immunoglobulin Light Chains Are Associated with Free Light Chain Levels in Patients with AL Amyloidosis

BackgroundThe amyloidoses are protein misfolding diseases characterized by the deposition of amyloid that leads to cell death and tissue degeneration. In immunoglobulin light chain amyloidosis (AL), each patient has a unique monoclonal immunoglobulin light chain (LC) that forms amyloid deposits. Somatic mutations in AL LCs make these proteins less thermodynamically stable than their non-amyloidogenic counterparts, leading to misfolding and ultimately the formation of amyloid fibrils. We hypothesize that location rather than number of non-conservative mutations determines the amyloidogenicity of light chains.Methodology/Principal FindingsWe performed sequence alignments on the variable domain of 50 κ and 91 λ AL light chains and calculated the number of non-conservative mutations over total number of patients for each secondary structure element in order to identify regions that accumulate non-conservative mutations. Among patients with AL, the levels of circulating immunoglobulin free light chain varies greatly, but even patients with very low levels can have very advanced amyloid deposition.ConclusionsOur results show that in specific secondary structure elements, there are significant differences in the number of non-conservative mutations between normal and AL sequences. AL sequences from patients with different levels of secreted light chain have distinct differences in the location of non-conservative mutations, suggesting that for patients with very low levels of light chains and advanced amyloid deposition, the location of non-conservative mutations rather than the amount of free light chain in circulation may determine the amyloidogenic propensity of light chains.

Anti-lambda-light chain-peptide antibodies are suitable for the immunohistochemical classification of AL amyloid.

We aimed to test whether antibodies raised against recombinant peptides corresponding to the variable region of immunoglobulin light chains are suitable for the immunohistochemical classification of amyloid. The Entrez database of the National Center for Biotechnology Information (NCBI) was searched for all protein sequence entries which met the search criteria "amyloid" and "lambda light chain". Sixty-four different lambda-light chain-derived amyloid protein sequences were retrieved, aligned and categorized into the V region subgroups of lambda-light chain detailed by the NCBI, i.e. subgroup I (21 protein sequences), II (14), III (6), IV (1), V (1) and VI (21). V region subgroup I was chosen for epitope sequence selection and two rabbits were immunized with the following peptides: NH2-ISCSGSSSNIGSNTV-CONH2 and NH2-QRPSG VPDRFSGSKSGTS-CONH2. Sensitivity and specificity of the IgG-purified antibodies was tested by Western blotting using amyloid A- (AA), ALlambda- and ALkappa-amyloid proteins, and by immunohistochemistry on tissue microarrays with 110 different amyloid containing tissue samples obtained at autopsy from 22 patients, and on 27 biopsy specimens from a series of 24 patients. Our peptide antibodies specifically stained AL amyloid lambda-light chain-origin, in both Western blots and formalin-fixed and paraffin-embedded tissue sections, confirming that peptide-antibodies directed against immunoglobulin-derived lambda-light chain proteins can be applied for the immunohistochemical classification of amyloid. This offers the opportunity to generate a large set of anti-lambda-light chain protein-antibodies for the immunohistochemical classification of amyloid independently from native human tissue sources.

Dysregulation of cyclin dependent kinase 6 expression in splenic marginal zone lymphoma through chromosome 7q translocations.

The increased or inappropriate expression of genes with oncogenic properties through specific chromosome translocations is an important event in the pathogenesis of B-cell lymphoproliferative diseases. Recent studies have found deletions or translocations of chromosome 7q to be the most common cytogenetic abnormality observed in SLVL, a leukemic variant of SMZL, with the q21-q22 region being most frequently affected. In three patients with translocations between chromosomes 2 and 7, the cloning of the breakpoints at 7q21 revealed that each was located within a small region of DNA 3.6 kb upstream of the transcription start site of cyclin dependent kinase 6 (CDK6). In each case the translocation event was consistent with aberrant VJ recombination between the immunoglobulin light chain region (Ig kappa) on chromosome 2p12 and DNA sequences at 7q21, resembling the heptamer recombination site. The t(7;21) breakpoint in an additional patient with splenic marginal zone lymphoma (SMZL), resided 66 kb telomeric to the t(2;7) breakpoints juxtaposing CDK6 to an uncharacterized transcript. In two of the SLVL patient samples, the CDK6 protein was found to be markedly over expressed. These results suggest that dysregulation of CDK6 gene expression contributes to the pathogenesis of SLVL and SMZL.

Fragments of the Constant Region of Immunoglobulin Light Chains Are Constituents of AL-Amyloid Proteins

Immunoglobulin light chains are the precursor proteins of AL-amyloidosis. In the fibril formation process properties of the variable part of the immunoglobulin light chains are believed to be of major importance. In this work it is shown that fragments of the constant part of the immunoglobulin light chain are a constituent of the AL-amyloid proteins of kappa type. A specific antiserum has identified these fragments in gel filtration fractions where the absorbance approached the base line after the main retarded peak. The fragments are small and have been overlooked previously in the purification process. The significance of the constant part in AL-proteins is unclear, but adds new aspects to the discussion of pre- or post-fibrillogenic cleavage of the immunoglobulin light chains.

Diversity of expressed V and J regions of immunoglobulin light chains in Xenopus laevis

In Xenopus laevis, two immunoglobulin light chain isotypes, designated L1 or rho and L2 or sigma, have been identified. The genomic organization of the L1 locus has been described previously: a constant (C) gene segment is preceded by a joining (J) gene segment; in addition, there are many cross-hybridizing variable (V) gene segments. To evaluate the extent of sequence diversity of L1 V regions, we screened three cDNA libraries, constructed from mitogen-stimulated Xenopus splenocytes, with probes for the C or the J gene segment. Eighteen cDNA clones that contain complete or truncated V regions were chosen for sequence analysis. The C regions of all clones are identical or nearly identical to the genomic C gene segment; the V regions are greater than 80% identical in nucleotide sequence and are presumably derived from a single family of V gene segments. Although framework regions are nearly identical, complementarity-determining regions are quite diverse. The expressed J segments fall into distinct groups, suggesting the presence of more than one germ-line J segment. Therefore, a genomic library was screened with a J region probe. A clone overlapping with the previously identified J-C clone, and containing four additional J gene segments, was isolated. All five J gene segments are very similar and three are identical in nucleotide sequence. Each of the three distinct germ-line J sequences is represented in the set of cDNA clones, suggesting that combinatorial diversification occurs; imprecision of V-J joining also appears to contribute to variability. Overall, these results suggest that the immunoglobulin repertoire in this species is not significantly restricted by a limitation in the diversity of light chain V regions.

Human autoantibodies reactive with synthetic autoantigens from T-cell receptor beta chain.

We used mapping with synthetic overlapping peptides in combination with molecular modeling to analyze the IgG antibodies that humans naturally produce against human T-cell receptor beta chains and to localize the recognized peptide autoantigens in the three-dimensional structure of the molecule. Healthy individuals produce low levels of antibodies against T-cell receptor peptides, and these can be increased in autoimmune diseases. We characterized the reactivities in detail because IgG molecules reactive with self peptides occur in preparations of intravenous immunoglobulin and can be isolated by immunoaffinity chromatography. Natural IgG antibodies were directed against three major peptides. One corresponds to the first complementarity-determining region of the variable region. A second corresponds to the third framework of the variable region. The third is located in the constant region and is predicted to be a loop that extends out of the beta-barrel structure. This peptide is one that would give a characteristic structural distinction between the beta-chain constant region and the constant regions of immunoglobulin light chains to which beta chains are homologous. The capacity to bind these peptides is found in small fractions of normal polyclonal IgG, which contains both kappa chains and lambda chains. The activity is antibody-like in being confined to the Fab fragment and in its capacity to discriminate among homologous synthetic peptides corresponding to distinct beta-chain variable-region genes. We propose that a recognition and regulatory process naturally occurs that parallels the immune network for the regulation of the production of antibodies.

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 κ and λ chains. At three positions (118, 119, and 181), residues that occur in all known κ and λ 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 λ and κ chains relative to the divergence of the lineages leading to amphibians and to mammals and birds.

A Sequence Restriction in the Variable Region of Immunoglobulin Light Chains from Sharks, Birds, and Mammals

Abstract A relatively conserved pentapeptide sequence is present in the variable region of normal immunoglobulin λ- and κ-chains from animals as diverse as sharks, birds, and mammals. This same conserved sequence is also present in most myeloma light chains from dog, mouse, and man. The conservation of this pentapeptide sequence over the more than 400 million years since sharks and mammals diverged suggests that this region must carry out a very important, but as yet unknown function. A homologous but less highly conserved stretch of sequence is present in the variable region of immunoglobulin heavy chains.