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Results for “Complementarity Determining Regions”

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PubMed2001

The diversity of rearranged immunoglobulin heavy chain variable region genes in peripheral blood B cells of preterm infants is restricted by short third complementarity-determining regions but not by limited gene segment usage.

The immunoglobulin diversity is restricted in fetal liver B cells. This study examined whether peripheral blood B cells of extremely preterm infants show similar restrictions (overrepresentation of some gene segments, short third complementarity-determining regions [CDR3]). DNA of rearranged immunoglobulin heavy chain genes was amplified by polymerase chain reaction, cloned, and sequenced. A total of 417 sequences were analyzed from 6 preterm infants (25-28 weeks of gestation), 6 term infants, and 6 adults. Gene segments from the entire V(H) and D(H) gene locus were rearranged in preterm infants, even though the D(H)7-27 segment was overrepresented (17% of rearrangements) compared to term infants (7%) and adults (2%). CDR3 was shorter in preterm infants (40 +/- 10 nucleotides) than in term infants (44 +/- 12) and adults (48 +/- 14) (P <.001) due to shorter N regions. Somatic mutations were exclusively found in term neonates and adults (mutational frequency 0.8% and 1.8%). We conclude that preterm infants have no limitations in gene segment usage, whereas the diversity of CDR3 is restricted throughout gestation.

Adult↗

PubMed2003

Selection of Ig mu heavy chains by complementarity-determining region 3 length and amino acid composition.

Although it is generally accepted that Ig heavy chains (HC) are selected at the pre-B cell receptor (pre-BCR) checkpoint, the characteristics of a functional HC and the role of pre-BCR assembly in their selection have remained elusive. We determined the characteristics of HCs that successfully passed the pre-BCR checkpoint by examining transcripts harboring V(H)81X and J(H)4 gene segments from J(H)(+/-) and lambda5(-/-)mice. V(H)81X-J(H)4-HC transcripts isolated from cells before or in the absence of pre-BCR assembly had no distinguishing complementarity-determining region 3 traits. In contrast, transcripts isolated subsequent to passage through the pre-BCR checkpoint had distinctive complementarity-determining regions 3 of nine amino acids in length (49%) and a histidine at position 1 (73%). Hence, our data define specific structural requirements for a functional HC, which is instrumental in shaping the diverse B cell repertoire.

Amino Acids↗

PubMed1994

Heterogeneity of T-cell receptor alpha-chain complementarity-determining region 3 in myelin basic protein-specific T cells increases with severity of multiple sclerosis.

The pathogenesis of multiple sclerosis (MS) is thought to involve a T-cell-mediated autoimmune process. Experimental allergic encephalomyelitis (EAE), an animal model resembling MS, can be induced by immunization with myelin antigens such as myelin basic protein. The T-cell antigen receptor (TCR) usage in EAE is highly restricted in some strains of animals and experimental treatments targeting the TCR have been successful in EAE. Examination of the TCR beta-chain variable-region (V beta) usage of MBP-specific T-cell lines in MS patients has produced conflicting results. Our previous studies of TCR alpha-chain variable-region usage in monozygotic twins demonstrated a general skewing of the TCR repertoire in individuals with MS. This skewing became apparent only after stimulation with antigens; in peripheral blood lymphocyte preparations from individuals with MS V alpha 8-bearing T cells were preferentially selected by stimulation with myelin basic protein. In the present study we examined complementarity-determining region 3 of those V alpha 8-positive TCRs. Marked sequence heterogeneity was found in all individuals with severe MS. In contrast, restricted areas of complementarity-determining region 3 were found in healthy control individuals and in individuals with a mild form of MS. Sequences from tetanus toxoid-specific V alpha 8-positive T cells generated from the same individuals were relatively homogeneous within individuals regardless of disease activity and were distinct from the sequences of complementarity-determining region 3 in myelin basic protein-stimulated lines. These findings suggest that disease severity may be associated with increased heterogeneity of myelin antigen-specific T cells and could reflect an impaired ability of the immune system to down-regulate these anti-self responses.

Adult↗

PubMed2004

Phage-displayed antibody libraries of synthetic heavy chain complementarity determining regions.

A structure-based approach was used to design libraries of synthetic heavy chain complementarity determining regions (CDRs). The CDR libraries were displayed as either monovalent or bivalent single-chain variable fragments (scFvs) with a single heavy chain variable domain scaffold and a fixed light chain variable domain. Using the structure of a parent antibody as a guide, we restricted library diversity to CDR positions with significant exposure to solvent. We introduced diversity with tailored degenerate codons that ideally only encoded for amino acids commonly observed in natural antibody CDRs. With these design principles, we reasoned that we would produce libraries of diverse solvent-exposed surfaces displayed on stable scaffolds with minimal structural perturbations. The libraries were sorted against a panel of proteins and yielded multiple unique binding clones against all six antigens tested. The bivalent library yielded numerous unique sequences, while the monovalent library yielded fewer unique clones. Selected scFvs were converted to the Fab format, and the purified Fab proteins retained high affinity for antigen. The results support the view that synthetic heavy chain diversity alone may be sufficient for the generation of high-affinity antibodies from phage-displayed libraries; thus, it may be possible to dispense with the light chain altogether, as is the case in natural camelid immunoglobulins.

Amino Acid Sequence↗

PubMed2002

Heavy chain V region diversity in the duck-billed platypus (Ornithorhynchus anatinus): long and highly variable complementarity-determining region 3 compensates for limited germline diversity.

In this work, to study the emergence of the H chain V region repertoire during mammalian evolution, we present an analysis of 25 independent H chain V regions from a monotreme, the Australian duck-billed platypus, Ornithorhynchus anatinus. All the sequences analyzed were found to form a single branch within the clan III of mammalian V region sequences in a distance tree. However, compared with a classical V gene family this branch was more diversified in sequence. Sequence analysis indicates that the apparent lack of diversity in germline V segments is well compensated for by relatively long and highly diversified D and N nucleotides. In addition, extensive sequence variation was observed in the framework region 3. Furthermore, at least five and possibly seven different J segments seem to be actively used in recombination. Interestingly, internal cysteine bridges in the complementarity-determining region (CDR)3 loop, or between the CDR2 and CDR3 loops, are found in approximately 36% of the platypus V(H) sequences. Such cysteine bridges have also been observed in cow, camel, and shark. Internal cysteine bridges may play a role in stabilizing long and diversified CDR3 and thereby have a role in increasing the affinity of the Ab-Ag interaction.

Amino Acid Sequence↗

PubMed2001

Mutation of a single conserved residue in VH complementarity-determining region 2 results in a severe Ig secretion defect.

During an immune response, somatic mutations are introduced into the VH and VL regions of Ig chains. The consequences of somatic mutation in highly conserved residues are poorly understood. Ile51 is present in 91% of murine VH complementarity-determining region 2 sequences, and we demonstrate that single Ile51-->Arg or Lys substitutions in the PCG1-1 Ab are sufficient to severely reduce Ig secretion (1-3% of wild-type (WT) levels). Mutant H chains, expressed in the presence of excess L chain, associate with Ig binding protein (BiP) and GRP94 and fail to form HL and H2L assembly intermediates efficiently. The mutations do not irreversibly alter the VH domain as the small amount of mutant H chain, which assembles with L chain as H2L2, is secreted. The secreted mutant Ab binds phosphocholine-protein with avidity identical with that of WT Ab, suggesting that the combining site adopts a WT conformation. A computer-generated model of the PCG1-1 variable region fragment of Ig (Fv) indicates that Ile51 is buried between complementarity-determining region 2 and framework 3 and does not directly contact the L chain. Thus, the Ile51-->Arg or Ile51-->Lys mutations impair association with the PCG1-1 L chain via indirect interactions. These interactions are in part dependent on the nature of the L chain as the PCG1-1 VH single Ile51-->Arg or Ile51-->Lys mutants were partially rescued when expressed with the J558L lambda1 L chain. These results represent the first demonstration that single somatic mutations in V(H) residues can impair Ig secretion and suggest one reason for the conservation of Ile51 in so many Ig VH.

Amino Acid Sequence↗

PubMed2003

Bovine IgM antibodies with exceptionally long complementarity-determining region 3 of the heavy chain share unique structural properties conferring restricted VH + Vlambda pairings.

Naturally occurring antibody repertoires of cattle (Bos taurus) include a group of IgMlambda antibodies with exceptionally long complementarity-determining region 3 of the heavy chain (CDR3H) segments, containing multiple Cys residues. These massive CDR3H segments will greatly influence the tertiary and quaternary structures of the bovine IgM combining sites. As an antibody's combining site is formed by both heavy and light chains, we have analyzed the nucleotide sequences and structural properties of the lambda-light chains that pair with micro -heavy chains containing exceptionally long CDR3H. There appears to be an exquisite selective pressure for the use of three V(lambda)1 genes (V(lambda)1x and two new V(lambda)1d and V(lambda)1e genes) in IgM with unusually long CDR3H. The V(lambda)1d and V(lambda)1e genes are similar to each other, but diverge from the other V(lambda)1 genes into two closely related subfamilies. The available bovine V(lambda) genes are classified into three V(lambda) gene families: V(lambda)1, V(lambda)2 and V(lambda)3 based on nucleotide similarity >/=80%. Further, analysis of total Ser content and positions of Ser residues in the sequences was found to be sufficient to classify the cattle V(lambda)1 subfamilies. Patterns of Ser residues differ for V(lambda) domains from ruminant species (e.g. cattle, sheep and goats) and other mammals (e.g. humans and mice). These 'Ser signatures' can be used to track divergent evolution in lambda-light chains. Interestingly, Ser90L in complementarity-determining region 3 of the light chain (CDR3L) occurred in all V(lambda) domains that pair with V(H) regions containing exceptionally long CDR3H. A structural role for Ser90L was revealed in homology models of V(lambda) domains, i.e. to hold the ascending polypeptide of CDR3L in a relatively tight space between the N-terminal segment and residues from CDR1L. The CDR3L of V(lambda) domains also occupied smaller volumes if paired to V(H) domains with extremely long CDR3H (>/=48 residues), and were more variable in their conformation and filled larger volumes if CDR3Hs were </=22 residues. Thus, the role of the lambda-light chains in these unusual cattle antibodies is probably to act as a relatively featureless supporting platform for the extremely long CDR3H regions, which undoubtedly are dominantly involved in binding to an antigen.

Amino Acid Sequence↗

PubMed1999

Thermodynamics of Fab-ssDNA interactions: contribution of heavy chain complementarity determining region 3.

The recombinant anti-ssDNA Fab, DNA-1, and 16 heavy chain complementarity determining region 3 (HCDR3) mutant variants were selected for thermodynamic characterization of ssDNA binding. The affinity of Fab to (dT)(15) under different temperatures and cation concentrations was measured by equilibrium fluorescence quenching titration. Changes in the standard Gibbs free binding energy (DeltaG degrees ), enthalpy (DeltaH degrees ), entropy (DeltaS degrees ), and the number of ionic pairs (Z) formed upon interaction were determined. All Fab possessed an enthalpic nature of interaction with ssDNA, that was opposite to the previously reported entropically driven binding to dsDNA [Tanha, J., and Lee, J. S. (1997) Nucleic Acids Res. 25, 1442-1449]. The contribution of separate residues of HCDR3 to ssDNA interaction was investigated. Analysis of the changes in DeltaH degrees and TDeltaS degrees, induced by substitutions in HCDR3, revealed a complete entropy/enthalpy compensation. Mutations R98A and D108A at the ends of the HCDR3 loop produced increases in TDeltaS degrees ( )()by 10.4 and 15.9 kcal/mol, respectively. Substitution of proline for arginine at the top of HCDR3 resulted in a new electrostatic contact with (dT)(15). The observed linear correlation of Z and DeltaG degrees ( )()of nonelectrostatic interactions (DeltaG degrees (nonel)) at the anti-ssDNA combining site was used for the estimation of the specific DeltaG degrees (nonel) [-20 to -25 cal/(mol.A(2))], the average contact area (450-550 A(2)), the maximal Z (6-7), and the limit in affinity under standard cation concentrations [(0.5-1) x 10(8) M(-)(1)] for this family of Fab. Results suggested that rational engineering of HCDR3 could be utilized to control the affinity and likely the specificity of Ab-DNA interactions.

Animals↗

PubMed2001

Novel secondary Ig VH gene rearrangement and in-frame Ig heavy chain complementarity-determining region III insertion/deletion variants in de novo follicular lymphoma.

Human germinal center B cell tumors retain the ability of their nontransformed counterparts to somatically hypermutate Ig V genes by nucleotide substitution. Among a survey of 60 primary previously untreated, clonal, follicular lymphomas we have identified a rare V(H) rearrangement variant and two other in-frame nucleotide insertion/deletion variants within complementarity-determining region III of the Ig heavy chain. The neoplastic origin of the V(H) rearrangement variant was directly demonstrated in cells isolated by microdissection from malignant follicles. In all three cases a common clonal origin for the variants was demonstrated by complementarity-determining region III nucleotide sequence homology and shared somatic mutations in germline encoded positions in framework region IV. The monoclonal nature of the tumors was independently confirmed by demonstrating a single t(14;18) translocation breakpoint in the two cases with a detectable translocation. All the variants occurred in functional V(H) rearrangements, which in two cases were directly shown to encode functional Ab molecules. Both recombination-activating genes 1 and 2 were expressed in lymph node tumor cells containing the V(H) rearrangement variant, although recombination-activating gene expression among a panel of lymphomas was not limited to this variant.

Amino Acid Sequence↗

PubMed1998

A particular characteristic of IgH complementarity-determining region 3 suggests autoreactive B-cell origin of primary gastric B-cell lymphomas.

To clarify the origin of tumor cells and the possible role of antigen in the pathogenesis of mucosa-associated lymphoid tissue (MALT) lymphoma, we analyzed the third complementarity determining region of IgH gene in nine primary gastric B-cell lymphomas by PCR. The presence of vacA gene of Helicobacter pylori was also determined. These cases were diagnosed histopathologically as three low (extranodal marginal zone B-cell lymphoma)- and three high-grade MALT lymphomas and three diffuse large-cell lymphomas without evidence of MALT lymphoma. All cases showed a monoclonally rearranged JH band. A single dominant clone was detected by sequencing the IgH CDR3 regions in eight cases. In the remaining cases. In the remaining case, two major sequences were identified. Complementarity-determining region 3 (CDR3) sequences in lymphoma cell clones of seven cases showed 61% to 81% homology with autoantibody-associated lymphocyte clones including rheumatoid factor. The N-D-N region in the low-grade MALT lymphoma group was significantly longer than in the high-grade lymphoma groups. Although vacA gene of H. pylori was detected in five of nine cases, all lymphoma cell clones showed no association with foreign antigen. These results indicate that autoantigen may be responsible for the clonal selection of lymphoma cells, and thus autoimmunity may play a role in the pathogenesis of primary gastric B-cell lymphomas.

Adult↗

PubMed2003

Complementarity-determining region 3 spectratyping analysis of the TCR repertoire in multiple sclerosis.

Multiple sclerosis (MS) is considered to be an autoimmune disease mediated by T cells reactive with Ags in the CNS. Therefore, it has been postulated that neuroantigen-reactive T cells bearing particular types of TCRs are expanded clonally during the course of the disease. However, there is a controversy with regard to the TCR usage by T cells associated with the development of MS. By the use of complementarity-determining region 3 spectratyping analysis that is shown to be a useful tool for identification of pathogenic TCR in autoimmune disease models, we tried to demonstrate that spectratype was T cells bearing particular types of TCR are activated in MS patients. Consequently, it was found that Vbeta5.2 were often oligoclonally expanded in peripheral blood of MS patients, but not of healthy subjects. Sequence analysis of the complementarity-determining region 3 region of spectratype-derived TCR clones revealed that the predominant TCR clone was different from patient to patient, but that similar results were obtained in a patient examined at different time points. More importantly, examination of cerebrospinal fluid T cells and longitudinal studies of PBLs from selected patients revealed that Vbeta5.2 expansion was detectable in the majority of patients examined. These findings suggest that Vbeta5.2 spectratype expansion is associated with the development of MS and that TCR-based immunotherapy can be applicable to MS patients if the TCR activation pattern of each patient is determined at different stages of the disease.

Adolescent↗

PubMed2006

[Complementarity-determining region 3 analysis of T cell receptor beta chain variable region in peripheral blood mononuclear cells of patients with systemic lupus erythematosus].

OBJECTIVE: To analyze the drift of the complementarity-determining region 3 (CDR3) of T cell receptor (TCR) beta chain variable region (TCR BV) in peripheral blood mononuclear cells (PBMCs) of patients with systemic lupus erythematosus. METHODS: Immunoscope spectratyping techniques was used to analyze the distribution of TCRbeta chain CDR3 in 5 normal blood donors and the dominant CDR3 in the PBMCs in 5 SLE patients. Sequence analysis of the CDR3 region in monoclonal or oligoclonal T cells was performed. RESULTS: The spectratypes of TCR BV gene CDR3 region showed Gaussian distribution in the 5 normal blood donors. The 5 SLE patients, however, displayed anomalous proliferation and oligoclonal expansion of the T cells was observed in different TCR BV families with different CDR3 sequences. CONCLUSION: Noticeable drift of TCRbeta chain CDR3 can be seen in active SLE, indicating possible association of selective expression of TCR with immune pathogenesis in SLE. Determination of specific TCR CDR3 sequence provides a new means for studying the pathogenesis and personalized treatment of SLE.

Amino Acid Sequence↗

PubMed2001

Third complementarity-determining region of mutated VH immunoglobulin genes contains shorter V, D, J, P, and N components than non-mutated genes.

The third complementarity-determining region (CDR3) of immunoglobulin variable genes for the heavy chain (VH) has been shown to be shorter in length in hypermutated antibodies than in non-hypermutated antibodies. To determine which components of CDR3 contribute to the shorter length, and if there is an effect of age on the length, we analysed 235 cDNA clones from human peripheral blood of VH6 genes rearranged to immunoglobulin M (IgM) constant genes. There was similar use of diversity (D) and joining (JH) gene segments between clones from young and old donors, and there was similar use of D segments among the mutated and non-mutated heavy chains. However, in the mutated heavy chains, there was increased use of shorter JH4 segments and decreased use of longer JH6 segments compared to the non-mutated proteins. The overall length of CDR3 did not change with age within the mutated and non-mutated categories, but was significantly shorter by three amino acids in the mutated clones compared to the non-mutated clones. Analyses of the individual components that comprise CDR3 indicated that they were all shorter in the mutated clones. Thus, there were more nucleotides deleted from the ends of VH, D, and JH gene segments, and fewer P and N nucleotides added. The results suggest that B cells bearing immunoglobulin receptors with shorter CDR3s have been selected for binding to antigen. A smaller CDR3 may allow room in the antibody binding pocket for antigen to interact with CDRs 1 and 2 as well, so that as the VDJ gene undergoes hypermutation, substitutions in all three CDRs can further contribute to the binding energy.

Adult↗

PubMed1996

Cloning of size-selected human immunoglobulin heavy-chain rearrangements from third complementarity-determining region fingerprint profiles.

Methods have been developed to rapidly visualize the size distribution of third complementarity-determining regions (CDR3) in immunoglobulin (Ig) and T-cell receptor (TCR) molecules. DNA fragments spanning the Ig or TCR CDR3 are generated by PCR using primers at fixed positions in the variable and constant segments. These fragments differ in length due to size variation of the CDR3s. Visualization of the amplification products in polyacrylamide gels as a "CDR3 fingerprint profile" is a rough measure for the complexity of the Ig and TCR antigen-binding specificities. We report an adaptation of this method for the analysis of human Ig heavy-chain genes that incorporates silver staining, which allows for the fine analysis of specific regions of the profiles. This is especially useful for the study of low-abundant transcripts.

Adult↗

PubMed2002

Analysis of IgE antibodies from a patient with atopic dermatitis: biased V gene usage and evidence for polyreactive IgE heavy chain complementarity-determining region 3.

To better understand V gene usage, specificity, and clonal origins of IgE Abs in allergic reactions, we have constructed a combinatorial Ab library from the mRNA of an adult patient with atopic dermatitis. Sequence analysis of random clones revealed that 33% of clones used the IGHV6-1 H chain V gene segment, the only member of the V(H)6 gene family. IGHV6-1 is rarely used in the expressed adult repertoire; however, it is associated with fetal derived Abs. Features of the V(H)6 rearrangements included short complementarity-determining region 3, frequent use of IGHD7-27 D gene, and little nucleotide addition at the D-J junction. There was also a low level of mutation compared with V(H)1, V(H)3, and V(H)4 rearrangements. The library was expressed as phage-Fab fusions, and specific phage selected by panning on the egg allergen ovomucoid. Upon expression as soluble IgE Fabs, 12 clones demonstrated binding to ovomucoid, skim milk, and BSA by ELISA. Nucleotide sequencing demonstrated that the IGHV6-1 V gene segment encoded each of the 12 multiply reactive IgE Fabs. A cyclic peptide was designed from the complementarity-determining region 3 of several of these clones. The cyclic peptide bound both self and nonself Ags, including ovomucoid, human IgG, tetanus toxoid, and human and bovine von Willebrand factor. These results suggest that some IgE Abs may bind more than one Ag, which would have important implications for understanding the multiple sensitivities seen in conditions such as atopic dermatitis.

Adult↗

PubMed2006

Amelioration of murine lupus by a peptide, based on the complementarity determining region-1 of an autoantibody as compared to dexamethasone: different effects on cytokines and apoptosis.

A peptide (hCDR1) based on the sequence of the complementarity-determining region-1 of an anti-DNA autoantibody ameliorates clinical manifestations of lupus. We analyzed the beneficial effects of hCDR1 when given alone or in combination with dexamethasone, while comparing the mechanisms of action of the latter. Treatment with either hCDR1 or dexamethasone, or a combination of the latter significantly reduced titers of dsDNA-specific autoantibodies, levels of proteinuria, and intensity of glomerular immune complex deposits. Both drugs down-regulated the secretion and expression of IFN-gamma and IL-10, but only treatment with hCDR1 up-regulated TGF-beta. While both drugs reduced the expression of Fas ligand (FasL) and caspase 8, treatment with hCDR1 resulted in reduced whereas dexamethasone administration resulted in increased rate of apoptosis. Furthermore, down-regulation of FasL appeared to play a role in cytokine modulation. We conclude that specific treatment with hCDR1 ameliorates murine lupus via distinct mechanisms of action than those of dexamethasone.

Animals↗

PubMed1998

Exploiting sequence space: shuffling in vivo formed complementarity determining regions into a master framework.

A novel approach in molecular design is presented, where in vivo formed complementarity determining regions (CDR) from antibody genes were shuffled into a specific framework region. A synthetic gene library of soluble VH-fragments was created and the complexity of the library was determined by sequencing. The synthetic genes were diverse and contained random combinations of CDR from different germlines. All CDR were randomised in one step and by using in vivo formed CDR, the length, sequence and combination were varied simultaneously.

Amino Acid Sequence↗

PubMed2004

A peptide based on the complementarity determining region 1 of a human monoclonal autoantibody ameliorates spontaneous and induced lupus manifestations in correlation with cytokine immunomodulation.

A peptide based on the sequence of the complementarity determining region (CDR) 1 of a human monoclonal anti-DNA autoantibody that bears the 16/6 idiotype (16/6Id) was synthesized as a potential candidate for the treatment of SLE patients. The peptide, designated hCDR1, did not induce experimental SLE upon active immunization of mice. The ability of the peptide to treat an already established lupus that was either induced in BALB/c mice or developed spontaneously in (NZB x NZW)F1 mice was tested. Ten weekly injections of hCDR1 (200, 50 microg/mouse) given subcutaneously mitigated disease manifestations (e.g., leukopenia, proteinuria and kidney damage) and resulted in a prominent reduction in the dsDNA specific antibody titers. Furthermore, treatment with hCDR1 resulted in reduced secretion and expression of the "pathogenic" cytokines [i.e., INFgamma, IL-1beta, TNFalpha (in the induced model) and IL-10], whereas the immunosuppressive cytokine TGFbeta was up-regulated. Thus, the significant ameliorating effects of hCDR1 are manifested at least partially via the immunomodulation of the cytokine profile. These results suggest that hCDR1 is a potential candidate for a novel treatment of SLE patients.

Animals↗