(1/1652) Induction of Ig light chain gene rearrangement in heavy chain-deficient B cells by activated Ras.
During B cell development, rearrangement and expression of Ig heavy chain (HC) genes promote development and expansion of pre-B cells accompanied by the onset of Ig light chain (LC) variable region gene assembly. To elucidate the signaling pathways that control these events, we have tested the ability of activated Ras expression to promote B cell differentiation to the stage of LC gene rearrangement in the absence of Ig HC gene expression. For this purpose, we introduced an activated Ras expression construct into JH-deleted embryonic stem cells that lack the ability to assemble HC variable region genes and assayed differentiation potential by recombination activating gene (RAG) 2-deficient blastocyst complementation. We found that activated Ras expression induces the progression of B lineage cells beyond the developmental checkpoint ordinarily controlled by mu HC. Such Ras/JH-deleted B cells accumulate in the periphery but continue to express markers associated with precursor B cells including RAG gene products. These peripheral Ras/JH-deleted B cell populations show extensive Ig LC gene rearrangement but maintain an extent of kappa LC gene rearrangement and a preference for kappa over lambda LC gene rearrangement similar to that of wild-type B cells. We discuss these findings in the context of potential mechanisms that may regulate Ig LC gene rearrangement. (+info)
(2/1652) Characterization of an immunoglobin cDNA clone containing the variable and constant regions for the MOPC 21 kappa light chain.
Nucleotide sequence analysis and restriction endonuclease mapping have been used to characterize a cDNA copy of immunoglobulin MOPC 21 Kappa mRNA clones in the bacterial plasmid pMB9. Three regions of the inserted cDNA of plasmid pL21-1 have been sequenced and match the known protein sequence at amino acid residues 1-24, 128-138 and 171-179. With these sequences to provide absolute correlations between the restriction map and the structural gene sequence it has been possible to exactly deduce the positions of all 11 of the insert restriction sites mapped within the structural gene. The pL21-1 insert contains the complete variable and constant regions as well as parts of the 3' untranslated and polypeptide leader coding sequences. (+info)
(3/1652) Recombinant DNA clones constructed from immunoglobulin kappa light chain messenger RNA.
Recombinant DNA clones have been generated from mouse myeloma MOPC 21 immunoglobulin kappa light chain mRNA. Complementary DNA (cDNA) synthesized on kappa light chain mRNA by reverse transcriptase was made double stranded and inserted into the bacterial plasmid vector, pMB9. Approximately 70 tetracycline-resistant transformed colonies containing kappa light chain mRNA sequences were identified by colony hybridization. Five of these recombinant clones were selected and characterized. Three clones contain both kappa light chain constant and variable region sequences. Two of these three recombinant clones have been shown to include all of the kappa light chain constant and variable region coding sequences. Another of the five selected recombinant clones contain kappa light chain constant region sequences. The remaining characterized clone appears to be derived from sequences at the 5'-end of kappa light chain mRNA, possibly extending to the terminal cap structure. (+info)
(4/1652) Novel mechanisms control the folding and assembly of lambda5/14.1 and VpreB to produce an intact surrogate light chain.
Surrogate light chain, which escorts the mu heavy chain to the cell surface, is a critical component of the pre-B cell receptor complex. The two proteins that comprise the surrogate light chain, VpreB and lambda5/14.1, contain both unique regions and Ig-like domains. The unique regions have been postulated to function in the assembly of the surrogate light chain. However, by using transient transfection of COS7 cells, we show that deletion of the unique regions of both proteins did not inhibit the assembly of surrogate light chain. Instead, in vivo folding studies showed that the unique region of lambda5/14.1 acts as an intramolecular chaperone by preventing the folding of this protein when it is expressed in the absence of its partner, VpreB. The Ig domains of both lambda5/14.1 and VpreB are atypical. The one in VpreB lacks one of the canonical beta strands whereas the one in lambda5/14.1 has an extra beta strand. Deletion of the extra beta strand in lambda5/14.1 completely abrogated the formation of the surrogate light chain, demonstrating that complementation of the incomplete Ig domain in VpreB by the extra beta strand in lambda5/14.1 was necessary and sufficient for the folding and assembly of these proteins. Our studies reveal two novel mechanisms for regulating surrogate light chain formation: (i) the presence of an intramolecular chaperone that prevents folding of the unassembled subunit but that remains part of the mature assembled protein, and (ii) splitting an Ig domain between two proteins to control their folding and assembly. (+info)
(5/1652) Assignment of genes for immunoglobulin kappa and heavy chains to chromosomes 6 and 12 in mouse.
Using somatic cell hybrids from fusions of lymphocytes of two different mouse stocks with the myeloma cell line X63-Ag8, we have assigned genes for the immunoglobulin heavy and kappa-type light chains to chromosomes 12 and 6, respectively. The two mouse stocks exhibit karyotypes consisting of nine pairs of metacentric chromosomes as a result of centric fusions of acrocentric chromosomes in different combinations. In the hybrid cells these metacentric chromosomes can be distinguished from the acrocentric chromosomes of myeloma origin, permitting correlation of Ig chain expression with mitotic loss of individual metacentric chromosomes. (+info)
(6/1652) The structure of an entire noncovalent immunoglobulin kappa light-chain dimer (Bence-Jones protein) reveals a weak and unusual constant domains association.
Monoclonal free light chains secreted in immunoproliferative disorders are frequently involved in renal complications, including a specific proximal tubule impairment, Fanconi's syndrome. The latter is characterized in most cases by intracellular crystallization including a light-chain variable-domain fragment which resists lysosomal proteases. Bence-Jones protein (BJP) DEL was isolated from a patient with myeloma-associated Fanconi's syndrome. The crystal structure of this human kappa immunoglobulin light-chain noncovalent dimer was determined using molecular replacement with the structure of molecule REI, as the variable domain, and that of BJP LOC as the constant domain. To our knowledge, DEL is the first complete kappa BJP structure described to date. The R-factor is 20.7% at 2.8 A resolution. The BJP DEL dimer was compared with other light-chain dimers and with Fab fragments with a kappa light chain. Although the domain-folding pattern was similar, the relative positions of the constant domains differed. BJP DEL showed a noncanonical quaternary structural arrangement which may be attributable to the poor CL-CL affinity and lack of an interchain disulfide bridge, combined with the conformational editing effect of the crystal-packing forces. Our results suggest that, in the absence of a disulfide bridge, most BJP CLs are probably mobile in solution. This may explain their high susceptibility to proteases and the absence of naturally occurring crystals for these dimers. Furthermore, these findings of an unusual quaternary structure of an immunoglobulin light-chain association extend our knowledge about the large and highly diverse structures of the immunoglobulin superfamily. (+info)
(7/1652) Physicochemical consequences of amino acid variations that contribute to fibril formation by immunoglobulin light chains.
The most common form of systemic amyloidosis originates from antibody light chains. The large number of amino acid variations that distinguish amyloidogenic from nonamyloidogenic light chain proteins has impeded our understanding of the structural basis of light-chain fibril formation. Moreover, even among the subset of human light chains that are amyloidogenic, many primary structure differences are found. We compared the thermodynamic stabilities of two recombinant kappa4 light-chain variable domains (V(L)s) derived from amyloidogenic light chains with a V(L) from a benign light chain. The amyloidogenic V(L)s were significantly less stable than the benign V(L). Furthermore, only the amyloidogenic V(L)s formed fibrils under native conditions in an in vitro fibril formation assay. We used site-directed mutagenesis to examine the consequences of individual amino acid substitutions found in the amyloidogenic V(L)s on stability and fibril formation capability. Both stabilizing and destabilizing mutations were found; however, only destabilizing mutations induced fibril formation in vitro. We found that fibril formation by the benign V(L) could be induced by low concentrations of a denaturant. This indicates that there are no structural or sequence-specific features of the benign V(L) that are incompatible with fibril formation, other than its greater stability. These studies demonstrate that the V(L) beta-domain structure is vulnerable to destabilizing mutations at a number of sites, including complementarity determining regions (CDRs), and that loss of variable domain stability is a major driving force in fibril formation. (+info)
(8/1652) Cells with clonal light chains are present in peripheral blood at diagnosis and in apheretic stem cell harvests of primary amyloidosis.
In primary systemic amyloidosis, small numbers of bone marrow plasma cells secrete monoclonal light chains that form extracellular fibrils (amyloid) in various organs. Evidence limited to a few cases suggests that rare clonal elements can also be found in the peripheral blood (PB), and this may be relevant in PB stem cell autotransplantation. Since up to 40% of amyloid clones do not synthesize heavy chains, in order to detect tumor cells with high specificity and sensitivity we developed a seminested allele-specific oligonucleotide polymerase chain reaction for tumor light chains. Clone-related sequences were detected in DNA and/or cDNA from the PB cells of eight of 10 patients at diagnosis and from apheretic collections of three of four cases undergoing PB progenitor autotransplantation. Since there are experimental data suggesting that circulating tumor cells may be involved in the growth of the amyloidogenic clone and may be chemoresistant, these findings are relevant to the use of leukapheresis purging strategies for PB progenitor autotransplantation in amyloidosis. (+info)