Rabbit variable kappa light chain regions: subgroups contain polypeptides encoded by multiple genes.

Two identical light chain variable regions were identified in anti-streptococcal Group A-variant antibodies elicited in litter-mate rabbits by hyperimmunization with vaccine. In addition, one rabbit produced two additional clonally restricted antibodies to this polysaccharide antigen. The partial amino acid sequence of the light chain of one of these antibodies was identical with the dominant antibody light chain sequence, while the light chain of the other antibody, also partially established, showed significant variations in the framework-associated regions with identical CDRI and II. Since all of these light chains were from a small subset of rabbit kappa light chain pools (b4 allotype) the data suggest, together with other light chains reported in the literature, that more than one copy of variable region genes are present in the germ-line per subgroup. Furthermore, framework associated amino acid substitutions are not random; this suggests the existence of some "ordered" mechanism for linked amino acid substitutions (presumably recombination). Furthermore one light chain can pair with more than one heavy chain to yield functional antibodies.     

Unfolding and refolding of a type kappa immunoglobulin light chain and its variable and constant fragments

By limited proteolysis of a type kappa immunoglobulin light chain (Oku) with clostripain, both the VL and CL fragments were obtained with a high yield. The unfolding and refolding by guanidine hydrochloride of light chain Oku and its VL and CL fragments were studied at pH 7.5 and 25 degrees C with circular dichroism and tryptophyl fluorescence. The concentration of guanidine hydrochloride at the midpoint of the unfolding curve was 1.2 M for the VL fragment and 0.9 M for the CL fragment. As in the case of the CL fragment of light chain Nag (type lambda) [Goto, Y., & Hamaguchi, K. (1982) J. Mol. Biol. 156, 891-910], the unfolding and refolding kinetics of the CL fragment could be explained in principle on the basis of the three-species mechanism U1 in equilibrium U2 in equilibrium N, where N is native protein and U1 and U2 are the slow-folding and fast-folding species, respectively, of unfolded protein. The unfolding and refolding kinetics of the VL(Oku) fragment were described by a single exponential term. Double-jump experiments, however, showed that two forms of the unfolding molecule exist. The equilibrium and kinetics of unfolding of light chain Oku were explained by the sum of those of the VL and CL fragments. On the other hand, the refolding kinetics of light chain Oku were greatly different from the sum of those of the VL and CL fragments.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of a mouse DNA clone containing an immunoglobulin variable region gene.

A 4.8 kilobase mouse embryo DNA fragment was inserted into a phage lambda genome and was subsequently characterized by electron microscopy, restriction enzyme mapping and partial DNA sequencing. This fragment contains a 400 base sequence which is homologous to that of an immunoglobulin light lambda chain mRNA which spans 3.3 to 3.7 kilobases from one end of the fragment. Restriction enzyme mapping as well as partial nucleotide sequencing of the 3' terminal of the homology region confirm the previous conclusion [Tonegawa, Brack, Hozumi and Schuller, Proc. Natl. Acad. Sci. USA. 74, 3518-3522 (1977)] that the cloned DNA fragment contains a Vlambda gene sequence which is separate from any Clambda sequence.     

Cloning of immunoglobulin kappa light chain genes from mouse liver and myeloma MOPC 173

The organization of the kappa chain constant region gene was compared in DNA from an immunoglobulin-producing mouse myeloma (MOPC 173) and from liver. In situ hybridization using the Southern blotting technique revealed constant region gene-containing EcoRI-DNA fragments of 14 and 20 kb in the myeloma tissue whereas one EcoRI-DNA fragment with a length of 15 kb was found in liver DNA. After enrichment by RPC-5 chromatography and preparative electrophoresis the 14 kb fragment from MOPC 173 DNA and the 15 kb fragment from liver DNA were cloned in the bacteriophage lambda vector Charon 4A using in vitro packaging. Extensive characterization of the two fragments by restriction endonuclease mapping, in situ hybridization, and electron microscopy (R-loop and heteroduplex) showed that both fragments contain the constant region but no MOPC 173 variable region gene. Both fragments are homologous over a length of 12.5 kb including the constant region but differ from one another starting about 2.7 kb from the 5' end of the constant region gene. This indicates that the 14 kb EcoRI-DNA fragment from the myeloma tissue clearly resulted from somatic DNA rearrangement although it does not seem to carry the MOPC 173 variable region gene. These observations suggest that somatic DNA rearrangement of immunoglobulin light chain genes can involve both homologous chromosomes.Images     

Protein L from Peptostreptococcus magnus binds to the kappa light chain variable domain.

Protein L is an immunoglobulin light chain-binding protein expressed by some strains of the anaerobic bacterial species Peptostreptococcus magnus. The major variable region subgroups of human kappa and lambda light chains were tested for protein L binding; V kappa I, V kappa III, and V kappa IV bound protein L, whereas no binding occurred with proteins of the V kappa II subgroup or with any lambda light chain subgroups. Studies of the protein L binding capacity of naturally occurring VL fragments, and VL- and CL-related trypsin- and pepsin-derived peptides prepared from a kappa I light chain, localized the site of interaction to the VL domain. The affinity constant for the binding to an isolated V kappa I fragment was comparable to that for the native protein (Ka 0.9 x 10(9) M-1 and Ka 1.5 x 10(9) M-1, respectively). No binding occurred with CL-related fragments. Extensive reduction and alkylation of the V kappa fragment or the native kappa chain resulted in complete loss of protein L binding. Although it is possible, from comparative amino acid sequence data, to identify certain VL-framework region residues that account for the selective binding of protein L by kappa I, kappa III, and kappa IV proteins, our studies indicate that this interaction is essentially dependent upon the tertiary structural integrity of the kappa chain VL domain.     

Identification of a novel substitution in the constant region of a gene coding for an amyloidogenic kappa1 light chain.

Current concepts regarding the association between immunoglobulin (Ig) light chain structure and AL amyloidosis (AL) emphasize Ig variable region amino acid substitutions because the majority of light chain amyloid fibrils that have been sequenced contain amino termini of the variable region with only small amounts of the constant region. In this report, we describe a patient with rapidly progressive AL whose amyloid deposits contained primarily monoclonal kappa light chain constant region fragments. We sequenced and analyzed this AL protein, determining that it was an O18-O8 kappa1 variant and that the constant region possessed an unusual Ser-->Asn substitution at position 177. Using pre-mortem bone marrow cells, we cloned and sequenced the cDNA for this AL protein (HCAK1) and, using DNA from post-mortem somatic tissue, we cloned and sequenced the patient's kappa germline O18-O8 donor and kappa constant region (Ckappa) gene segments. The cDNA that coded for HCAK1 contained a variable region that was derived from O18-O8, showing 96.1% homology to germline, and a Ckappa that had a nucleotide substitution (AGC to AAC), resulting in the 177Ser-->Asn replacement. Two Ckappa genes were cloned from somatic tissue DNA, one identical to a known Ckappa sequence and another containing this substitution which likely is a new Ckappa allotype. Our findings indicate that further investigation is warranted into the contributions genetic polymorphisms and light chain constant regions may make to amyloidogenesis.     

Characterization and bacterial expression of the Dictyostelium myosin light chain kinase cDNA. Identification of an autoinhibitory domain.

A full-length cDNA corresponding to the Dictyostelium myosin light chain kinase gene has been isolated and characterized. Sequence analysis of the cDNA confirms conserved protein kinase subdomains and reveals that the Dictyostelium sequence is highly homologous to those of calcium/calmodulin-dependent protein kinases, including myosin light chain kinases from higher eukaryotes. Despite the high homologies to calcium/calmodulin-dependent protein kinases, there is no recognizable calmodulin-binding domain within the Dictyostelium sequence. However, the Dictyostelium myosin light chain kinase possesses a putative auto-inhibitory domain near its carboxyl terminus. To further characterize this domain, the full-length enzyme as well as a truncated form lacking this domain were expressed in bacterial cells and purified. The full-length enzyme expressed in bacteria exhibits essentially the same biochemical characteristics as the enzyme isolated from Dictyostelium. The truncated form however exhibits a Vmax that is approximately ten times greater than that of the native enzyme. In addition, unlike the native kinase and the full-length kinase expressed in bacteria, the truncated enzyme does not undergo autophosphorylation. These results suggest that the Dictyostelium enzyme, like myosin light chain kinases from higher eukaryotes, is regulated by an autoinhibitory domain but that the specific molecular signals necessary for activation of the Dictyostelium enzyme are entirely distinct.     

Nucleotide sequence of a mouse kappa light chain cDNA cloned in a bacterial plasmid.

A mouse MOPC21 cDNA previously cloned in plasmid pMB9(Higuchi $\text{et}\text{al.}$, Proc. Natl. Acad. Sci. 73 (1976) 2136–2140; Wall $\text{et}\text{al.}$, Nucleic Acid Res. 5 (1978) 3113–3128) and is designated pL21-3 has been extensively characterized. Cleavage of pL21-3 with Hpall has shown the insert to be 910 basepairs long, consistent with the length of the entire variable and constant regions and the untranslated regions. Digestion of pL21-3 with various restriction endonucleases has established that the insert sequence starts from parts of the 5′ leader region and extends downstream to include the untranslated 3′ terminus. 131 nucleotides in the variable region corresponding to amino acids 49–91 have been determined.     

Unfolding and refolding of the constant fragment of the immunoglobulin light chain containing an intramolecular mercury bridge

The conformation of the constant fragment of the immunoglobulin light chain in which the intrachain disulfide bond is replaced by the bond S-Hg-S (CL-Hg fragment), was as compact as that of the intact CL fragment, but its stability to guanidine hydrochloride was lower than that of the intact CL fragment [Goto, Y. & Hamaguchi, K. (1986) Biochemistry in press]. The kinetics of reversible unfolding and refolding of the CL-Hg fragment by guanidine hydrochloride were studied and compared with those for the intact CL and reduced CL fragments [Goto, Y. & Hamaguchi, K. (1982) J. Mol. Biol. 156, 891-910, 911-926]. The unfolding kinetics were explained on the basis of a three-species mechanism, U1----U2----F, where U1 and U2 are respectively slow-folding and fast-folding species of unfolded protein, and F is folded protein. However, an additional isomerization, though its contribution to the overall reaction process is small, had to be taken into account to explain the refolding kinetics. The kinetic properties of interconversion between U1 and U2 were similar to those for the intact CL and reduced CL fragments. This suggested that the same prolyl residue is involved in the isomerization reactions in the unfolded states of the intact CL, reduced CL, and CL-Hg fragments. The rate constant for the unfolding process, F to U2, was about 20 times greater than those for the intact CL and reduced CL fragments, while the rate constant for the refolding process, U2 to F, lay between the values for the intact CL and the reduced CL fragment. The free energy profiles of unfolding and refolding of the intact CL, reduced CL, and CL-Hg fragments were compared.     

Conformation and stability of the constant fragment of the immunoglobulin light chain containing an intramolecular mercury bridge

The constant fragment of the immunoglobulin light chain in which the intramolecular disulfide bond is reduced (reduced CL fragment) assumes a conformation very similar to that of the intact CL fragment and contains two sulfhydryl groups buried in the interior of the molecule [Goto, Y., & Hamaguchi, K. (1979) J. Biochem. (Tokyo) 86, 1433-1441]. In order to understand the role of the disulfide bond, a derivative in which the disulfide bond is replaced by an S-Hg-S bond was prepared and its conformation and stability were studied. The derivative was prepared by reacting the reduced CL fragment with mercuric chloride. Kinetic studies showed that the reaction is rate-limited by the unfolding process of the reduced CL fragment. The mercury derivative was as compact as the intact CL or reduced CL fragment, and a tryptophyl residue was found to be buried near the S-Hg-S bond in the interior of the protein molecule. Judging from the circular dichroic spectrum, however, the beta-structure characteristic of the immunoglobulin fold was disturbed. The stability of the derivative to guanidine hydrochloride was lower than that of the intact CL fragment, but the unfolding transition was reversible and cooperative. Decreased stability of the mercury derivative is due to its folded conformation being distorted by introduction of the S-Hg-S bond.     

Evolutionary relationship between human and mouse immunoglobulin kappa light chain variable region genes

Similar to the Igh-V multigene family, the human or mouse Igk-V repertoirer is a distorted continuum of homologous genes that may be grouped into families displaying >80% nucleic acid sequence similarity among their members. systematic interspecies sequence comparisons reveal that most human Igk-V gene families exhibit clear homology to mouse Ogk-V families (sequence similarity >74%). A hypothetical phylogenetic tree of Igk-V genes predicts that a minimum of seven Igk-V genes/families predate mammalian radiation. In two cases, several interrelated mouse Igk-V families exhibit phylogenetic equidistance with just one human Igk-V family, implying a more pronounced divergence for the elevated number of Igk-V gene families in the mouse. Mouse-human Igk-V comaprisons, moreover, illustrate how expansion, contraction, and perhaps deletion of Igk-V gene families shape the Igk-V repertoire during mammalian evolution.     

Serologic and chemical differentiation of human lambda III light chain variable regions

Human lambda L chains of a major V lambda subgroup, V lambda III, have been differentiated serologically and chemically into three V lambda III sub-subgroups designated V lambda IIIa, V lambda IIIb, and V lambda IIIc. Antisera prepared against lambda III Bence Jones proteins were obtained that recognized distinctive V lambda III-related epitopes expressed by monoclonal lambda III L chains. After appropriate absorption, these reagents were rendered specific for three distinct populations of lambda III proteins--lambda IIIa, lambda IIIb, and lambda IIIc. The antisera were used in comparative immunodiffusion analyses of 28 monoclonal lambda III L chains, 10 of which were classified as lambda IIIa, 4 as lambda IIIb, and 14 as lambda IIIc. The isotypic nature of the three lambda III sub-subgroups was demonstrated serologically through analyses of lambda-chains derived from the serum IgG molecules of normal individuals. The amino acid sequences of five serologically classified lambda III chains, which included members of the three V lambda III sub-subgroups, had been previously determined. This information, in addition to our establishment of the complete (or virtually complete) V region sequence of 15 and the partial sequence of eight other lambda IIIa, lambda IIIb, and lambda IIIc proteins, made it possible to correlate chemical data with serologic classification. Proteins within each of the three serologically-classified lambda III sub-subgroups typically possessed a high degree (approximately 83%) of intra-sub-subgroup sequence homology that included both framework and complementarity determining region residues. Furthermore, within the framework and complementarity determining regions, sub-subgroup-specific residues were identified. Taken together, these data reveal that the human V lambda III genome consists of (at least) three distinct V lambda IIIa, V lambda IIIb, and V lambda IIIc germline genes that encode for lambda IIIa, lambda IIIb, and lambda IIIc L chains, respectively.