Sequence similarities and cross-idiotypic specificity of L chains among human monoclonal IgM kappa with anti-gamma-globulin activity

The complete amino acid sequence of five light chain variable (V) regions of human monoclonal IgM kappa rheumatoid factors (RF) was determined, and their cross-reactive idiotypes (CRI) were characterized with antibodies induced by immunization with synthetic peptides PSL2 and PSL3, corresponding to the second and third complementarity-determining regions (CDR) of the SIE light chain. Together with two additional RF studied previously, all seven RF belong to the V kappa IIIb sub-subgroup. The region encoded by the V kappa gene segment (positions 1 to 95) in all seven proteins was virtually identical in primary structure, whereas the sequence from positions 96 to 108 defined the usage of the J kappa 1 gene in three proteins and the J kappa 2 gene in four of them. Position 96 contributed by the recombination of the V kappa and J kappa gene segments showed the presence of four different amino acid residues. Both anti-PSL2 and anti-PSL3 bind efficiently to all separated L chains when analyzed by the Western blot technique, and the binding was inhibited specifically by the corresponding peptides. The results reveal that the majority of human IgM-RF light chains are derived from a single germ line V kappa gene or a family of closely related V kappa III germ line genes, and express two "primary structure-dependent" CRI, which are largely dependent on the amino acid sequence of the second and third light chain CDR.     

Preferential association of kappa IIIb light chains with monoclonal human IgM kappa autoantibodies

The predominance of the relatively uncommon V region subgroup isotype kappa III among the light chains of human monoclonal (IgM kappa) anti-IgG antibodies, (i.e., rheumatoid factors), was further documented through sequence analyses of ten such autoantibodies isolated from IgM-anti-IgG cold-insoluble immune complexes (mixed cryoglobulins). The amino-terminal sequence of all ten kappa-chains was characteristic for kappa III proteins and virtually identical to that of a prototype kappa III light chain. Similar sequence identity was found for kappa-chains isolated from three IgM kappa autoantibodies that formed cold-insoluble immune complexes with low-density lipoprotein (LDL). The thirteen light chains were found to be virtually identical in sequence for the first framework region (FR); ten of these proteins sequenced through the first complementarity-determining region (CDR) and into the second FR were markedly similar. The second CDR of five proteins was almost identical in sequence to that of the prototype kappa III-chain. Concordance was also demonstrated between the structural classification of the light chains as kappa III and their immunochemical classification as members of this V region subgroup. Serologic analyses of light chains isolated from seven IgM kappa autoantibodies (six anti-IgG, one anti-LDL) and of one intact IgM kappa anti-LDL antibody showed that each had antigenic determinants common to kappa II proteins. These light chains also expressed the antigenic determinant(s) of a V-region sub-subgroup of kappa III proteins designated kappa IIIb. Our studies confirm the preferential association of kappa III (and kappa IIIb) light chains with IgM kappa anti-IgG antibodies and demonstrate a similar association for IgM kappa anti-LDL antibodies. The finding that these and other types of IgM kappa autoantibodies, e.g., cold agglutinins, have remarkably similar light chains suggests an inherent restriction in the immune response to self-antigens.     

Novel V genes encode virtually identical variable regions of six murine monoclonal anti-bromelain-treated red blood cell autoantibodies

The variable (V) region sequences of six immunoglobulin M (IgM, kappa) monoclonal autoantibodies that recognize bromelinized isologous red blood cells, obtained by fusions of peritoneal cells from NZB or CBA/J nonimmunized mice with BALB/c myeloma cells, were determined by direct mRNA sequencing. The V regions of the light chains (VL) are almost identical with one another, as are the V regions of the heavy chains (VH), which, however, differ by six linked-base substitutions, depending on the strain of mice producing the autoantibodies. Such variations may reflect allelic differences. The VH segments determined have no obvious correspondence to any VH genes identified so far. They may belong to the small VH group 4, where 73% homology, at the most, can be calculated at the protein level for codons 1 to 94. Alternatively, the VH regions may be members of a new group of VH sequences not previously found. The V kappa regions appear closely homologous to members of the V kappa-9 subgroup of myeloma proteins of unknown antigen-binding specificity. The joining segments, J kappa and JH, used by the autoantibodies investigated, originate from the J kappa 2 and JH1 germ-line gene segments, respectively. The nine base-long diversity segments, D, derive from one member of the germ-line D gene SP2 family.     

H chain V region sequences of three human monoclonal IgM with anti-myelin-associated glycoprotein activity.

The amino acid sequence corresponding to the V region H chain gene used by three monoclonal IgM directed to the myelin-associated glycoprotein (MAG) is presented. They all belonged to the VHIII variability subgroup, but each may well represent a new member of this family inasmuch as their homology with previously sequenced VHIII genes was less than 80%. Strikingly, there was no greater homology between the H chain V regions of the anti-MAG IgM. Partial amino acid sequence data indicated that these V regions were joined to as yet unidentified DH segments; however, two H chains used very similar DH, possibly indicating that this sequence was involved in the fine specificity of the IgM for MAG. All H chains included a JHIV region. These data, together with results obtained from the sequence of the three kappa L chains of the same IgM molecules (Mihaesco, E., H. Ayadi, N. Congy, M. C. Gendron, J. P. Roy, H. Heyermann, B. Frangione, and J. C. Brouet. 1989. J. Biol. Chem. 264:21481), indicate that the repertoire of VL and VH gene segments used by anti-MAG IgM is quite diverse, in contrast to previous structural data obtained for other human monoclonal IgM autoantibodies. Possibly, these differences reflect distinct pathogenesis.     

Structural studies of monoclonal human cryoprecipitable immunoglobulins

The light chain type, immunoglobulin class and when possible, heavy chain subclass of eleven monoclonal human cryoglobulins were correlated with the variable region subgroup of their light chains. The variable region subgroups were assigned by determining the primary amino acid sequence for the first 15 amino-terminal residues of these light chains. $\text{5}\text{5}$ IgM cryoglobulins which react with human IgG had light chains of the variable region-III kappa chain subgroup (vK-III). $\text{4}\text{4}$ IgG and $\text{2}\text{2}$ IgM cryoglobulins with undefined antibody specificity had both lambda and kappa light chains none of which were vK-III. The data support the concept that there is marked restriction of the IgM anti-IgG antibody response to the IgG auto-antigen.     

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.     

Shared N-terminal sequences in monclonal IgMkappa and IgGkappa proteins from a patient with a complex multiple paraprotein disorder.

The N-terminal sequence analyses were performed on the heavy (H) and light (L) chains of the idiotypically identical IgM kappa and IgG kappa paraproteins isolated from the serum of patient, Cam. The N-terminal 39 residues of the kappa chains of the IgM and IgG were identical and belonged to the human V kappa III subgroup. This sequenced stretch included the first L chain hypervariable region. The N-terminal 27 residues of the variable regions (VH) of the respective mu and gamma heavy chains were also identical and belonged to the human VHIII subgroup. These identical VH sequences were unique with lysine residues at positions 13 and 19. This dual lysine substitution has not been seen in 37 other human VHIII sequences reported in the literature. This N-terminal sequence homology in the V-regions of Cam IgM kappa and IgG kappa paraproteins and the shared idiotypy expressed by Cam IgM, IgG, and IgA proteins strongly suggest the existence of complete structural homology in the variable regions of the and L chains of these Ig molecules of three separate Ig classes. At the cellular and genetic level, these results point toward a common clonal origin for the idiotypically related Ig molecules and suggest that identical V-region (VH and VL) genes were utilized by the Cam lymphoid clone in the biosynthesis of the respective IgM, IgC, and IgA proteins.     

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.     

Primary structures and chain dominance of anti-DNA antibodies

Using several anti-DNA autoantibodies, we analyzed the relative involvement of heavy and light chains in their interactions with DNA. We previously obtained eight hybridomas producing monoclonal anti-DNA autoantibodies by fusing spleen cells from an MRL-lpr/lpr mouse with myeloma cells. The chain dominance was analyzed by UV cross-linking experiments, in which the antibodies were covalently cross-linked with radioisotope-labeled oligonucleotides by short-wavelength UV-light, and the cross-linked H and L chains were analyzed by SDS-PAGE and densitometric scanning. Among these, three were found to be heavy chain dominant antibodies in which heavy chains are dominantly involved in DNA binding. The other five were co-dominant antibodies in which both heavy and light chains are involved in DNA binding. To determine the factor(s) that can explain the chain dominance in DNA binding, we determined the amino acid sequences of the variable regions of both heavy (VH) and light (VL) chains of all eight monoclonal antibodies. By analyzing the data, we were able to draw the following conclusions: (1) The arginine residues are found in the CDR3 regions of both VH and VL of the co-dominant antibodies; whereas, the same residues are found only in the CDR3s of VH, but not in VL, of the heavy chain dominant antibodies. (2) The net charges of the V regions affect the chain dominance. From the results of this study it is suggested that the presence of arginine residue in CDR3 is a critical factor in determining chain-dominance, as well as DNA binding of anti-DNA antibodies in general.     

The critical role of arginine residues in the binding of human monoclonal antibodies to cardiolipin

Previously we reported that the variable heavy chain region (VH) of a human beta2 glycoprotein I-dependent monoclonal antiphospholipid antibody (IS4) was dominant in conferring the ability to bind cardiolipin (CL). In contrast, the identity of the paired variable light chain region (VL) determined the strength of CL binding. In the present study, we examine the importance of specific arginine residues in IS4VH and paired VL in CL binding. The distribution of arginine residues in complementarity determining regions (CDRs) of VH and VL sequences was altered by site-directed mutagenesis or by CDR exchange. Ten different 2a2 germline gene-derived VL sequences were expressed with IS4VH and the VH of an anti-dsDNA antibody, B3. Six variants of IS4VH, containing different patterns of arginine residues in CDR3, were paired with B3VL and IS4VL. The ability of the 32 expressed heavy chain/light chain combinations to bind CL was determined by ELISA. Of four arginine residues in IS4VH CDR3 substituted to serines, two residues at positions 100 and 100 g had a major influence on the strength of CL binding while the two residues at positions 96 and 97 had no effect. In CDR exchange studies, VL containing B3VL CDR1 were associated with elevated CL binding, which was reduced significantly by substitution of a CDR1 arginine residue at position 27a with serine. In contrast, arginine residues in VL CDR2 or VL CDR3 did not enhance CL binding, and in one case may have contributed to inhibition of this binding. Subsets of arginine residues at specific locations in the CDRs of heavy chains and light chains of pathogenic antiphospholipid antibodies are important in determining their ability to bind CL.     

Amino acid sequence of the N-terminal 139 residues of light chain derived from a homogeneous rabbit antibody

The amino acid sequence of the N-terminal 139 residues of the L (light) chain derived from a homogeneous rabbit antibody (designated BS-1) to type III pneumococci was determined. A combination of methods involving tryptic cleavage restricted to the 2 arginine residues of the molecule and mild acid hydrolysis of a labile peptide bond between the V (variable) and C (constant) regions of the L chain (Fraser et al., 1972) allowed the isolation of two large peptides comprising the entire V region (residues 1-109); these peptides were suitable for automated Edman degradation. The complete sequence analysis of the V region was carried out with only 4mumol of L chain. This material was homogeneous, although minor variant sequences, if present at the 10% value, would not have been detected. The L chain contains 3 intrachain disulphide bridges, whose pairing was established by diagonal electrophoresis: there is one V-region bridge between positions 23 and 88 and one C-region bridge between positions 134 and 194; the third one connects V and C domains between positions 80 and 171. When compared with the basic sequence of human kappa chains, rabbit L chain BS-1 appears to be more similar to the V(KI) prototype sequence than to V(KII) or V(KIII) sequences, where V(KI), V(KII) and V(KIII) represent subgroups I, II and III respectively of V regions of kappa light chains. The V regions of rabbit heavy and light chains are homologous to each other. The presence of two clusters of 3 glycine residues in positions 94-96 and 99-101 respectively is remarkable. Residues 94-96 may be related to antibody complementarity whereas residues 99-101 function probably as a pivot permitting the combining region of the L chain to make optimal contact with the antigenic determinant (Wu & Kabat, 1970).     

Structural similarities in the kappa light chains of human rheumatoid factor paraproteins and serum immunoglobulins bearing a cross-reactive idiotype

The monoclonal antibody (MoAb) 17.109 recognizes a cross-reactive idiotype (CRI) associated with the light chains of Waldenstrom's macroglobulins with rheumatoid factor (RF) activity. The MoAb also reacts with a proportion of IgM-RF molecules from the sera of rheumatoid arthritis and primary Sjogren's syndrome patients, and from the sera of seropositive normal human subjects. In the present experiments, we used affinity chromatography to purify the 17.109 CRI-positive immunoglobulin from serum and have analyzed the isolated material by Western blotting. The purified 17.109 CRI-positive material from the sera of rheumatoid arthritis patients, Sjogren's syndrome patients, and normal subjects contained exclusively kappa light chains, and had demonstrated RF activity. In every case the 17.109 CRI-positive isolates reacted with antibodies against synthetic peptides corresponding to both the conserved second and third complementarity-determining regions (CDR) of the monoclonal kappa IgM-RF paraprotein Sie. The binding was inhibited specifically by the free peptides in solution. The antipeptide antibodies did not react appreciably with unfractionated human immunoglobulin. The data establish that the 17.109 CRI-positive immunoglobulin from diverse human sera have similar or identical second and third light chain CDR. These results suggest i) that the MoAb 17.109 identifies the protein product of a single or a very few V kappa genes, ii) that the ability to make kappa light chains with the 17.109-associated variable region is widespread in the human population, and iii) that the 17.109-defined kappa variable region segment is associated with IgM-RF autoantibodies.     

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.     

The majority of human monoclonal IgM rheumatoid factors express a "primary structure-dependent" cross-reactive idiotype

Genetic studies of human immunoglobulin variable regions have been hampered by the lack of anti-idiotypic antibodies that recognize specific heavy and light chain variable region sequences. Sixty percent of human monoclonal IgM anti-IgG autoantibodies (rheumatoid factors [RF]) from unrelated individuals share a cross-reactive idiotype (CRI) termed Wa. In previous experiments in which we used an enzyme-linked immunosorbent assay, we reported that a synthetic peptide (PSL2), corresponding to the second hypervariable region in the kappa light chain of a monoclonal IgM-RF (Sie), induced rabbit antibodies reactive with several RF paraproteins. In the present experiments, to avoid interference due to the human IgM-RF binding toward rabbit IgG, the reactivity of the anti-PSL2 antibody to the separated heavy and light chains of multiple IgM proteins and Bence-Jones proteins was assessed by the Western blot technique. The PSL2-induced anti-CRI reacted well with the separated kappa chains from 10 out of 12 IgM-RF, zero out of four light chains from IgM proteins lacking anti-IgG activity, and one out of six kappa Bence-Jones proteins. The results show that the PSL2-CRI is associated with RF and is not a kappa subgroup marker. Furthermore, a comparison of the reported light chain sequences of the PSL2-CRI-positive IgM-RF suggests that the majority of human IgM-RF light chains derive from a single germ-line VK gene or from a family of closely related VK genes that is highly conserved in the human population. Synthetic peptide-induced anti-CRI provide a potent tool for analyzing the genetic basis of CRI and abnormal autoantibody production in humans.     

Three-dimensional structure of an Fv from a human IgM immunoglobulin.

An IgM(kappa) immunoglobulin from a patient (Pot) with Waldenstrom's macroglobulinemia was hydrolyzed with pepsin to release a fragment consisting of the 'variable' (V) domains of the light and heavy chains plus eight residue 'tails' from the 'constant' (C) domains. The crystal structure of this fragment was determined at 2.3 A resolution by molecular replacement and crystallographic refinement methods. When examined separately, the light chain component closely resembles another human kappa chain (Rei) in both the beta-pleated sheet regions and the 'hypervariable' loops. The conserved pleated sheets in the heavy chain are similar to those in the human Kol IgG1 protein, but the third hypervariable loop in particular is different from that in any immunoglobulin structure described to date. As in the Kol protein, this loop blocks the access to any internal active site along the light-heavy chain interface. Unlike the Kol protein, however, the loop does not protrude beyond the boundaries of a conventional antigen combining site. Instead, it forms a very compact structure, which fills almost all residual space between the domains. This is an example of one dominant complementarity-determining region (CDR) essentially negating the diversity possible with five other CDRs in the two chains. Ordered water molecules are associated with light chain constituents along the interface, but not with CDR3 of the heavy chain. In screening exercises the Pot IgM failed to bind a wide variety of peptides. Together, the results suggest that ligand binding can only occur on external surfaces of the protein. These surfaces carry a limited number of side chains usually assigned to CDRs in more typical antibodies.     

The limited diversity of the mouse gamma-chains anti-GAT repertoire does not seem to be noticeably amplified upon class switch

NH2-terminal sections of H and L chains isolated from five monoclonal anti-GAT antibodies derived from BALB/c mice have been sequenced upon to residue 43. Four among these five antibodies, sharing similar public idiotypic determinants, possess extremely conserved sequences, both for the H, which is apparented to the VH II type, and the L chains, which belong to the V kappa I subgroup. VH sequences are identical up to residue 43 and contain the common sequences (residues 1 to 32) defined for the H chains derived from the DBA/2 IgM anti-GAT monoclonal antibodies. Light chains are also remarkably conserved, a rather unusual situation for kappa-chains. The fifth antibody that expresses only part of the public idiotypic determinants contains very distinctive H and L chains. Its heavy chains are close to the VH I subgroup, whereas its kappa-chains permit definition of a new V kappa subgroup. The repertoire appears to be highly conserved between BALB/c and DBA/2 mice, and does not seem larger in IgG than in IgM antibodies. This latter observation does not speak in favor of a switch-linked amplification of diversity.     

Sequences of variable regions of hybridoma antibodies to alpha (1----6) dextran in BALB/c and C57BL/6 mice

The variable region sequences of light and heavy chains of three hybridoma antibodies to alpha (1----6) dextran, two from BALB/c and one from C57BL/6 mice, were determined by cloning and sequencing their cDNA. The three kappa-light chains are identical in nucleotide and amino acid sequences, except for the use of different J by BALB/c and C57BL/6; all three had the germ-line sequence of antibodies to 2-phenyloxazolone (20). Nevertheless, 2-phenyloxazolone BSA did not cross-react in gel with antidextrans, nor did dextran react with anti-2-phenyloxazolone ascitic fluids. The heavy chains differed, the BALB/c hybridomas having only three amino acid differences in CDR2 and two in CDR3; the C57BL/6 hybridoma differed throughout the variable region. All three VH are members of the J558 family. The three identical V kappa sequences suggest a significant role in dextran binding, with the differences in CDR of VH and the various J mini-genes of VL and VH being responsible for only fine differences in specificity. Alternatively, the role of V kappa might be minor, with most of the complementarity ascribable to VH. Additional sequences are needed to evaluate whether these data are typical of the repertoire of anti-alpha (1----6) dextran-combining sites.