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.     

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.     

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.     

Immunogenic and antigenic epitopes of Ig. XXV. Monoclonal antibodies that differentiate the Mcg+/Mcg- and Oz+/Oz- C region isotypes of human lambda L chains

The C region of human lambda L chains is specified by multiple C lambda genes of which three--C lambda 1, C lambda 2, and C lambda 3--encode for the isotypes designated Mcg+, Kern- Oz-, and Kern- Oz+, respectively. The Mcg, Kern, and Oz factors have been characterized by sequence differences involving specific C lambda amino acid residues. They have also been recognized serologically by polyclonal antisera but, with rare exception, these reagents are no longer available. We have obtained two murine anti-human lambda-chain mAb, 14G1 and 14D1, that recognize antigenic determinants specific for the C lambda isotypes Mcg and Oz, respectively. These antisera have been used to classify as Mcg+/Mcg- or Oz+/Oz- monoclonal lambda-chains (Bence Jones proteins) and intact Ig lambda proteins. There was complete concordance between the chemical and serologic assignment of lambda-chains as Mcg+/Mcg- or as Oz+/Oz-; no single protein expressed both isotypes. There was no evident association between the C region isotype Mcg or Oz and the V region subgroup of the protein tested. However, our finding that four of seven amyloid-associated lambda VI Bence Jones proteins were Oz+ suggests a predominant expression of the C lambda 3 gene product among proteins of this uncommon V lambda subgroup.     

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.     

Novel human light chain V kappa segment: serologic and structural analyses of the kappa III-like Bence Jones protein and IgG kappa light chain REE

Immunochemical and sequence analyses of kappa light chain REE (Bence Jones protein REE and the light chain isolated from IgG kappa myeloma protein REE) revealed antigenic and structural features not previously described for human kappa-chains. Although closely related to proteins of the V kappa III subgroup, light chain REE is readily distinguished from light chains classified serologically as members of the kappa IIIa or kappa IIIb sub-subgroups. Light chains REE (Bence Jones protein REE and light chain REE) are identical in sequence and differ from kappa III proteins by at least 10 uncommon amino acid substitutions in the first three framework regions. Further, kappa-chain REE is unique by virtue of a four-residue deletion in the third complementarity-determining region. The deletion encompasses the three carboxyl-terminal residues in the V kappa-encoded segment and the first residue at the site of V-J recombination. Urine specimens from patient REE also contained a light chain fragment that lacked the first (amino-terminal) 85 residues of the native light chain but otherwise was identical in sequence to the light chain REE. The extensive amino acid differences and unique length of the V kappa segment in light chain REE indicate that this kappa-chain is the product of an unusual V kappa III gene or, alternatively, represents a rarely expressed and novel human V kappa gene.     

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.     

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.     

Characterization of a variable fragment of a human immunoglobulin lambda chain obtained by peptic cleavage in urea

The digestion of a human dimeric lambda Bence Jones protein with pepsin in 8 M urea without prior reduction and alkylation produced a fragment of molecular weight 11,000, designated as upV lambda, corresponding to almost the entire variable region of an immunoglobulin light chain. The C-terminal amino acid residue of fragment upV lambda was shown to correspond to leucine at position 104 of the immunoglobulin lambda light chain. Fragment upV lambda was isolated in the yield of 30% by ion exchange chromatography after gel filtration. When another lambda Bence Jones protein was treated in the same way, similar results were obtained.     

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.     

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.     

A cross-reactive idiotype on anti-DNA antibodies defines a H chain determinant present almost exclusively on IgG antibodies

We have previously reported two anti-idiotypic antibodies, 3I and 8.12, that recognize L chain determinants on anti-DNA antibodies. We have generated a new anti-idiotypic antibody, F4, that recognizes a H chain determinant on cationic anti-DNA antibodies. F4 reactivity is present in high titer in serum of approximately 60% of SLE patients and on 84 of 706 myeloma proteins. It is preferentially associated with 3I reactive L chains. Furthermore, antibodies bearing both the F4 and 3I idiotypic determinants preferentially bind DNA. Amino acid sequencing of H chains isolated from four F4-reactive myeloma proteins suggests that they derive from two currently identified VH gene families. F4 reactivity is restricted almost exclusively to Ig of the IgG isotype suggesting that F4 may recognize either a somatically mutated hypervariable region or a variable region used late in the immune response. F4, therefore, represents a new idiotypic family preferentially associated with auto-Ag specificity and having features of an Ag-driven immune response.     

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.     

Infrared spectroscopy of human amyloid fibrils and immunoglobulin proteins

The presence of the antiparallel-β-pleated sheet coformation io isolated human amyloid protein fibrils has been confirmed by infrared spectroscopy. In most amyloid samples, this conformation was enhanced by acidic solution conditions. Infrared spectroscopy (Amide I and Amide V absorption bands) and x-ray diffraction methods were also used to examine the immunoglobulin molecule for solid state-β-structure. It was found that both heavy chains and Bence Jones proteins exhibited some β-pleated sheet content upon acid and/or heat treatment. Furthermore, pepsin digests comprising either the variable-rich region (Fd′) of the immunloglobulin heavy chain or in particular, filamentous variable segments of κ and λ Bence Jones proteins were, as isolated, very similar to amyloid in β-structure content. Data from other immunoglobulin-derived sample did not exhibit extensive β-pleated sheet content. On the other hand, most amyliod and immunoglobulin-derived samples did display some β-structure when cast from 50% HCOOH solution. Under these conditions, however, filamentous light chain-variable segments exhibited well-defined infrared patterns rich in antiparallel-β-pleated sheet structure and gave a “cross-β” x-ray diffraction pattern.     

Idiotypic variation in a human B lymphoma cell line

The Ig Id of a B cell lymphoma serves as a distinct marker of the malignant clone and thus as a tumor-specific target for antibody therapy. Somatic variation of the Ig genes expressed by B cell tumors can lead to loss of reactivity with anti-Id antibodies and escape of tumors from the therapeutic effects of such antibodies. In our study, we have used anti-Id antibodies to screen for variants within a cell line derived from a patient with a large cell lymphoma of the B cell type. Cells were simultaneously stained on their surface for idiotypic and for isotypic Ig determinants using reagents labeled with different fluorochromes. Tumor cells expressing intact Ig molecules with alteration of their idiotypic determinants were isolated with the fluorescence activated cell sorter. Idiotypic variation was an ongoing process in vitro with Id- variants being generated at a rate of 2.7 x 10(-4)/cell per generation and Ig- cells being produced at a rate of 1.31 x 10(-5)/cell per generation. Subcloned variants expressed subtle differences in reactivity with a panel of three non-cross-blocking anti-Id antibodies. Analysis of Ig gene rearrangements by the Southern blotting technique using a JH probe established that the variants and the original tumor cells were all clonally related. Immunoprecipitation of surface labeled Ig molecules from the variant subclones disclosed major alterations of the lambda-L chains with no gross alterations of the mu-H chains. Related studies have established that the tumor cells undergo rearrangement and expression of new lambda-L chain genes.     

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 light chain derived from a rabbit anti-p-azobenzoate antibody of restricted heterogeneity

The amino acid sequence of the light chain from a specifically purified rabbit (No. 2717) anti-p-azobenzoate antibody preparation (b4 allotype) of restricted heterogeneity has been determined. This light chain is composed of 216 residues, including seven half-cystine residues located at positions 23, 80, 88, 134, 171, 194 and 216. Three intrachain disulfide bonds appear to be present in contrast to only two disulfide bonds as has been so far described for Bence Jones protein and light chains of human and mouse. This light chain was sequenced by isolating the tryptic peptides, sequencing the peptides and establishing their order within the molecule. Unambiguous identification of the overlaps was achieved by taking into account the partially characterized tryptic peptides from citraconic anhydride-treated light chains and chymotryptic and peptic peptides from digests of both untreated and citraconylated light chains. Comparison of this amino acid sequence with the amino acid sequence of the car?ylterminal half of the b4 light chains from unimmunized rabbits reveals differences at positions 165, 166, 169 and 176 indicating the existence of more than one sequence in the b4 “constant” region. There is substantial sequence homology between the variable half of 2717 light chain and human Bence Jones protein. Indeed, 46 positions in the V region (42%) are occupied by the same residues in this light chain and in human subgroup V_κIII.     

Structural characterization of a cross-reactive idiotype shared by monoclonal antibodies specific for the human CD4 molecule.

A panel of mouse monoclonal anti-CD4 antibodies was characterized in terms of idiotypic expression by using specific anti-idiotypic antibody (anti-Id) reagents generated in rabbits immunized with anti-Leu3a, a monoclonal anti-CD4 which inhibits the human immunodeficiency virus (HIV) gp120 binding to CD4. Direct binding and competitive inhibition assays demonstrate that the majority of monoclonal anti-CD4 antibodies able to recognize CD4 epitopes overlapping the epitope recognized by anti-Leu3a expressed an antigen-combining site-related cross-reactive idiotype (IdX). Western blot analysis was used to demonstrate that this IdX is associated primarily with the light (L) chain of the monoclonal anti-CD4 antibodies. To further characterize the structural basis of the IdX, the nucleotide sequence of the variable region of the L kappa chain of anti-Leu3a was determined. Peptides corresponding to the first, second, and third complementarity determining regions (CDRs) of the L chain of anti-Leu3a were synthesized and used to immunize rabbits. All anti-peptide antisera recognized the immunizing peptide, the cognate anti-Leu3a molecule, and several other monoclonal anti-CD4 antibodies by direct binding assays. Western blot analysis utilizing the anti-CDR peptide reagents demonstrates that the reactivity to the monoclonal anti-CD4 antibodies was L chain-specific. The anti-Id generated by immunizing with the intact anti-Leu3a molecule failed to recognize the three L chain-derived CDR synthetic peptides, suggesting that the IdX requires the presence of the three-dimensional configuration of the L chain for its expression. The broad range of reactivity exhibited by the antipeptide antisera indicates that the majority of mouse monoclonal anti-CD4 antibodies characterized in this study utilize L chains encoded by a single germ line variable (V) region kappa (V kappa) chain gene or by V kappa genes that belong to the same gene family.     

Phage display and peptide mapping of an immunoglobulin light chain fibril-related conformational epitope

Amyloid fibrils and partially unfolded intermediates can be distinguished serologically from native amyloidogenic precursor proteins or peptides. In this regard, we previously had reported that mAb 11-1F4, generated by immunizing mice with a thermally denatured variable domain (VL) fragment of the human kappa4 Bence Jones protein Len, bound to a non-native conformational epitope located within the N-terminal 18 residues of fibrillar, as well as partially denatured, Ig light chains (O'Nuallain, B., et al. (2006) Biochemistry 46, 1240-1247). To define further the antibody binding site, we used random peptide phage display and epitope mapping of VL Len using wild-type and alanine-mutated Len peptides where it was shown that the antibody epitope was reliant on up to 10 of the first 15 residues of protein Len. Comparison of Vkappa and Vlambda N-terminal germline consensus sequences with protein Len and 11-1F4-binding phages indicated that this antibody's cross-reactivity with light chains was related to an invariant proline at position(s) 7 and/or 8, bulky hydrophobic residues at positions 11 and 13, and additionally, to the ability to accommodate amino acid diversity at positions 1-4. Sequence alignments of the phage peptides revealed a central proline, often flanked by aromatic residues. Taken together, these results have provided evidence for the structural basis of the specificity of 11-1F4 for both kappa and lambda light chain fibrils. We posit that the associated binding site involves a rare type VI beta-turn or touch-turn that is anchored by a cis-proline residue. The identification of an 11-1F4-related mimotope should facilitate development of pan-light chain fibril-reactive antibodies that could be used in the diagnosis and treatment of patients with AL amyloidosis.     

Analysis of urinary proteins from 50 patients with multiple myeloma by discelectrophoresis (author's transl)]

Urinary proteins from 50 patients with multiple myeloma (37 Ig G, 6 Ig A, 7 Bence Jones) were investigated by discelectrophoresis in polyacrylamidgels containing sodium dodecylsulfat. All samples were also characterized by immunelectrophoresis. Quantitatively and qualitatively normal proteinuria was found in 13 patients (26%). 22 patients (44%) had monoclonal free light chains in the urine, kappachains were eliminated mainly in the monomeric form, lambdachains in all samples in the dimeric form. In 2 patients were found to exist light chains as monomers and dimers. 11 other patients (22%) had peaks of monoclonal Ig G or Ig A in the urine, always associated with the elimination of other nonmonoclonal proteins. Non-specific proteinuria was found in the remaining 4 patients. The clinical validity of the findings is discussed.