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Results for “Complementarity Determining Regions”

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PubMed2001

Normal B cells express 51p1-encoded Ig heavy chains that are distinct from those expressed by chronic lymphocytic leukemia B cells.

51p1 is an allele of V(H)1-69 that frequently is expressed by chronic lymphocytic leukemia (CLL) B cells with little or no somatic mutation. The rearranged 51p1 genes expressed by CLL B cells have a distinctive use of D segments D3-3/DXP4 and D3-10/DXP'1, a favored use of J(H)6, and a longer third complementarity-determining region than the rearranged Ig genes used by CLL B cells that express V(H)1 genes other than V(H)1-69. We examined the 51p1-encoded Ig expressed by blood B cells of healthy donors. In contrast to the infrequent use of J(H)4 by 51p1-expressing CLL (e.g., 4%), 36% of the rearranged 51p1 sequences from normal blood B cells used J(H)4. Furthermore, the D segment use of the rearranged 51p1 sequences from normal blood B cells was not restricted, but reflected the D segment use of nonselected IgH of normal B cells. Finally, the mean length of the third complementarity-determining region for the 51p1 genes of normal blood B cells was 14.6 +/- 4.3 (SD) codons. This is significantly shorter than that noted for 51p1-expressing CLL B cells (18.8 +/- 3.2; p < 0.0001, n = 51). This study demonstrates that the 51p1-encoded IgH expressed in CLL are not representative of the 51p1-encoded IgH expressed by normal blood B cells, indicating that CLL B cells express IgH that are distinctive from those found in the normal adult blood B cell repertoire.

Adult↗

PubMed1999

T cell repertoire alterations of vascularized xenografts.

The role of T cells in the rejection of vascularized xenografts has been little explored. Because of the high potential diversity of xenoantigens, it has been suggested that xenotransplantation could induce a strong cellular response that could contribute to delayed rejection. Alternatively, alterations in molecular interactions could impair the T cell response. Because the analysis of TCR repertoire in vivo indirectly reflects the nature and the magnitude of T cell xenorecognition, we took advantage of the possibility of obtaining long term survival of hamster heart xenografts in rat recipients treated with a combination of cobra venom factor and cyclosporin A (CsA), to analyze T cell infiltration and, for the first time, V beta TCR usage, at the complementarity-determining region 3 level, in accommodated and rejected xenografts, compared with allografts. After withdrawal of CsA (on day 40), the analysis of V beta family expression and corresponding complementarity-determining region 3 lengths in rejected xenografts revealed a Gaussian pattern, in contrast to a much more restricted pattern in rejected allografts (p = 0.002), suggesting that, after withdrawal of CsA, all the underrepresented T cell clones are rapidly expanded in xenografts. These results correlate with the rapid kinetics of rejection (4 +/- 1 days), the high number of T cells, the rapid expression of markers of activation (IL-2 receptor alpha-chain and class II receptor), and the strong deposit of IgG Abs in rejected xenografts. Taken together, these results suggest that the intensity and diversity of the T cell response to xenografts could be stronger than the response to allografts in vivo.

Animals↗

PubMed1999

Gene conversion and hypermutation during diversification of VH sequences in developing splenic germinal centers of immunized rabbits.

The young rabbit appendix and the chicken bursa of Fabricius are primary lymphoid organs where the B cell Ab repertoire develops in germinal centers (GCs) mainly by a gene conversion-like process. In human and mouse, V-gene diversification by somatic hypermutation in GCs of secondary lymphoid organs leads to affinity maturation. We asked whether gene conversion, somatic hypermutation, or both occur in rabbit splenic GCs during responses to the hapten DNP. We determined DNA sequences of rearranged heavy and light chain V region gene segments in single cells from developing DNP-specific GCs after immunization with DNP-bovine gamma-globulin and conclude that the changes at the DNA level that may lead to affinity maturation occur by both gene conversion and hypermutation. Selection was suggested by finding some recurrent amino acid replacements that may contribute increased affinity for antigen in the complementarity-determining region sequences of independently evolved clones, and a narrower range of complementarity-determining region 3 lengths at day 15. Some of the alterations of sequence may also lead to new members of the B cell repertoire in adult rabbits comparable with those produced in gut associated lymphoid tissues of young rabbits.

Amino Acid Sequence↗

PubMed1997

V delta 1 T cells expanded in the blood throughout HIV infection display a cytotoxic activity and are primed for TNF-alpha and IFN-gamma production but are not selected in lymph nodes.

We have previously reported significant alterations of gamma delta subset distribution in the peripheral blood of HIV-infected donors. These modifications are characterized by the depletion of the V delta 2 subset associated with a strong increase in peripheral V delta 1 T cells. In addition, the latter exhibit ex vivo an activated phenotype and show a restricted complementarity-determining region 3 (CDR3) repertoire. In the present report we first address the question of the origin of these expanded cells. The lack of expansion and the Gaussian complementarity-determining region 3 size distribution of lymph node V delta 1 T cells suggest that lymph nodes do not represent the site of specific activation of this subset. The function of blood V delta 1 T cells was also explored. We report that patients' V delta 1 T cells express high levels of perforin and display an in vitro cytotoxic activity, whose characteristics are different from those of NK and LAK cells. In addition, single cell analysis of cytokine production revealed that, in contrast to V delta 1 T cells from control donors, patients' V delta 1 T cells are primed in vivo for IFN-gamma and TNF-alpha production. Together, these results indicate that in the course of HIV infection, expanded blood V delta 1 T cells are differentiated into a functional subset and raise the question of the contribution of this subset to AIDS pathogenesis.

Cell Differentiation↗

PubMed2001

Unusual germline DSP2 gene accounts for all apparent V-D-D-J rearrangements in newborn, but not adult, MRL mice.

Anti-dsDNA autoantibodies in MRL mice contain a higher than average frequency of atypical complementarity-determining regions 3, including those made with D-D rearrangements. It has been reported that MRL mice have an intrinsically high frequency of creating VDDJ rearrangements; however, we show in this study that the majority of these apparent D-D rearrangements in B cell progenitors can be accounted for by a very novel germline D(H) gene in mice of the Igh(j) haplotype. This gene has the appearance of a D to D rearrangement due to the duplication of 9 bp common to most DSP2 genes. Germline DSP2 genes from Igh(j) mice were amplified, cloned, and sequenced, showing the presence of this novel gene as well as a new allele of a conventional DSP2 gene. Sequencing of D-J rearrangements revealed that Igh(j) mice also have a different allele of DFL16.1 and apparently lack DFL16.2. Despite the existence of this new DSP gene, analysis of VDJ rearrangements from adult bone marrow pre-B cells of MRL/lpr mice still revealed the presence of complementarity-determining region 3 containing apparent D-D joinings in 4.6% of the sequences. C3H pre-B cells had 4.2% of sequences with apparent VDDJ rearrangements, and BALB/c pre-B cells had approximately 2%. DDJ intermediates were also observed, but at a lower frequency. However, strikingly, no VDDJ rearrangements were observed in newborn sequences, suggesting the process of assembly of VDJ rearrangements is fundamentally different in newborn mice vs adult mice.

Age Factors↗

PubMed2000

Alterations in the spinal cord T cell repertoire during relapsing experimental autoimmune encephalomyelitis.

The CNS T cell repertoire was analyzed by RT-PCR, spectratyping, and nucleotide sequencing of the amplified products at different times following adoptive transfer of a CD4+, Th1, VB2+ encephalitogenic SJL/J proteolipid protein peptide 139-151-specific T cell clone. The third complementarity-determining region of TCR B chains in the spinal cord was used as an indicator of T cell heterogeneity. Spectratypic analysis revealed that a single peak corresponding to the third complementarity-determining region of the initiating T cell clone predominated during the acute phase. During recovery and relapse the complexity of the spectratype increased. DNA sequence analysis revealed that the donor clone predominated at the acute phase. By the first relapse the donor clone, although represented most frequently, was a minority of the total. Spectratypic analysis of the same samples for several other VB families revealed their presence during acute disease or relapses but, with the exception of VB17, their absence during the recovery stage.

Amino Acid Sequence↗

PubMed2003

Structure, genomic organization, and phylogenetic implications of six new VH families in the channel catfish.

To define members of previously unknown VH gene families, a channel catfish immunoglobulin heavy chain cDNA library was constructed and screened with probes specific for the seven known catfish VH families. Reiterative screening and sequence studies defined six new VH families, designated VH8-VH13, which brings the total number of VH families in the catfish to 13. This is the highest number of VH families presently defined in a lower vertebrate. Sequence comparisons indicate there is extensive diversity between members of different families with the greatest variability encoded within the complementarity determining regions. Genomic libraries were screened, and germline VH segments representing each of these new families were identified. The VH segments are closely linked and interspersed with members of different VH families. Each of these germline gene segments shared characteristic structural features: an upstream region that contained transcriptional regulatory elements, a leader sequence split by a short intron, an open reading frame encoding readily identified framework and complementarity determining regions, and a terminal recombination signal sequence consisting of a consensus heptamer, a 22-24 bp spacer with conserved 5'- and 3'-ends, and a consensus A-rich nonamer. Southern blot analyses estimate the number of members within these new families ranges from small (2-7 members in VH9, VH10, and VH12) to medium (9-13 members in VH8, VH11, and VH13). Thus, there are between 165 and 200 germline VH segments represented by these combined 13 families with present analyses indicating that perhaps one-half of these are pseudogenes. Phylogenetic comparisons indicate that members of these different catfish VH families cluster within Groups C and D of vertebrate VH genes. These analyses also indicate that Group D is represented by two different branches and both branches include VH families from different lineages of bony fish that diverged early in vertebrate phylogeny.

Amino Acid Sequence↗

PubMed1993

Length distribution of CDRH3 in antibodies.

Sequences of the third complementarity determining region of antibody heavy chains (CDRH3s) are listed according to their length. Human sequences vary from 2 to 26 amino acids residues, but less extensively in other species. When combined with the other five complementarity determining regions, this enormous length variation of CDRH3, together with amino acid substitutions in their sequences, can provide a very large number of antibody specificities and can influence the shape of antibody combining sites.

Amino Acid Sequence↗

PubMed1997

Structure and genomic organization of a second class of immunoglobulin light chain genes in the channel catfish.

Earlier studies distinguished two classes of catfish light (L) chain (designated F and G). The cDNA structure and genomic organization of G L chain gene clusters has also been characterized previously. In this study, full length cDNA encoding F L chain was derived using PCR strategies based on the determined amino-terminal protein sequence. The encoded V region is readily delineated into framework regions (FR) and complementarity-determining regions (CDR). Multiple sequence alignments indicate that the F V(L) is closely related to kappa gene families. The F C(L) cannot be generally classified but it is structurally distinct from the C(L) regions of G: the amino acid sequence similarity is <35%. cDNA sequences representing processed sterile F transcripts of different loci were identified. Each sequence begins within the J(L) recombination signal sequence and extends downstream through the I(L)-C(L) segments. Genomic blots hybridized with C(L) probes indicate that there are at least 50 different C(L) segments. Based upon V(L) hybridization studies, different families of V(L) segments appear to be associated with closely related F C(L) segments. In characterized genomic clones, F gene segments are arranged in closely linked clusters with single copies of V(L), J(L), and C(L) segments within each cluster. The V(L) segments are located in opposite transcriptional polarity relative to the J(L) and C(L) segments, which indicates that V(L) segments rearrange by inversion. These combined studies establish that two structurally distinct classes of L chains are present in teleost fish and that both of the L chain classes evolved within a common organizational pattern of clustered segmental genes.

Amino Acid Sequence↗

PubMed1990

Resolution of hypervariable regions in T-cell receptor beta chains by a modified Wu-Kabat index of amino acid diversity.

The Wu-Kabat variability coefficient is a well-established descriptor of the susceptibility of an amino acid position to evolutionary replacements. It conveniently highlights stretches of accentuated amino acid variation that, for example, in an antibody molecule account for most of the antigen contacts (complementarity-determining regions). Diverse opinion are held as to why the index yields unclear results when applied instead to the polypeptide sequences of the T-cell antigen receptor. We show that a simple modification enhances the resolving power of the index by increasing the weight on the frequency distribution of the amino acids in the formula. Application of the improved index to T-cell receptor beta chains highlights four unambiguous hypervariable regions, three of which are positioned similar to immunoglobulin complementarity-determining regions along the chain. In a Fab-like three-dimensional model of the T-cell receptor, the four hypervariable regions coincide with the four loops on the surface of the domain and form a contiguous area available for binding.

Amino Acid Sequence↗

PubMed2000

Prediction of the interacting surfaces in a trimolecular complex formed between the major dust mite allergen Der p 1, a mouse monoclonal anti-Der p 1 antibody, and its anti-idiotype.

BACKGROUND: Two mouse monoclonal antibodies (mAbs) have been described recently; namely, mAb 2C7 (IgG2b kappa), which is directed against the major house dust mite allergen Der p 1, and mAb 2G10 (IgG1 kappa), which is an anti-idiotypic antibody raised against mAb 2C7. The anti-idiotype mAb 2G10 does not block the binding of mAb 2C7 to Der p 1, which means that mAb 2C7 can simultaneously bind to Der p 1 and to mAb 2G10, thereby generating a trimolecular complex consisting of antigen-idiotype-anti-idiotype. AIMS: To sequence and model the V region of the anti-idiotypic antibody mAb 2G10 to enable the prediction of the interacting surfaces in the trimolecular complex consisting of Der p 1-mAb 2C7-mAb 2G10. METHODS: DNA sequencing of mAb 2G10 was carried out and the Swiss Model and Swiss PDB-Viewer programs were used to build a three dimensional model of the trimolecular complex. RESULTS: Complementarity of shape and charge was revealed when comparing the protrusion of the previously determined Der p 1 epitope (Leu147-Gln160) with the cavity formed by the complementarity determining regions (CDRs) of mAb 2C7. Such complementarity was also observed between the mAb 2C7 epitope predicted to be recognised by mAb 2G10 (residues Lys19 from framework region 1 (FRW1) and Ser74-Gln81 from FRW3) and residues from the CDRs of mAb 2G10 (a negatively charged patch flanked by the residues Asp55H/Glu58H and Glu27L/Glu27cL). As expected, the location of the mAb 2C7 epitope recognised by mAb 2G10 does not appear to interfere with the binding of Der p 1 to mAb 2C7. CONCLUSION: Although the results obtained represent only an approximation, they nevertheless provide a rare insight into how an antigen (Der p 1) might bind to its antibody (mAb 2C7) while in complex with an anti-idiotype (mAb 2G10).

Allergens↗

PubMed1986

Three-dimensional structure of an antigen-antibody complex at 2.8 A resolution.

The 2.8 A resolution three-dimensional structure of a complex between an antigen (lysozyme) and the Fab fragment from a monoclonal antibody against lysozyme has been determined and refined by x-ray crystallographic techniques. No conformational changes can be observed in the tertiary structure of lysozyme compared with that determined in native crystalline forms. The quaternary structure of Fab is that of an extended conformation. The antibody combining site is a rather flat surface with protuberances and depressions formed by its amino acid side chains. The antigen-antibody interface is tightly packed, with 16 lysozyme and 17 antibody residues making close contacts. The antigen contacting residues belong to two stretches of the lysozyme polypeptide chain: residues 18 to 27 and 116 to 129. All the complementarity-determining regions and two residues outside hypervariable positions of the antibody make contact with the antigen. Most of these contacts (10 residues out of 17) are made by the heavy chain, and in particular by its third complementarity-determining region. Antigen variability and antibody specificity and affinity are discussed on the basis of the determined structure.

Animals↗

PubMed1998

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.

Antibodies, Monoclonal↗

PubMed1992

Chimeric and humanized antibodies with specificity for the CD33 antigen.

L and H chain cDNAs of M195, a murine mAb that binds to the CD33 Ag on normal and leukemic myeloid cells, were cloned. The cDNAs were used in the construction of mouse/human IgG1 and IgG3 chimeric antibodies. In addition, humanized antibodies were constructed which combined the complementarity-determining regions of the M195 antibody with human framework and constant regions. The human framework was chosen to maximize homology with the M195 V domain sequence. Moreover, a computer model of M195 was used to identify several framework amino acids that are likely to interact with the complementarity-determining regions, and these residues were also retained in the humanized antibodies. Unexpectedly, the humanized IgG1 and IgG3 M195 antibodies, which have reshaped V regions, have higher apparent binding affinity for the CD33 Ag than the chimeric or mouse antibodies.

Amino Acid Sequence↗

PubMed1988

Matching nucleotide sequences of human antibodies with other known sequences.

From an evolutionary point of view, the complementarity-determining regions of antibodies are distinct from other proteins including the framework regions of antibodies. A search for identical nucleotide sequences of eighty-four 15 consecutive bp in the complementary-determining regions of human antibody heavy chains with other known sequences yielded four matches: two sequential 15-bp matches, or one 16-bp match, with the coding region of a sea-urchin testis histone H2b-2, one 15-bp match with the promotor region of a cauliflower mosaic virus inclusion body protein, and a 15-bp match with an intron between exons 1 and 2 of human factor IX. As a control, an identical search of eighty-four 15 consecutive bp in the framework regions of human antibody heavy chains yielded no matches with other sequences except those from other antibody framework regions. Since the currently available nucleotide sequence database used in the search consisted of about 1 x 10(7) bp, finding such matches in the complementarity-determining regions might not be random.

Antibodies↗

PubMed1999

DNA binding by the VH domain of anti-Z-DNA antibody and its modulation by association of the VL domain.

mAb Z22 is a highly selective IgG anti-Z-DNA Ab from an immunized C57BL/6 mouse. Previous studies showed that heavy chain CDR3 amino acids are critical for Z-DNA binding by the single chain variable fragment (scFv) comprising both V region heavy chain (VH) and V region light chain (VL) of mAb Z22 and that the VH domain alone binds Z-DNA with an affinity similar to that of whole variable fragment (Fv). To determine whether Z-DNA binding by VH alone and by Fv involves identical complementarity determining region residues, we tested effects of single or multiple amino acid substitutions in recombinant VH, scFv, and associated VH-VL heterodimers. Each recombinant product was a fusion protein with a B domain of Staphylococcal protein A (SPA). Z22VH-SPA alone was not highly selective; it bound strongly to other polynucleotides, particularly polypyrimidines, and ssDNA as well as to Z-DNA. In contrast, scFv-SPA or associated VH-VL dimers bound only to Z-DNA. VL-SPA domains bound weakly to Z-DNA; SPA alone did not bind. Introduction of multiple substitutions revealed that the third complementarity determining region of the heavy chain (CDR3H) was critical for both VH and scFv binding to Z-DNA. However, single substitutions that eliminated or markedly reduced Z-DNA binding by scFv instead caused a modest increase or no reduction in binding by VH alone. Association of VH-SPA with Z22VL-SPA restored both the effects of single substitutions and Z-DNA selectivity seen with Fv and intact Ab. Polypyrimidine and ssDNA binding by the isolated VH domain of immunization-induced anti-Z-DNA Ab resembles the activity of natural autoantibodies and suggests that VH-dependent binding to a ligand mimicked by polypyrimidines may play a role in B cell selection before immunization with Z-DNA.

Amino Acid Substitution↗

PubMed1997

Structural analysis of human antibodies to proteinase 3 from patients with Wegener granulomatosis.

We determined the structure of five IgM autoAbs to proteinase-3 (PR3). These Abs are highly specific for Wegener's granulomatosis (WG) and may be involved in the pathogenesis of vasculitis in WG. Five clonal lymphoblastoid cell lines secreting Abs to PR3 were derived from four patients' B cells. From 3 to 5% of supernatants from wells contained detectable anti-PR3 Abs, indicating that anti-neutrophil cytoplasmic Ab specificity represents a sizable part of the IgM B cell repertoire in patients with WG. Mu heavy chains of WG1, WG4-1, and WG4-2 clones belonged to the VH3 subgroup. WG4-1 and WG4-2 heavy chains were identical, indicating an oligoclonal expansion of autoreactive B cells in this patient. WG4-1 (and WG4-2) used the VH3-23 V(H) gene, the product of which was shown to directly bind PR3. Heavy chains of WG2 and WG3 derived from VH4-59 and VH1-2 genes, respectively. Comparison with germline sequences showed that three of the five V(H) genes from clonal lines were somatically mutated with a R:S ratio in complementarity-determining regions of 3:0, 5:1, and 5:1, respectively. Three kappa light chains derived from the Vkappa4 gene, and one derived from a Vkappa1 gene. In these four Vkappa genes, there were overall R:S ratios of mutation of 8:1 and 0:7 in complementarity-determining regions and framework regions, respectively. These data suggest that the production of these autoantibodies, which are increasingly important in the diagnosis and management of WG, are influenced by an Ag-driven process.

Amino Acid Sequence↗

PubMed1995

Monoclonal thyroglobulin autoantibodies: variable region analysis and epitope recognition.

A panel of human monoclonal thyroglobulin (Tg) autoantibodies (TgAAb) has been used to analyze autoantigenic determinants on human Tg and to investigate the relationship between variable (V) region gene sequences and epitope specificity. Two monoclonal TgAAb bound to the same (or closely related) epitope on Tg, and these were defined as type I TgAAb. Three other monoclonals bound to a different site and were defined as type II TgAAb. Inhibition studies with mixtures of type I and type II monoclonal TgAAb (Fab)2 preparations indicated that a mixture of the (Fab)2s almost completely inhibited (> 75%) labeled Tg binding to intact TgAAb in the sera of apparently healthy blood donors and patients with autoimmune thyroid disease (AITD). Type I TgAAb predominated in apparently healthy blood donors' sera, whereas type II TgAAb predominated in AITD sera. Analysis of V region gene sequences of the TgAAb indicated that a range of light chain and heavy chain genes from different gene families was used. Furthermore, the same germline genes that are used by TgAAb are also well represented in the genes coding for other self- and nonself-reactive antibodies. No homology in terms of light chain and heavy chain gene families, germline gene usage, or complementarity determining region sequences was observed in TgAAb directed to the same or closely related epitopes. Our studies show that TgAAb are directed to two major conformational epitopes on the Tg molecule and that the proportion of TgAAb directed to these epitopes in apparently healthy blood donors and that in patients with AITD appear to be different. TgAAb derived from different germline genes and with different complementarity determining region sequences can display similar epitope specificity, and this indicates that AAb directed to the same or a closely related epitope show considerable heterogeneity at the molecular level.

Amino Acid Sequence↗