Association of human lambda light chain V/J/C segments: serologic analysis and primary structure of the lambda VI Bence Jones protein THO

The complete amino acid sequence of the human monoclonal lambda VI light chain Bence Jones protein THO was determined. We have found it to have remarkable similarities to the previously sequenced lambda VI Bence Jones protein SUT. Immunochemical analyses demonstrated that both lambda VI chains belong to a V lambda VI sub-subgroup. The 98-residue V gene-encoded segments of proteins THO and SUT are closely homologous and are distinguished from other lambda VI chains by a one-residue deletion at the V-J recombination site. Proteins THO and SUT have identical 13-residue J segments and therefore are encoded by the same J lambda gene. Further, both proteins have identical 105-residue C regions that by sequence represent products of the C lambda 3 (Kern-, Oz+) gene. The primary structure and serologic properties of proteins THO and SUT imply at the protein level of association between certain types of V lambda, J lambda, and C lambda segments.     

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.     

Serologically defined V region subgroups of human lambda light chains

The availability of numerous antisera prepared against lambda-type Bence Jones proteins and lambda chains of known amino acid sequence has led to the differentiation and classification of human lambda light chains into one of five V lambda subgroups. The five serologically defined subgroups, V lambda I, V lambda II, V lambda III, V lambda IV, and V lambda VI, correspond to the chemical classification that is based on sequence homologies in the first framework region (FR1). Proteins designated by sequence as lambda V react with specific anti-lambda II antisera and are thus included in the V lambda II subgroup classification. The isotypic nature of the five V lambda subgroups was evidenced through analyses of lambda-type light chains that were isolated from the IgG of normal individuals. Based on analyses of 116 Bence Jones proteins, the frequency of distribution of the lambda I, lambda II/V, lambda III, lambda IV, and lambda VI proteins in the normal lambda chain population is estimated to be 27%, 37%, 23%, 3%, and 10%, respectively. This distribution of V lambda subgroups was comparable to that found among 82 monoclonal Ig lambda proteins. Considerable V lambda intragroup antigenic heterogeneity was also apparent. At least two sub-subgroups were identified among each of the five major V lambda subgroups, implying the existence of multiple genes in the human V lambda genome. The V lambda classification of 54 Ig lambda proteins obtained from patients with primary or multiple myeloma-associated amyloidosis substantiated the preferential association of lambda VI light chains with amyloidosis AL and the predominance of the normally rare V lambda VI subgroup in this disease.     

Cloning and sequence analysis of an Ig lambda light chain mRNA expressed in the Burkitt''s lymphoma cell line EB4.

A cDNA library was constructed from the mRNA of the Ig lambda producing Burkitt's lymphoma cell line, EB4. Overlapping clones encompassing the coding sequence of the Ig lambda mRNA were isolated and sequenced. The predicted amino acid sequence shows a short hydrophobic leader peptide and a mature polypeptide of 217 residues in which V, J and C regions can be distinguished. The V region belongs to subgroup VI and has greatest homology (80%) with the Amyloid-AR protein. The constant region is the Kern- Oz+ isotype. Probing normal human DNA with the subcloned V lambda coding sequence detects one gene at high stringency and a family of 11 members at low stringency. To date, no restriction enzyme site polymorphisms have been detected. The V lambda VI gene is rearranged on both chromosomes of EB4 and is deleted on both chromosomes in the Burkitt's lymphoma cell line BL2.     

Primary structure of the variable region of a human lambda VI light chain: Bence Jones protein SUT

To ascertain if lambda VI light chains have unique structural features that account for the preferential association of these proteins with primary or multiple myeloma-related amyloidosis (amyloidosis AL) we have determined the complete amino acid sequence of the variable (V) region of the lambda VI Bence Jones protein SUT. This protein, obtained from a patient with amyloidosis AL, represents a complete light chain consisting of 216 residues and it has structural and serologic properties characteristic for lambda VI light chains. The sequence of the joining segment (J) (positions 100 to 111) of protein SUT is identical to that of the J lambda I segment of the mouse IG lambda light chain gene. V region SUT is closely homologous in sequence to that of another lambda VI amyloid fibrillar protein, AR, differing by 21 residues. The V regions of proteins SUT and AR contain a two-residue insertion at positions 68 and 69 that has also been found in two other lambda VI human light chains but not in the lambda-chains of other V region subgroups.     

Unraveling of the polymorphic C lambda 2-C lambda 3 amplification and the Ke+Oz- polymorphism in the human Ig lambda locus

Two polymorphisms of the human Ig(lambda) (IGL) locus have been described. The first polymorphism concerns a single, 2- or 3-fold amplification of 5.4 kb of DNA in the C(lambda)2-C(lambda)3 region. The second polymorphism is the Mcg(-)Ke(+)Oz(-) isotype, which has only been defined via serological analyses in Bence-Jones proteins of multiple myeloma patients and was assumed to be encoded by a polymorphic C(lambda)2 segment because of its high homology with the Mcg(-)Ke(-)Oz(-) C(lambda)2 isotype. It has been speculated that the Mcg(-)Ke(+)Oz(-) isotype might be encoded by a C(lambda) gene segment of the amplified C(lambda)2-C(lambda)3 region. We now unraveled both IGL gene polymorphisms. The amplification polymorphism appeared to result from a duplication, triplication, or quadruplication of a functional J-C(lambda)2 region and is likely to have originated from unequal crossing over of the J-C(lambda)2 and J-C(lambda)3 region via a 2.2-kb homologous repeat. The amplification polymorphism was found to result in the presence of one to five extra functional J-C(lambda)2 per genome regions, leading to decreased Ig(kappa):Ig(lambda) ratios on normal peripheral blood B cells. Via sequence analysis, we demonstrated that the Mcg(-)Ke(+)Oz(-) isotype is encoded by a polymorphic C(lambda)2 segment that differs from the normal C(lambda)2 gene segment at a single nucleotide position. This polymorphism was identified in only 1.5% (2 of 134) of individuals without J-C(lambda)2 amplification polymorphism and was not found in the J-C(lambda)2 amplification polymorphism of 44 individuals, indicating that the two IGL gene polymorphisms are not linked.     

Bence Jones proteins and light chains of immunoglobulins. XIII. Effect of elastase-like and chymotrypsin-like neutral proteases derived from human granulocytes on Bence Jones proteins.

Bence Jones proteins can be cleaved specifically by several types of endopeptidases into fragments corresponding to the amino-terminal, variant (VL) portion and to the carboxyl-terminal, constant (CL) portion of the light polypeptide chain. Two types of neutral proteases, designated elastase-like (ELP) and chymotrypsin-like (CLP), have been isolated and purified from human polymorphonuclear leukocytes. Because these proteases have defined proteolytic activity under physiologic conditions for several types of human proteins, we investigated their effect on human Bence Jones proteins. Incubation of kappa-type or lambda-type Bence Jones proteins with ELP or CLP under appropriate conditions resulted in cleavage of both types of light chains as evident by immunochemical and electrophoretic analyses. Treatment with ELP or CLP of one kappa Bence Jones protein resulted in the formation of a single component that had antigenic and electrophoretic properties similar to the VL fragment derived from pepsin digestion of the native protein. No component corresponding to the CL could be detected immunochemically or electrophoretically. Studies of isolated pepsin-labile (37 degrees C) and pepsin-stable (55 degrees C) CL fragments demonstrated the marked susceptibility of the carboxyl-terminal half of the light chain to proteolysis by the leukocyte-derived neutral proteases. Incubation with ELP of three other kappa Bence Jones proteins and three reduced-alkylated lambda Bence Jones proteins resulted, in each case, in the formation of a homogeneous component which was electrophoretically and immunochemically distinct from the pepsin-derived VL fragment. An identical component could also be formed by incubating a pepsin-derived VL fragment with ELP. In the ELP-treated samples, no CL-related material was detected electrophoretically or immunochemically with antisera possessing specificity for CL antigenic determinants present on the unfolded light polypeptide chain or on the isolated CL. The component formed by ELP or CLP treatment of certain Bence Jones proteins thus appears to be VL-related, but lacks the idiotypic antigenic determinant present on the native protein. In this respect, these neutral protease-derived light chain components are similar to the amyloid-like VL fragments generated in vitro from certain endopeptidase-treated Bence Jones proteins.     

Comparative studies on the structure of the light chains of human immunoglobulins. IV. Assignment of a subsubgroup

Amino acid sequence analysis was done on a human lambda Bence Jones protein NIG-64 with the major objective of determining the sequence of the variable region. Nineteen tryptic peptides covering 216 residues were isolated from the completely reduced and aminoethylated protein, and 17 of these were completely sequenced. These comprised the entire variable region and 11 from the constant region. For the remaining peptides covering the rest of the constant region, only partial sequences or the amino acid compositions were determined. All the tryptic peptides could be arranged in order on the basis of the above results and homology with other human lambda light chains of the same isotype. The sequence of the variable region of the protein is highly homologous with that of protein New of subgroup V lambda I as compared with other proteins of the same subgroup, suggesting that subgroup V lambda I may be further divided into subsubgroups, namely subsubgroups V lambda I-1 and V lambda I-2.     

Primary structure of a human IgA1 immunoglobulin. V. Amino acid sequence of a human IgA lambda light chain (Bur).

The sequence of the lambda light chain of the Bur IgA1 molecule has been determined. It comprises 214 amino acid residues with a blocked NH2 terminus and lacks carbohydrate. The V-region sequence is of the VlambdaII subgroup and contains the coupled interchanges Arg-7 and Cys-87. The Lv3 region is comparatively short and hydrophobic in nature and lends support for the designation of this area as a hypervariable deletion region. The C-region exhibits the Mcg+ Kren+ Oz- isotypes. These appear coupled with substitution at position 100 (in the V-region). The pattern of nonrandom association of V- and C-regions and H and L chains is discussed in terms of the generation of antibody diversity. With the companion papers in this series, the complete primary structure of a human IgA1 molecule is established.     

Study of the antigenic structure of human immunoglobulin lambda-chain using monoclonal antibodies]

Nine monoclonals against human Ig lambda chains were produced, 4 antibodies react with C-domain, 5--with V-domain of the lambda chain. Anti-C lambda domain antibodies recognize not less than 3 epitopes and one of them is expressed only on the isolated chain. Anti-V lambda antibodies bind both isolated lambda chain and intact IgG, IgM, IgA. Four epitopes are expressed by few lambda Bence Jones proteins of the III subgroup, the immunogen possessing the same isotype. The 4 mentioned epitopes represent private idiotypic determinants. The epitope 3E10 is characteristic of 50% Bence Jones proteins of the II and III V lambda-subgroups thus representing a common idiotypic determinant. Using anti-V lambda antibodies germ line variability of V lambda III proteins was analysed and the similarity of antigenic structure of normal and myeloma human Ig lambda chains was demonstrated.     

Amino acid sequence of an amyloidogenic Bence Jones protein in myeloma-associated systemic amyloidosis

The complete amino acid sequence of an amyloidogenic Bence Jones protein (NIG-84) from an individual with myeloma-associated systemic amyloidosis has been determined. The protein, with a blocked N-terminus, represents a complete light chain consisting of 217 residues and it has a structural feature characteristic of the V lambda II subgroup. In addition to a two-residue insertion at positions 28 and 29, it has an additional rare insertion of alanine at position 100. NIG-84 is an example of the first complete sequence presented for the amyloidogenic Bence Jones protein of the V lambda II subgroup.     

Primary structure of the variable region of an amyloidogenic Bence Jones protein NIG-77

The complete amino acid sequence of the variable region of a Bence Jones protein NIG-77 from an individual with myeloma-associated systemic amyloidosis has been determined. This protein represents a complete light chain consisting of 216 residues and it has a sequence characteristic of V lambda I subgroup, which is closely homologous to that of another amyloidogenic V lambda I Bence Jones protein NIG-51, differing by 20 of 111 residues (82% homology). In contrast, it differs by 29 residues (74% homology) to that of non-amyloidogenic V lambda I light chain NIG-64. This finding shows that, in accordance with our previous report(1), the V lambda I-related light chains can further be divided into two distinct subsubgroups, V lambda I-1 and V lambda I-2, and the latter property seems to be more prone in association with the amyloid process.     

The amino acid sequence of a lambda light chain presenting abnormal physicochemical and antigenic features

The amino acid sequence of the light chain of a human monoclonal IgA1 (Mem) was established, in part by analogy with already known sequences. By homology its variable part was shown to belong to the V lambda I subgroup while the isotype-associated amino acid residues characterized it as Mcg+, Kern+ and Oz-. The normal primary structure of this chain was in contrast to its abnormal physical and antigenic properties: (a) its apparent molecular mass estimated by SDS/polyacrylamide gel electrophoresis, by gel filtration chromatography and by gradient ultracentrifugation was found to be lower by approximately equal to 10% than the values (23.5 kDa) of 'normal' light chain used as controls; (b) the lambda I chain Mem, when tested in native state was not antigenically reactive. These abnormalities were reverted when the chain was treated with 8 M urea. These data suggest that the abnormal behaviour of lambda I chain Mem is at a conformational level.     

Comparative study on the structure of the light chains of human immunoglobulins. II. Assignment of a new subgroup.

The primary structure of the variable region of the human lambda type Bence Jones protein NIG-48 was determined by analysis of the N-terminal sequence of the completely reduced and aminoethylated protein, as well as of five cyanogen bromide fragments. The variable region of NIG-48 contains 112 amino acid residues. The protein NIG-48, having a unique sequence of the variable region, a low degree of homology (about 50%) with lambda chains of the five other subgroups and the addition of two residues around 65, may represent a new subgroup, namely V lambda VI.     

Amino acid sequence of the variable region of the light (lambda) chain from human myeloma cryoimmunoglobulin IgG Hil.

We have determined the complete amino acid sequence of the variable region of the light (lambda) chain from a human myeloma cryoimmunoglobulin (IgG Hil), the Fab fragment from which has been previously crystallized. The presence of unblocked alpha-amino terminal residue and the isolation of a CNBr fragment starting at position 46 and of a maleylated tryptic fragment spanning residues 61 to 189 provided three suitable starting points for automatic Edman degradation. In addition, tryptic peptides and chymotryptic subpeptides covering the whole extension of the light chain were obtained and characterized to further verify the sequence of the variable region and the established sequence of the constant region. The proposed sequence of the variable region indicates that it may be assigned to subgroup III. Positions 152 (serine) and 189 (arginine) correspond to the isotypic markers Kern- and Oz-, respectively. In addition, a novel substitution has been detected in the constant region where at position 155 isoleucine replaces the usually occurring valine.     

The human lambda L chain Ig locus. Recharacterization of JC lambda 6 and identification of a functional JC lambda 7

The human lambda L chain Ig gene complex consists of multiple JC gene segments. A seventh human lambda C region gene segment, C lambda 7, was found 2.7 kb downstream of C lambda 6 in this gene complex. A J lambda gene segment, J lambda 7, was found 1.2 kb upstream of C lambda 7 and contains potentially functional nonamer and heptamer recombination sites, an RNA splice site and J coding region. C lambda 7 maintains an open reading frame and encodes a new lambda isotype. C lambda 7 encodes Kern+ and Oz- determinants, but does not encode any of the Kern+Oz- myeloma proteins published to date. Nevertheless, we present evidence that JC lambda 7 is transcribed in normal lymphocytes and is functional. In contrast, we present new data that the C lambda 6 gene segment, reported by others to encode the Kern+Oz- protein, is non-functional due to a 4-bp insertion in our cosmid clone. The 4-bp insertion was characterized further in 32 genomic DNA samples by producing a distinctive restriction fragment length and verified by the DNA sequences of the polymerase chain reaction products of two different cell lines. We discuss the possibility that the Kern+Oz- myeloma proteins do not define an isotype and are not encoded by JC lambda 7 nor other non-allelic genes, and we discuss the level of expression of JC lamba 7 as compared to that of JC lambda 2 and JC lambda 3.     

Rule of antibody structure. Primary structure of a human monoclonal IgAl-immunoglobulin (myeloma protein Tro). VI. Amino acid sequence of the L-chain, lambda-type, subgroup II]

The primary structure of the L-chain of an IgA1-immunoglobulin (Myeloma protein Tro) has been determined by means of cleavage with trypsin and, if necessary, with alpha-chymotrypsin. The tryptic peptides of the variable part were characterized by amino acid analysis, Dansyl-Edman degradation and cleavage with carboxypeptidase; the peptides of the constant part were identified by amino acid analyses and determination of its N- and C-terminal residues. The sequence of the remaining amino acids and the arrangement of the peptides were established in homology to known structures. The protein comprises 216 amino acids. The homology of the variable part clearly characterizes it as belonging to subgroup II of lambda-chains. In positions 27a, b and c, there are the subgroup-specific additional residues and in position 96 is the characteristic deletion. The constant part of the chain is Kern- and Oz- which indicates that it has serine in position 154 and arginine in position 191.     

Refolding of the immunoglobulin light chain.

The kinetics of the refolding reactions of type lambda Bence Jones proteins from 4 M GuHCl were studied by CD, ultraviolet absorption, and fluorescence spectrophotometry. The kinetics were complex and consisted of at least three phases, an undetectable fast phase, a detectable fast phase, and a slow phase. The slow phase followed first-order kinetics and the three experimental methods used gave similar rate constants for all the Bence Jones proteins (about 3 X 10(-3) s-1). The refolding reaction of VL fragment was too fast to be measured in the present experiments. The refolding process of CL fragment was very similar to those of Bence Jones proteins except that the detectable fast phase was less significant. The rate constant of the slow phase observed for the CL fragment was similar to those of the slow phase observed for Bence Jones proteins. The activation energy of the slow phase was the same for a Bence Jones protein and its CL fragment. These results indicate that the refolding kinetics of the CL domain are very similar to those of isolated CL fragment and that refolding of the VL domain precedes refolding of the CL domain, even though both domains have similar immunoglobulin folds. However, the results of refolding experiments on Bence Jones proteins, and VL and CL fragments in the presence of ANS, as well as the other lines of evidence, indicate that the refolding kinetics of the Bence Jones protein molecule cannot be expressed as simple sum of the refolding reactions of isolated VL and CL fragments.     

The amino acid sequence of a carbohydrate-containing immunoglobulin-light-chain-type amyloid-fibril protein.

The amino acid sequence of an amyloid-fibril protein Es492 of immunoglobulin-lambda-light-chain origin (AL) was elucidated. The amyloid fibrils were obtained from the spleen of a patient who died from systemic amyloidosis. The amino acid sequence was elucidated from structural studies of peptides derived from digestion of the protein with trypsin, thermolysin, chymotrypsin and Staphylococcus aureus V8 proteinase and from cleavage of the protein with CNBr and BNPS-skatole. A heterogeneity in the length of the polypeptide was seen in the C-terminal region. The protein was by sequence homology to other lambda-chains shown to be of the V lambda II subgroup. Although an extensive homology was seen, some amino acid residues in positions 26, 31, 32, 40, 44, 93, 97, 98 and 99 have not previously been reported in these positions of V lambda II proteins. The significance of these residues in the fibril formation is unclear. The protein was found to contain carbohydrate, with glycosylation sites in two of the hypervariable regions.     

Complete amino acid sequence determinations demonstrate identity of the urinary Bence Jones protein (BJP-DIA) and the amyloid fibril protein (AL-DIA) in a case of AL-amyloidosis.

The complete primary structures of both the main amyloid fibril protein component (AL-DIA) and the soluble Bence Jones protein (BJP-DIA) obtained from the same patient with AL-amyloidosis are reported for the first time. The amino acid sequences were determined by automated Edman degradation following proteolytic digestion of the isolated proteins and HPLC separation of the resulting fragments and by amino-terminal sequencing after treatment with pyroglutamate aminopeptidase. Sequencing data were confirmed by amino acid analysis and plasma desorption mass spectrometry (PDMS). Molecular weights of the complete proteins were determined by laser desorption mass spectrometry. The amyloid fibril preparation contained a complete monoclonal lambda immunoglobulin light chain (subgroup 1.2) as well as different-sized fragments thereof which were identified by immunoblotting and amino-terminal sequencing following immobilization of electrophoretically-separated proteins on poly(vinylidene difluoride) (PVDF) membranes. The soluble urinary Bence Jones protein (BJP-DIA) was a dimer of monoclonal L-chains with a primary structure identical to that of the amyloid L-chain (AL-DIA) and thus represented the amyloid precursor protein.