Light Chain Complementarity-determining Regions Research Articles

Chronic lymphocytic leukemia antibodies with a common stereotypic rearrangement recognize nonmuscle myosin heavy chain IIA

AbstractLeukemic B lymphocytes of a large group of unrelated chronic lymphocytic leukemia (CLL) patients express an unmutated heavy chain immunoglobulin variable (V) region encoded by IGHV1-69, IGHD3-16, and IGHJ3 with nearly identical heavy and light chain complementarity-determining region 3 sequences. The likelihood that these patients developed CLL clones with identical antibody V regions randomly is highly improbable and suggests selection by a common antigen. Monoclonal antibodies (mAbs) from this stereotypic subset strongly bind cytoplasmic structures in HEp-2 cells. Therefore, HEp-2 cell extracts were immunoprecipitated with recombinant stereotypic subset-specific CLL mAbs, revealing a major protein band at approximately 225 kDa that was identified by mass spectrometry as nonmuscle myosin heavy chain IIA (MYHIIA). Reactivity of the stereotypic mAbs with MYHIIA was confirmed by Western blot and immunofluorescence colocalization with anti-MYHIIA antibody. Treatments that alter MYHIIA amounts and cytoplasmic localization resulted in a corresponding change in binding to these mAbs. The appearance of MYHIIA on the surface of cells undergoing stress or apoptosis suggests that CLL mAb may generally bind molecules exposed as a consequence of these events. Binding of CLL mAb to MYHIIA could promote the development, survival, and expansion of these leukemic cells.

Broadening of Neutralization Activity to Directly Block a Dominant Antibody-Driven SARS-Coronavirus Evolution Pathway

Phylogenetic analyses have provided strong evidence that amino acid changes in spike (S) protein of animal and human SARS coronaviruses (SARS-CoVs) during and between two zoonotic transfers (2002/03 and 2003/04) are the result of positive selection. While several studies support that some amino acid changes between animal and human viruses are the result of inter-species adaptation, the role of neutralizing antibodies (nAbs) in driving SARS-CoV evolution, particularly during intra-species transmission, is unknown. A detailed examination of SARS-CoV infected animal and human convalescent sera could provide evidence of nAb pressure which, if found, may lead to strategies to effectively block virus evolution pathways by broadening the activity of nAbs. Here we show, by focusing on a dominant neutralization epitope, that contemporaneous- and cross-strain nAb responses against SARS-CoV spike protein exist during natural infection. In vitro immune pressure on this epitope using 2002/03 strain-specific nAb 80R recapitulated a dominant escape mutation that was present in all 2003/04 animal and human viruses. Strategies to block this nAb escape/naturally occurring evolution pathway by generating broad nAbs (BnAbs) with activity against 80R escape mutants and both 2002/03 and 2003/04 strains were explored. Structure-based amino acid changes in an activation-induced cytidine deaminase (AID) “hot spot” in a light chain CDR (complementarity determining region) alone, introduced through shuffling of naturally occurring non-immune human VL chain repertoire or by targeted mutagenesis, were successful in generating these BnAbs. These results demonstrate that nAb-mediated immune pressure is likely a driving force for positive selection during intra-species transmission of SARS-CoV. Somatic hypermutation (SHM) of a single VL CDR can markedly broaden the activity of a strain-specific nAb. The strategies investigated in this study, in particular the use of structural information in combination of chain-shuffling as well as hot-spot CDR mutagenesis, can be exploited to broaden neutralization activity, to improve anti-viral nAb therapies, and directly manipulate virus evolution.

Complex of a Protective Antibody with Its Ebola Virus GP Peptide Epitope: Unusual Features of a Vλ x Light Chain

13F6-1-2 is a murine monoclonal antibody that recognizes the heavily glycosylated mucin-like domain of the Ebola virus virion-attached glycoprotein (GP) and protects animals against lethal viral challenge. Here we present the crystal structure, at 2.0 Å, of 13F6-1-2 in complex with its Ebola virus GP peptide epitope. The GP peptide binds in an extended conformation, anchored primarily by interactions with the heavy chain. Two GP residues, Gln P406 and Arg P409, make extensive side-chain hydrogen bond and electrostatic interactions with the antibody and are likely critical for recognition and affinity. The 13F6-1-2 antibody utilizes a rare Vλ x light chain. The three light-chain complementarity-determining regions do not adopt canonical conformations and represent new classes of structures distinct from Vκ and other Vλ light chains. In addition, although Vλ x had been thought to confer specificity, all light-chain contacts are mediated through germ-line-encoded residues. This structure of an antibody that protects against the Ebola virus now provides a framework for humanization and development of a postexposure immunotherapeutic.

Structure of the Malaria Antigen AMA1 in Complex with a Growth-Inhibitory Antibody

Identifying functionally critical regions of the malaria antigen AMA1 (apical membrane antigen 1) is necessary to understand the significance of the polymorphisms within this antigen for vaccine development. The crystal structure of AMA1 in complex with the Fab fragment of inhibitory monoclonal antibody 1F9 reveals that 1F9 binds to the AMA1 solvent-exposed hydrophobic trough, confirming its importance. 1F9 uses the heavy and light chain complementarity-determining regions (CDRs) to wrap around the polymorphic loops adjacent to the trough, but uses a ridge of framework residues to bind to the hydrophobic trough. The resulting 1F9-AMA1–combined buried surface of 2,470 Å2 is considerably larger than previously reported Fab–antigen interfaces. Mutations of polymorphic AMA1 residues within the 1F9 epitope disrupt 1F9 binding and dramatically reduce the binding of affinity-purified human antibodies. Moreover, 1F9 binding to AMA1 is competed by naturally acquired human antibodies, confirming that the 1F9 epitope is a frequent target of immunological attack.

Autoantibodies, lupus and the science of sabotage.

Anti-double-stranded DNA antibodies (anti-dsDNA) and antiphospholipid antibodies (APL) are important in the pathogenesis of systemic lupus erythematosus (SLE) and the antiphospholipid syndrome (APS) respectively. Not all anti-dsDNA or APL antibodies can cause clinical effects. Those that are particularly likely to cause tissue damage tend to be of IgG isotype and to possess particular binding properties. Rigorous statistical analysis of published sequences of human monoclonal anti-DNA and APL antibodies showed that IgG antibodies with binding properties characteristic of pathogenicity tend to have multiple somatic mutations in their variable regions. The distribution of these mutations suggests that they have been selected by antigen. This leads to accumulation of certain residues at the antigen-binding sites of these antibodies. Arginine residues are especially important. A computer-generated model of the pathogenic human monoclonal anti-DNA antibody B3 predicted that arginines in the heavy and light chain complementarity-determining regions (CDRs) would interact with dsDNA. We expressed cloned sequences encoding the B3 heavy and light chains in vitro to produce whole IgG. The cloned sequences of the heavy and light chains were manipulated to express a range of variant IgG antibodies. Binding assays on the expressed antibodies showed that altering specific arginine residues reduced binding to dsDNA in a way consistent with computer generated structural models. Changing the pattern of somatic mutations in the light chain altered binding to both dsDNA and histones, but in different ways. A single arginine-to-serine mutation in light-chain CDR1 of B3 reduced binding to both those antigens and may also have reduced the pathogenicity of the expressed antibodies in severe combined immunodeficiency (SCID) mice. Monoclonal human APL were expressed using the same system. Nineteen different heavy-light combinations were expressed. The ability to bind cardiolipin correlated well with the presence of exposed arginine residues in the heavy- and light-chain CDRs. The heavy chain of the pathogenic APL antibody IS4 contains four exposed arginines in CDR3. The results of mutagenesis studies suggested that two of these promote binding to cardiolipin whereas the other two have no such effect.

Structure of an anti-HIV monoclonal Fab antibody fragment specific to a gp120 C-4 region peptide.

Structure of an anti-HIV monoclonal Fab antibody fragment specific to a gp120 C-4 region peptide.

Sequence analysis of the immunoglobulin antigen receptor of hepatitis C virus–associated non-Hodgkin lymphomas suggests that the malignant cells are derived from the rheumatoid factor–producing cells that occur mainly in type II cryoglobulinemia

Analysis of the immunoglobulin receptor (IGR) variable heavy- and light-chain sequences on 17 hepatitis C virus (HCV)-associated non-Hodgkin lymphomas (NHLs) (9 patients also had type II mixed cryoglobulinemia [MC] syndrome and 8 had NHL unrelated to MC) and analysis of intraclonal diversity on 8 of them suggest that such malignant lymphoproliferations derive from an antigen-driven pathologic process, with a selective pressure for the maintenance of a functional IgR and a negative pressure for additional amino acid mutations in the framework regions (FRs). For almost all NHLs, both heavy- and light-chain complementarity-determining regions (CDR3) showed the highest similarity to antibodies with rheumatoid factor (RF) activity that have been found in the MC syndrome, thus suggesting that a common antigenic stimulus is involved in MC syndrome and in HCV-associated lymphomagenesis. Moreover, because HCV is the recognized pathologic agent of MC and the CDR3 amino acid sequences of some HCV-associated NHLs also present a high homology for antibody specific for the E2 protein of HCV, it may be reasonable to speculate that HCV E2 protein is one of the chronic antigenic stimuli involved in the lymphomagenetic process. Finally, the use of specific segments, in particular the D segment, in assembling the IgH chain of IgR seems to confer B-cell disorders with the property to produce antibody with RF activity, which may contribute to the manifestation of an overt MC syndrome.

Sequence analysis of the immunoglobulin antigen receptor of hepatitis C virus–associated non-Hodgkin lymphomas suggests that the malignant cells are derived from the rheumatoid factor–producing cells that occur mainly in type II cryoglobulinemia

AbstractAnalysis of the immunoglobulin receptor (IGR) variable heavy- and light-chain sequences on 17 hepatitis C virus (HCV)-associated non-Hodgkin lymphomas (NHLs) (9 patients also had type II mixed cryoglobulinemia [MC] syndrome and 8 had NHL unrelated to MC) and analysis of intraclonal diversity on 8 of them suggest that such malignant lymphoproliferations derive from an antigen-driven pathologic process, with a selective pressure for the maintenance of a functional IgR and a negative pressure for additional amino acid mutations in the framework regions (FRs). For almost all NHLs, both heavy- and light-chain complementarity-determining regions (CDR3) showed the highest similarity to antibodies with rheumatoid factor (RF) activity that have been found in the MC syndrome, thus suggesting that a common antigenic stimulus is involved in MC syndrome and in HCV-associated lymphomagenesis. Moreover, because HCV is the recognized pathologic agent of MC and the CDR3 amino acid sequences of some HCV-associated NHLs also present a high homology for antibody specific for the E2 protein of HCV, it may be reasonable to speculate that HCV E2 protein is one of the chronic antigenic stimuli involved in the lymphomagenetic process. Finally, the use of specific segments, in particular the D segment, in assembling the IgH chain of IgR seems to confer B-cell disorders with the property to produce antibody with RF activity, which may contribute to the manifestation of an overt MC syndrome.

Study of B72.3 combining sites by molecular modeling and site-directed mutagenesis.

A B72.3 Fab/sTn(2) complex was modeled from the known structure of B72.3 Fab and the dimeric Tn-serine cluster (sTn(2)). In the complex model, the side chains of 15 heavy- and light-chain complementarity-determining region (CDR) residues and the main chains of two light-chain CDR residues contact the sTn(2) epitope. Among 15 CDR residues which contact sTn(2) in the model, two heavy-chain residues (Ser95 and Tyr97) and light-chain CDR residue (Tyr96) have been confirmed in a previous study. To test the accuracy of the computational model, further site-directed mutagenesis was performed by alanine scanning on the remaining 12 residues that are predicted in the model to have side-chain interactions with sTn(2). Of these 12 mutants, eight that are all from the heavy-chain (His32Ala, Ala33Leu, Tyr50Ala, Ser52Ala, Asn52Ala, Asp56Ala, Lys58Ala and Tyr96Ala) had significantly reduced sTn(2) affinities, and four consisting of three light-chain mutations (Asn32Ala, Trp92Ala and Thr94Ala) and one heavy-chain mutation (His35Ala) retained wild-type sTn(2) affinity. On the whole, this evidence suggests that the complex model, although not perfect, is correct in many of its features. In a more general vein, these results lend credibility to the computational modeling approach for the study of the molecular basis of antigen-antibody complexes.

Antibody binding defines a structure for an epitope that participates in the PrPC → PrPSc conformational change

The X-ray crystallographic structures of the anti-Syrian hamster prion protein (SHaPrP) monoclonal Fab 3F4 alone, as well as the complex with its cognate peptide epitope (SHaPrP 104–113), have been determined to atomic resolution. The conformation of the decapeptide is an Ω-loop. There are substantial alterations in the antibody combining region upon epitope binding. The peptide binds in a U-shaped groove on the Fab surface, with the two specificity determinants, Met109 and Met112, penetrating deeply into separate hydrophobic cavities formed by the heavy and light chain complementarity-determining regions. In addition to the numerous contacts between the Fab and the peptide, two intrapeptide hydrogen bonds are observed, perhaps indicating the structure bound to the Fab exists transiently in solution. This provides the first structural information on a portion of the PrP N-terminal region observed to be flexible in the NMR studies of SHPrP 90–231, SHaPrP 29–231 and mouse PrP 23–231. Antibody characterization of the antigenic surfaces of PrPC and PrPSc identifies this flexible region as a component of the conformational rearrangement that is an essential feature of prion disease.

Antiendotoxin Agents Share Molecular Homology within Their Lipopolysaccharide Binding Domains

Background. The purpose of this study was to determine whether antiendotoxin agents exhibit molecular homology within their lipopolysaccharide (LPS) binding domains, suggesting a common mechanism of action. We hypothesized that the presence of positively charged basic amino acids or a paucity of negatively charged acidic amino acids, or both, would be a critical characteristic of that portion of the molecule that binds to the highly negatively charged deep core/lipid A (DCLA) region of LPS.Materials and methods. We analyzed the amino acid sequences of the variable light (VL) and heavy (VH) chain complementarity-determining regions (CDRs) of anti-DCLA monoclonal antibodies (mAbs) 1B6, 5A5, and 7C5 and compared them with (1) the CDRs of three irrelevant control mAbs and (2) the LPS binding region of bactericidal permeability-increasing protein (BPI). We purified and amplified the specific nucleotide sequences of the variable regions using reverse transcriptase polymerase chain reaction. DNA was sequenced by dideoxy termination, and protein sequences were deduced and analyzed. The percentages of acidic, basic, polar, and hydrophobic amino acids within VH and VL chain CDRs were determined.Results. We identified a paucity of negatively charged acidic amino acids exclusively within VL chain CDRs of anti-DCLA mAbs (P < 0.005). Although increased, the number of positively charged basic residues was not statistically significantly different; neither was the number of polar or hydrophobic amino acids.Conclusions. Our data suggest that the near absence of negatively charged acidic residues is critical for LPS binding. This characteristic appears to reside exclusively in the VL chain CDRs of anti-DCLA mAbs.

Rheumatoid Factor Specificity of a VH3-Encoded Antibody Is Dependent on the Heavy Chain CDR3 Region and Is Independent of Protein A Binding

Rheumatoid factors (RF) recognize conformational determinants located within the Fc portion of IgG. By analyzing a panel of monoclonal rheumatoid arthritis (RA)-derived RFs, we previously demonstrated that the somatically generated light chain complementarity-determining region 3 (CDR3) contributes to RF specificity. We have now generated a panel of heavy chain mutants of the B'20 Ab, a high affinity RA-derived IgM RF. B'20 also binds avidly to protein A and weakly to ssDNA and tetanus toxoid. B9601, a RF negative Ab that is highly homologous to B'20 but does not bind any of the Ags tested, and RC1, a low affinity polyreactive RF, were used to generate heavy chain mutants with framework (FR) and CDR switches. The mutated heavy chains were cotransfected into a myeloma cell line with the germline counterpart of the B'20 light chain, and the expressed Ig tested for antigenic specificity. We show that both RF specificity and polyreactivity of B'20 is dependent on its unique heavy chain CDR3 region. Replacement with a B9601 CDR3 shortened to the same length as the B'20 CDR3, and with only 5 amino acid differences, did not restore Fc binding. Conversely, absence of protein A binding of B9601 is due to the presence of a serine residue at position 82a in the B9601 heavy chain FR3 region. Together, our data suggest that Ig gene recombination events can generate B cells with autoantibody specificities in the preimmune repertoire. Abnormal release, activation, expansion, or mutation of such cells might all contribute to the generation of a high titer RF response in patients with RA.

Stepwise in vitro affinity maturation of Vitaxin, an alphav beta3-specific humanized mAb.

A protein engineering strategy based on efficient and focused mutagenesis implemented by codon-based mutagenesis was developed. Vitaxin, a humanized version of the antiangiogenic antibody LM609 directed against a conformational epitope of the alphav beta3 integrin complex, was used as a model system. Specifically, focused mutagenesis was used in a stepwise fashion to rapidly improve the affinity of the antigen binding fragment by greater than 90-fold. In the complete absence of structural information about the Vitaxin-alphav beta3 interaction, phage-expressed antibody libraries for all six Ig heavy and light chain complementarity-determining regions were expressed and screened by a quantitative assay to identify variants with improved binding to alphav beta3. The Vitaxin variants in these libraries each contained a single mutation, and all 20 amino acids were introduced at each complementarity-determining region residue, resulting in the expression of 2,336 unique clones. Multiple clones displaying 2- to 13-fold improved affinity were identified. Subsequent expression and screening of a library of 256 combinatorial variants of the optimal mutations identified from the primary libraries resulted in the identification of multiple clones displaying greater than 50-fold enhanced affinity. These variants inhibited ligand binding to receptor more potently as demonstrated by inhibition of cell adhesion and ligand competition assays. Because of the limited mutagenesis and combinatorial approach, Vitaxin variants with enhanced affinity were identified rapidly and required the synthesis of only 2,592 unique variants. The use of such small focused libraries obviates the need for phage affinity selection approaches typically used, permitting the use of functional assays and the engineering of proteins expressed in mammalian cell culture.

Design and construction of a hybrid immunoglobulin domain with properties of both heavy and light chain variable regions.

The complementarity-determining regions (CDRs) of a human kappa light chain were replaced with CDRs from a murine gamma-1 heavy chain and, by use of molecular modeling, key heavy chain framework residues were identified and thus included to preserve the native conformation of the heavy chain CDRs. Co-expression of this hybrid human kappa chain (V[HB]C[L]) with a human kappa chain counterpart (V[L]C[L], engineered to contain murine light chain CDRs) resulted in the secretion of high levels of a heterodimeric protein (V[HB]C[L]::V[L]C[L]) termed 'kappabody'. This protein also had equivalent affinity for antigen as the Fab' of the parent murine IgG1. High-level secretion was also observed for the hybrid chain as homodimers (V[HB]C[L]::V[HB]C[L]), which is not observed for chimeric chains consisting of a heavy chain variable region and light chain constant region, i.e. V[H]C[L] homodimers or single chains are not secreted. This indicates that regions within the variable domain, required for secretion of light chains, reside outside of the hypervariable regions (CDRs) and that the heavy chain CDRs and supporting residues do not prevent secretion. These results demonstrate the possibility of designing small, single-domain molecules possessing a given binding activity which may be secreted at high levels from mammalian cells.

Characterization of horse (Equus caballus) immunoglobulin mu chain-encoding genes.

Horse (Equus caballus) immunoglobulin mu chain-encoding (IgM) variable, joining, and constant gene segments were cloned and characterized. Nucleotide sequence analyses of 15 cDNA clones from a mesenteric lymph node library identified 7 unique variable gene segments, 5 separate joining segments, and a single constant region. Based on comparison with human sequences, horse variable segments could be grouped into either family 1 of immunoglobulin (Ig) clan I or family 4 of Ig clan II subclan IV. All horse sequences had a relatively conserved 16 base pair (bp) segment in framework 3 which was recognized with high specificity in polymerase chain reaction by a degenerate oligonucleotide primer. Horse complementarity determining regions (CDR) had considerable variability in predicted amino acid content and length but also included the presence of relatively conserved residues and several canonical sequences that may be necessary in formation of the beta chain main structure and conformation of antigen-binding sites through interaction with light chain CDR. Sequence analysis of joining regions revealed the presence of nearly invariant 3' regions similar to those found in human and mouse genes. A single horse IgM constant region comprising 1472 bp and encoding 451 residues was also identified. Direct comparison of the horse constant region predicted amino acid sequence with those from eleven other species revealed the presence of 53 invariant residues with particularly conserved sequences within the third and fourth exons. Phylogenetic analysis using a neighbor-joining algorithm showed closest similarity of the horse mu chain-encoding constant region gene to human and dog sequences. Together, these findings provide insights into the comparative biology of IgM as well as data for additional detailed studies of the horse immune system and investigation of immune-related diseases.

1.85 A structure of anti-fluorescein 4-4-20 Fab.

The crystal complex of fluorescein bound to the high-affinity anti-fluorescein 4-4-20 Fab (Ka = 10(10) M-1 at 2 degrees C) has been determined at 1.85 A. Isomorphous crystals of two isoelectric forms (pI = 7.5 and 7.9) of the anti-fluorescein 4-4-20 Fab, an IgG2A [Gibson et al. (1988) Proteins: Struct. Funct. Genet., 3, 155-160], have been grown. Both complexes crystallize with one molecule in the asymmetric unit in space group P1, with a = 42.75 A, b = 43.87 A, c = 58.17 A, alpha = 95.15 degrees, beta = 86.85 degrees and gamma = 98.01 degrees. The final structure has an R value of 0.188 at 1.85 A resolution. Interactions between bound fluorescein, the complementarity-determining regions (CDRs) of the Fab and the active-site mutants of the 4-4-20 single-chain Fv will be discussed. Differences were found between the structure reported here and the previously reported 2.7 A 4-4-20 Fab structure [Herron et al. (1989) Proteins: Struct. Funct. Genet., 5, 271-280]. Our structure determination was based on 26,328 unique reflections--four times the amount of data used in the previous report. Differences in the two structures could be explained by differences in interpreting the electron density maps at the various resolutions. The r.m.s. deviations between the variable and constant domains of the two structures were 0.77 and 1.54 A, respectively. Four regions of the light chain and four regions of the heavy chain had r.m.s. backbone deviations of > 4 A. The most significant of these was the conformation of the light chain CDR 1.

X-ray Structures of the Antigen-binding Domains from Three Variants of Humanized anti-p185 HER2 Antibody 4D5 and Comparison with Molecular Modeling

The X-ray structures of 1 Fv and 2 Fab humanized anti-p185 HER2 antibody fragments (IgG 1-κ) have been determined at a resolution between 2·7 Å and 2·2 Å. The antibodies are three different versions of a human antibody framework onto which the antigen recognition loops from a murine antibody (4D5) have been grafted. The sequences of the three versions differ in the framework region at positions L55, H78 and H102. The version 8 Fv fragment crystallizes in space group P 2 1 with cell parameter a= 37·6 A, b = 63·4 Å, c = 90·2 Å, β = 98·2°, with two molecules per asymmetric unit, and has refined against data 10·0 Å-2·5 Å resolution to an R-factor of 17·9%. Version 7 has been refined against data 10 Å-2·7 Å to an R-factor of 17·1%. The X-ray structures have been used to assess the accuracy of structural predictions made via molecular modeling, and they confirm the structural role of certain framework residues and the conformations of five of six complementarity determining regions (CDRs). The average deviation of the model from the X-ray structures is within the range observed among the X-ray structures for 81% of the C α atoms. Of the hydrogen bonds common to the X-ray structures, 94% of he main-chain-main-chain and 79% of the main-chain-side-chain ones were predicted by the model. The side-chain conformation was predicted correctly for 79% of the buried residues. The third CDR in the heavy chain is variable, differing by up or 8 Å between molecules within an asymmetric unit. The structural relationship between variable domains of light and heavy chains is not significantly altered by the absence of constant domains in the Fv molecule. The antigen-binding potential of an unusual light chain sequence has been confirmed. The arginine at position 66 interacts with the first light chain CDR, but in a fashion somewhat different than predicted. A substitution of a leucine for an alanine side-chain directed between the β-sheets has only relatively small and local effects. There is no direct contact between tyrosines at positions 55 of the light chain and 102 of the heavy chain of version 8, despite their proximity and the synergism seen in their effects on binding affinity and biological activity.

Crystal structure of a streptococcal protein G domain bound to an Fab fragment.

Protein G is a cell-surface protein from Streptococcus which binds to IgG molecules from a wide range of species with an affinity comparable to that of antigen. The high affinity of protein G for the Fab portion of IgG poses a particular challenge in molecular recognition, given the variability of heavy chain subclass, light chain type and complementarity-determining regions. Here we report the crystal structure of a complex between a protein G domain and an immunoglobulin Fab fragment. An outer beta-strand in the protein G domain forms an antiparallel interaction with the last beta-strand in the constant heavy chain domain of the immunoglobulin, thus extending the beta-sheet into the protein G. The interaction between secondary structural elements in Fab and protein G provides an ingenious solution to the problem of maintaining a high affinity for many different IgG molecules. The structure also contrasts with Fab-antigen complexes, in which all contacts with antigen are mediated by the variable regions of the antibody, and to our knowledge provides the first details of interaction of the constant regions of Fab with another protein.

Selection of immunoglobulin variable regions in autoimmunity to DNA.

Results from our analyses of variable region gene usage among spontaneous anti-DNA antibodies in autoimmune mice have indicated that both the early IgM and later-appearing IgG autoantibodies to DNA are generated by clonally selected B cells. The recurrent usage of particular variable region genes among all the anti-DNA hybridomas analyzed and reported to date supports this hypothesis. The preferential expression of particular light and heavy chain variable region genes among selected populations of both IgM and IgG anti-DNA hybridomas likewise supports the hypothesis. Both IgM and IgG antibody-producing B cells are derived from the same clonal precursor population and may be derived from the same B cell clonal precursor within an individual mouse. The selective and recurrent expression of germline and somatically-derived structures that would be expected to promote protein binding to DNA within anti-DNA antibody variable regions, particularly arginines in both light and heavy chain complementarity-determining regions, indicates that DNA or DNA-containing complexes may be the antigen that stimulates anti-DNA antibody in autoimmune mice. The progressive increase in the specificity of spontaneous anti-DNA antibodies for native DNA as the autoimmune response matures from IgM to IgG likewise suggests that DNA may be the antigenic stimulus for spontaneous anti-DNA in autoimmune mice. A hypothetical, computer-generated model of anti-DNA antibody binding to DNA provides an interesting paradigm for the molecular basis of antibody specificity for DNA.

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.