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PubMed2002

The 'just-right' signaling model: APC somatic mutations are selected based on a specific level of activation of the beta-catenin signaling cascade.

According to the classical interpretation of Knudson's 'two-hit' hypothesis for tumorigenesis, the two 'hits' are independent mutation events, the end result of which is loss of a tumor suppressing function. Recently, it has been shown that the APC (adenomatous polyposis coli) gene does not entirely follow this model. Both the position and type of the second hit in familial adenomatous polyposis (FAP) polyps depend on the localization of the germline mutation. This non-random distribution of somatic hits has been interpreted as the result of selection for more advantageous mutations during tumor formation. However, the APC gene encodes for a multifunctional protein, and the exact cellular function upon which this selection is based is yet unknown. In this study, we have analyzed somatic APC point mutations and loss of heterozygosity (LOH) in 133 colorectal adenomas from six FAP patients. We observed that when germline mutations result in truncated proteins without any of the seven beta-catenin downregulating 20-amino-acid repeats distributed in the central domain of APC, the majority of the corresponding somatic point mutations retain one or, less frequently, two of the same 20-amino-acid repeats. Conversely, when the germline mutation results in a truncated protein retaining one 20-amino-acid repeat, most second hits remove all 20-amino-acid repeats. The latter is frequently accomplished by allelic loss. Notably, and in contrast to previous observations, in a patient where the germline APC mutation retains two such repeats, the majority of the somatic hits are point mutations (and not LOH) located upstream and removing all of the 20-amino-acid repeats. These results indicate selection for APC genotypes that are likely to retain some activity in downregulating beta-catenin signaling. We propose that this selection process is aimed at a specific degree of beta-catenin signaling optimal for tumor formation, rather than at its constitutive activation by deletion of all of the beta-catenin downregulating motifs in APC.

Adenomatous Polyposis Coli↗
PubMed1998

Analysis of VH genes in follicular and diffuse lymphoma shows ongoing somatic mutation and multiple isotype transcripts in early disease with changes during disease progression.

Investigations of VH gene mutational patterns in B-cell tumors are often performed at an arbitrary time point of disease. To assess the effects of disease progression, tumor-derived VH genes have been monitored from presentation through treatment and relapse in one patient with follicle center lymphoma (FCL), and two patients with primary diffuse large B-cell lymphoma (DLCL). The patient with FCL and one patient with DLCL both achieved clinical remission, although this was only partial in the FCL. However, both subsequently relapsed, and the second patient with DLCL was refractory to radiotherapy and chemotherapy. In each case, the tumor-derived VH sequence was identified, and the CDR3 "clonal signature" was used to track tumor cell sequences in subsequent biopsies. All cases showed somatic mutations, with intraclonal heterogeneity evident at presentation, and some sequences were aberrant. The VH sequences of the DLCL which responded to treatment became homogeneous at relapse. The sequences of both the FCL and the refractory DLCL remained heterogeneous. In all cases, transcripts of multiple Ig isotypes could be identified, and there was immunophenotypic evidence for expression of several Ig isotypes. The case of refractory DLCL had identifiable transcripts from IgM, IgD, IgA, IgG, and IgE, but appeared to lose the ability to produce alternative isotype transcripts and protein at the late stage of disease. These cases indicate that VH gene analysis can be used to probe tumor cell behavior in cases of lymphoma and that perturbations caused by therapy and disease progression can occur.

Amino Acid Sequence↗
PubMed1982

Somatic mutation in genes for the variable portion of the immunoglobulin heavy chain.

The size of the gene pool potentially encoding antibodies to p-azophenyl arsonate has been examined. A heavy chain-specific full-length complementary DNA clone has been constructed with the use of messenger RNA from a hybridoma that produces antibodies to the arsonate hapten and bears nearly a full complement of the determinants comprising the cross-reactive idiotype (CRI). The sequences of both the complementary DNA clone and the corresponding immunoglobulin heavy chain have been independently determined. A probe for the variable region gene was prepared from the original heavy chain complementary DNA clone and used to analyze, by Southern filter hybridization, genomic DNA from both A/J (CRI positive) and BALB/c (CRI negative) mice. Approximately 20 to 25 restriction fragments containing "germline" variable region gene segments were detected in both strains, and many are shared by both, Since 35 CRI-positive heavy chains have been partially sequenced thus far and 31 are different, the results of the hybridization analysis suggest that somatic mutation events involving the variable region gene segments of the heavy chain play a role in the origin of the amino acid sequence diversity seen in this system.

Amino Acid Sequence↗
PubMed1996

Four IgG anti-islet human monoclonal antibodies isolated from a type 1 diabetes patient recognize distinct epitopes of glutamic acid decarboxylase 65 and are somatically mutated.

The selective destruction by an autoimmune process of the beta cells in the pancreas is the hallmark of the type 1 insulin-dependent diabetes mellitus. What triggers islet cell-specific autoreactive T and B cells, however, remains unclear. Identification of the targets of the anti-islet cell autoantibodies frequently found in insulin-dependent diabetes mellitus patients and analysis of their sequences should provide some insights into the nature of this disease. We have combined EBV transformation with CD40 activation of peripheral B cells from one patient with insulin-dependent diabetes mellitus to isolate four B cell clones that secrete islet cell-specific autoantibodies, These four human monoclonal autoantibodies are of the IgG1 isotype, and they each recognize a different epitope of the glutamic acid decarboxylase enzyme. Analysis of their variable gene sequences shows that, while clonally unrelated, three of the four human monoclonal autoantibodies use a member of the VH4 family, and two have rearranged the same delta light chain variable gene. The IgG1 isotype of the four autoantibodies as well as the presence of somatic mutations in both heavy and light chain genes provide concrete evidence for their derivation by a T cell-dependent immune response.

Adult↗
PubMed1994

Hodgkin's disease with a B-cell phenotype often shows a VDJ rearrangement and somatic mutations in the VH genes.

The nature of Hodgkin and Reed-Sternberg (HRS) cells remains in question. Immunophenotypic studies favor a relation to the lymphoid lineage with the existence of B- and T-cell types. However, studies on the detection of antigen (Ag) receptor gene rearrangements provided inconsistent results. They concur in that rearranged Ig and T-cell receptor (TCR) genes are not demonstrable in most Hodgkin's disease (HD) cases. To clarify whether this is because of the insensitivity of the method of detection or a real absence of clonal Ig heavy chain (IgH) rearrangements, a polymerase chain reaction (PCR) method with high sensitivity was applied, allowing the detection of less than 50 cells with clonally rearranged IgH genes in a mixture of 100,000 germline or individually rearranged cells. In 67 cases of HD, most of those (67%) with B-Ag+ HRS cells express clonal VDJ rearrangements of the IgH gene. No cases with T-cell Ag+ HRS cells harbored detectable clonal VDJ rearrangements. Of 10 sequenced rearranged IgH genes, the VH segment of six contained considerable somatic mutations. These results suggest that the demonstrated VDJ rearrangements stem from the HRS cells themselves and that the HRS cells of cases with rearranged IgH genes are B-cell related and correspond in their differentiation stage either to naive pregerminal center B cells or (more commonly) to germinal center/postgerminal center-derived memory B cells.

Amino Acid Sequence↗
PubMed1999

Molecular IgV(H) analysis demonstrates highly somatic mutated B cells in synovialitis of osteoarthritis: a degenerative disease is associated with a specific, not locally generated immune response.

In osteoarthritis (OA), the synovial tissue exhibits a nonfollicular inflammatory infiltration with a characteristic arrangement of lymphocytes and plasma cells. These arrangements are either small perivascular aggregates with plasma cells surrounding the lymphocytes or small groups of plasma cells, located in the vicinity of small blood vessels. These patterns suggest that B lymphocytes directly differentiate into plasma cells. To understand the B-cell response in OA, we analyzed the V(H) genes from B cells of synovial tissue of nine OA patients (average age, 71.5+/-10.5 years; six female and three male). V(H) gene repertoires were determined from RNA prepared from tissue cryosections and from DNA of single isolated B lymphocytes and plasma cells. The inflammatory infiltrate was analyzed immunohistochemically by detecting CD20, Ki-M4 (follicular dendritic cells), CD4, IgG, IgM, IgA, Ki-67, and by simultaneous demonstration of the plasma-cell-specific antigen CD138 (syndecan-1) and factor VIII. The molecular data demonstrate B cells with a high number of somatic mutations (average, 16.5 to 19.8), and high ratios of replacement to silent mutations in the small lymphocytic/plasmacellular aggregates of OA. In the tissue cryosections, the values of the sigmaR/sigmaS at the complementarity determining regions were 5.3 and 2.0 in the framework regions. For both the isolated B lymphocytes and plasma cells, the value of this ratio in the complementarity determining regions was 3.5. In the framework regions, the values of this ratio were 2.0 for the isolated B cells and 1.8 for the plasma cells. B lymphocytes and plasma cells exhibited a distribution not described thus far. Two patterns of B-cell distribution could be observed: (a) Centrally located CD20+ B and CD4+ and CD8+ T lymphocytes were surrounded directly by IgG (predominantly) or IgA and IgM plasma cells. No proliferating Ki-67-positive cells and no follicular dendritic cells (germinal centers) could be detected in the aggregates; (b) Plasma cells (predominantly IgG) were located directly near endothelial cells of small blood vessels. The finding of highly mutated V(H) genes in B lymphocytes and the characteristic arrangement of B lymphocytes and plasma cells suggests that B cells, which participate in OA synovialitis, have undergone germinal center reaction at different sites. This may explain the low inflammatory infiltration without germinal centers in OA, which is a feature of this primarily degenerative joint disease.

Aged↗
PubMed1998

Shift from Rh-positive to Rh-negative phenotype caused by a somatic mutation within the RHD gene in a patient with chronic myelocytic leukaemia.

We report a female patient whose Rh phenotype shifted from RhD-positive to RhD-negative over a 3-year period (1991-94), during which time she was treated with mastectomy (1992) and local irradiation for a low-grade recurrent breast cancer. She was diagnosed with chronic myeloid leukaemia in 1994, and has since then received chemotherapy. The patient was repeatedly typed as O, RhD-positive between 1965 and 1991 and was repeatedly found RhD-negative after 1994. Bcr-Abl transcripts typical of Ph1 chromosome were detected. Molecular analysis indicated that the patient was heterozygous at the RH locus, carrying one haplotype in which the RHD gene exhibited a single nucleotide deletion (G600) resulting in a frameshift and premature stop codon, and a normal RHCE gene (allele Ce). The second haplotype contained only the RHCE gene (allele ce) and was normal. Further analysis carried out on total leucocytes, purified neutrophils, EBV-lymphoblastoid cell line and cultured erythroblasts indicated that the G600 deletion was restricted to the myeloid lineage. No modification of other blood group antigens could be detected. These findings suggest a somatic mutation which most probably occurred in a stem cell common to the myeloid lineage.

Aged↗
PubMed2000

Lack of somatic mutation in the coding sequence of SIAH1 in tumors hemizygous for this candidate tumor suppressor gene.

The human homolog of the Drosophila seven in absentia gene (SIAH1) may play an important role in apoptosis and tumor suppression. Transcription of SIAH1 is up-regulated in non-tumorigenic clonal populations of cells derived from 2 different tumorigenic parental cell lines. Intracellular SIAH1 mRNA concentration increases in intestinal cells as they migrate from the bottom of the crypt to the lumen, where they undergo apoptosis. Finally, SIAH1 is located on chromosome 16q12-q13, a region that is frequently deleted in a large variety of human tumors. These observations suggest SIAH1 as a candidate tumor suppressor gene that may be inactivated during tumorigenesis. To test this hypothesis, a search for mutation in the coding sequence of this gene was initiated in tumors exhibiting loss of heterozygosity (LOH) at 16q12-q13. No difference was found in 12 hepatocellular carcinomas, 19 breast carcinomas, 9 prostate carcinomas, 7 colon carcinomas and 5 human cell lines derived from colon cancer. One silent sequence variant (C to T transition at amino acid 270) was observed in the FET colon carcinoma cell line. It was subsequently found once in a group of 100 unrelated individuals from the CEPH families. A rapid real-time quantitative RT-PCR fluorescent method shows that SIAH1 remained transcriptionally active in the 6 colon cancer-derived cell lines, and the expression is comparable to the normal colon tissue. Taken together, these observations suggest that although many tumors may have lost one SIAH1 allele, the second allele would not be the site of frequent somatic mutations and may even remain functional.

Alleles↗
PubMed1982

Human lymphocytes resistant to 6-thioguanine: restrictions in the use of a test for somatic mutations arising in vivo studied by flow-cytometric enrichment of resistant cell nuclei.

A method is presented that facilitates the enumeration of 6-thioguanine(TG)-resistant human lymphocytes from peripheral blood. By the use of flow cytometry, nuclei from cells stimulated to divide in vitro and having reached their late S and G2 phases of growth, were sorted out from cultures incubated for 48 h, the last 6 h in medium containing [3H]thymidine. The out-sorted nuclei were subjected to autoradiography, and the labelling index was determined. The frequency of TG-resistant cells was calculated from the quotient (labelling index in the presence of TG/labelling index in the absence of TG). Over a period of several months, two blood donors serving as internal standards showed a variation of 20% in their frequencies of resistant lymphocytes. In a referent group of 48 blood donors, inter-individual differences in the frequency of resistant cells (range 10(-5)-10(-4) ) were established. There was a positive correlation between an increased frequency of resistant cells and the age of the blood donors. Patients treated with cytostatics or psoralen and ultraviolet radiation (PUVA) showed elevated frequencies of resistant cells (about 10(-3)-10(-2) ). We suggest that at least a part of the resistant cells scored are phenocopies and thus have not originated from somatic mutation. The high number of phenocopies after treatments with cytostatics or PUVA may depend on a temporary depletion from the cells of functioning HGPRT enzyme owing to a transcriptional block caused by the agents combined with a rather fast turnover of the enzyme. We conclude that there are restrictions in the use of this system for determining genotoxic effects in vivo.

Cell Separation↗
PubMed1996

IgM-producing chronic lymphocytic leukemia cells undergo immunoglobulin isotype-switching without acquiring somatic mutations.

The malignant B cells in chronic lymphocytic leukemia (CLL) typically express low-density membrane IgM or IgM/IgD. In vitro experiments have shown that the CLL cells can be induced to differentiate into cells that secrete immunoglobulin (Ig) and can occasionally undergo heavy (H) chain class switching. We now show that the CLL cells also undergo isotype-switching in vivo, since gamma and/or alpha H chain transcripts with identical FW3/CDR3/FW4 regions as the mu CLL transcripts were detected in all of the 13 investigated patients with IgM+ CLL. In most cases switching had occurred to alpha1 and gamma3, but CLL transcripts corresponding to the other gamma chain isotypes were also detected. In one case both the productively and nonproductively rearranged allele were found to undergo H chain class switching. CLL gamma transcripts were also present in surface IgG+ sorted B cells, demonstrating that a small subset of the CLL cells express membrane IgG. In addition, transcripts encoding secretary gamma2 and gamma3 H chains were detected in two cases, which suggests that some serum IgG could be produced by the leukemic clone. Analysis of sorted PBL showed that isotype-switching occurs in CLL cells that express the CD5 antigen. Finally, nucleotide sequence analysis showed that the mu, alpha, and gamma CLL transcripts are identical, demonstrating that the CLL cells do not accumulate somatic mutations in their variable region genes after the H chain class switching. These data provide in vivo evidence that isotype-switching is a frequent phenomenon in CLL, and indicate that a subset of the CLL lymphocytes progress to later stages of B cell differentiation.

Base Sequence↗
PubMedDate not supplied

[Long-term effects of chronic radiation exposure on the level of somatic mutations in peripheral blood cells].

The level of comatic mutations was studied at GPA locus and in the TCR system at late times since the onset of radiation exposure in persons exposed to chronic radiation accidents at the Mayak Production Association in the period 1949-1952. Individuals with predominantly external gamma-exposure and those with predominantly internal (mainly due to 90Sr) exposures have been identified among the exposed population. The average dose to red bone marrow made up 121.5 cSv with individual values ranging from 11.0 to 462.7 cSv. An increased frequency of TCR-mutant lymphocytes was noted for exposed subjects. The level of GPA-variant erythrocytes of different types in the study group did not differ from the respective value registered in control. The analysis of TCR status showed no dependence of mutant cell frequency on exposure dose. The assumption is made that the rate of exposure dose and the nature of its formation (chronic exposure) exert a decisive influence on the level of somatic mutations in peripheral blood cells in humans.

Glycophorins↗
PubMed1993

A minor group of rheumatoid factors isolated from a patient with rheumatoid arthritis is derived from somatically mutated Vk1 genes further evidence that rheumatoid factors during autoimmune diseases undergo an antigen driven maturation.

To better understand the structural basis for rheumatoid factor [RF] activity and the origin of autoantibodies in human autoimmune diseases, we isolated the RF producing B cells from the peripheral blood and from the synovial fluid of a patient suffering from rheumatoid arthritis [RA]. We previously demonstrated that a significant fraction of these RF were derived from three V kappa III genes known to encode most of the monoclonal RF light chain variable regions. To get more insight into the actual repertoire of RF-V kappa genes during RA, we analyzed the nucleotide sequences of RF light chain variable regions of other V kappa families. Using two sets of polymerase chain reactions in order to amplify the cDNA derived from RF producing cells from the same patient KRA, we isolated only three different rearranged V kappa-J kappa complexes: slkv5, slkv7 and bkv42, all derived from V kappa I germ-line genes not previously known to be associated with RF activity; this suggests that the repertoire of VL genes coding for RF during RA is more diverse than the one involved in the generation of paraprotein RF during monoclonal lymphoid proliferations, although there remains a possible bias in favor of the V kappa III family. Moreover, each of these genes is somatically mutated with a pattern suggesting a selective pressure of the antigen. Particularly interesting is the additional proline residue at the V kappa-J kappa junction of bkv42, an unorthodox feature that we found previously in more than 50% of RF V kappa III-J kappa gene complexes. Finally, the homogeneity of some non conservative mutations suggests the existence of a restricted set of pathogenic epitopes driving the production of RF during RA.

Amino Acid Sequence↗
PubMed1981

Heavy chain variable region contribution to the NPb family of antibodies: somatic mutation evident in a gamma 2a variable region.

To examine germ line genes of the heavy chain variable region (VH) that might contribute to formation of antibodies of the NPb family, we have derived cDNA clones from two hybridomas making NPb antibodies. One, B1-8, made an IgM protein and was derived during a primary response; the other, S43, made an IgG2a protein and was derived during a hyperimmune response. Sequence comparison of the two clones showed that they differed by only 10 bp in the VH region, had very different D segments and had identical J segments (J2). A set of closely related germ line VH genes was then cloned from a partial Eco RI library of C57Bl/6 DNA. By comparing the germ line VH regions to the cDNA VH regions, we identified seven potential candidates for encoding the VH regions of NPb antibodies. The seven VH regions were sequenced, and one V(186-2) contained exactly the DNA sequence found in the clone derived from B1-8. None of the DNA sequence differences that distinguished the S43-derived clone from the B1-8 clone was found in any of the other six germ line genes. Because the S43 sequence was more closely related to the V(186-2) germ line sequence than to any of the other VH genes, we conclude that the differences between the genes resulted from somatic mutation and that the two hybridomas derived their VH regions from the same germ line gene. Certain of the sequenced VH genes contain crippling mutations; the repertoire of germ line VH genes that can contribute to the diversity of antibodies may therefore be less than the total number of genes detectable by hydridization.

Animals↗
PubMed2001

A stochastic model of cell replicative senescence based on telomere shortening, oxidative stress, and somatic mutations in nuclear and mitochondrial DNA.

Human diploid fibroblast cells can divide for only a limited number of times in vitro, a phenomenon known as replicative senescence or the Hayflick limit. Variability in doubling potential is observed within a clone of cells, and between two sister cells arising from a single mitotic division. This strongly suggests that the process by which cells become senescent is intrinsically stochastic. Among the various biochemical mechanisms that have been proposed to explain replicative senescence, particular interest has been focussed on the role of telomere reduction. In the absence of telomerase--an enzyme switched off in normal diploid fibro-blasts-cells lose telomeric DNA at each cell division. According to the telomere hypothesis of cell senescence, cells eventually reach a critically short telomere length and cell cycle arrest follows. In support of this concept, forced expression of telomerase in normal fibroblasts appears to prevent cell senescence. Nevertheless, the telomere hypothesis in its basic form has some difficulty in explaining the marked stochastic variations seen in the replicative lifespans of individual cells within a culture, and there is strong empirical and theoretical support for the concept that other kinds of damage may contribute to cellular ageing. We describe a stochastic network model of cell senescence in which a primary role is played by telomere reduction but in which other mechanisms (oxidative stress linked particularly to mitochondrial damage, and nuclear somatic mutations) also contribute. The model gives simulation results that are in good agreement with published data on intra-clonal variability in cell doubling potential and permits an analysis of how the various elements of the stochastic network interact. Such integrative models may aid in developing new experimental approaches aimed at unravelling the intrinsic complexity of the mechanisms contributing to human cell ageing.

Cell Culture Techniques↗
PubMed2002

Spiradenocylindroma of the kidney: clinical and genetic findings suggesting a role of somatic mutation of the CYLD1 gene in the oncogenesis of an unusual renal neoplasm.

We describe the morphology and comparative genomic hybridization findings in a tumor for which we propose the term "spiradenocylindroma" of the kidney. The tumor arose in the wall of a renal cyst in an otherwise healthy male patient who had a favorable clinical course after nephrectomy. Tumor cells formed either large nodules exhibiting a solid or trabecular architecture with conspicuous perivascular spaces or cylindromatous small tumor cell islands arranged in a jigsaw pattern. Focally, there were interspersed tubular structures and tumor cell rosettes with central deposits of periodic acid-Schiff-positive material. A minor tumor component showed epidermoid differentiation. The tumor cells were strongly positive for cytokeratins 5/6, high molecular weight cytokeratins 34betaE12 and AE1/3, and E-cadherin, but only weakly positive for cytokeratins 7, 8, 18, 19, and epithelial membrane antigen. Focal reactivity for actin, vimentin, and S-100 protein or lysozyme and alpha 1 -antichymotrypsin within tubular and cylindromatous areas suggested myoepithelial and apocrine differentiation, respectively. By comparative genomic hybridization, the only abnormality was loss of the long arm of chromosome 16 and gain of genetic material on the short arm of chromosome 16, suggesting isochromosome i(16p). This finding is unique among renal neoplasms and implies loss of heterozygosity at 16q12-13 of the CYLD1 gene that is critically involved in the oncogenesis of familial cylindromatosis and some sporadic spiradenocylindromas. We conclude that somatic mutation of the CYLD1 gene outside the skin can have a role in the oncogenesis of tumors with cylindromatous features.

Carcinoma, Adenoid Cystic↗
PubMed2001

Association of CYP1B1 codon 432 mutant allele in head and neck squamous cell cancer is reflected by somatic mutations of p53 in tumor tissue.

Tobacco use is causally associated with head and neck squamous cell cancer (HNSCC). Here, we present the results of a case-control study that investigated the effects that the genetic variants of the cytochrome (CYP)1A1, CYP1B1, glutathione-S-transferase (GST)M1, GSTT1, and GSTP1 genes have on modifying the risk of smoking-related HNSCC. Allelisms of the CYP1A1, GSTT1, GSTM1, and GSTT1 genes alone were not associated with an increased risk. CYP1B1 codon 432 polymorphism was found to be a putative susceptibility factor in smoking-related HNSCC. The frequency of CYP1B1 polymorphism was significantly higher (P < 0.001) in the group of smoking cases when compared with smoking controls. Additionally, an odds ratio (OR) of 4.53 (2.62-7.98) was discovered when investigating smoking and nonsmoking cases for the susceptible genotype CYP1B1*2/*2, when compared with the presence of the genotype wild type. In combination with polymorphic variants of the GST genes, a synergistic-effect OR was observed. The calculated OR for the combined genotype CYP1B1*2/*2 and GSTM1*2/*2 was 12.8 (4.09-49.7). The calculated OR for the combined genotype was 13.4 (2.92-97.7) for CYP1B1*2/*2 and GSTT1*2/*2, and 24.1 (9.36-70.5) for the combination of CYP1B1*2/*2 and GSTT1-expressors. The impact of the polymorphic variants of the CYP1B1 gene on HNSCC risk is reflected by the strong association with the frequency of somatic mutations of the p53 gene. Smokers with susceptible genotype CYP1B1*2/*2 were 20 times more likely to show evidence of p53 mutations than were those with CYP1B1 wild type. Combined genotype analysis of CYP1B1 and GSTM1 or GSTT1 revealed interactive effects on the occurrence of p53 gene mutations. The results of the present study indicate that polymorphic variants of CYP1B1 relate significantly to the individual susceptibility of smokers to HNSCC.

Alleles↗
PubMed1995

Comprehensive epitope analysis of monoclonal anti-proenkephalin antibodies using phage display libraries and synthetic peptides: revelation of antibody fine specificities caused by somatic mutations in the variable region genes.

Filamentous phage libraries, displaying 6, 12 or 20 amino acid residue peptides at the N terminus of coat protein pIII were used to define and localize the epitopes of 15 monoclonal antibodies raised against human proenkephalin, a neuropeptide precursor. Eight monoclonal antibodies (PE14 to PE19, PE23 and PE25), which inhibit each other's binding to proenkephalin, recognized phage clones selected by PE14, PE15, PE19, PE23 and PE25. With the peptide sequences DLL(X)(X)LL (12mer library) and DLL(X)(X)L (6mer library) shared by most of the phage clones it was possible to define the putative antibody epitope 155DLLKELL161 on human proenkephalin. For five antibodies (PE13, PE20 to PE22 and PE24) belonging to another inhibition group, a common consensus motif G(X)D(X)E(X)(X)V(X)(X)R could be defined with help of a 20mer library. The corresponding minimum epitope sequence has been found to be 175GSDNEEEVSKR185. Antibody PE1, raised in a separate fusion, was able to select phage clones from a 12mer and 20mer library, revealing that the sequence 187GGFMRG192 is probably the antibody epitope. The assumed localization of the epitopes was confirmed by screening a set of overlapping synthetic peptides, covering the region of human proenkephalin thought to contain all antibody binding sites. It was found that antibodies, although recognizing the same epitope, gave different binding patterns with the selected phage clones. By analysing the VH chain sequences of these antibodies it could be shown that a varying number of somatic mutations is likely to be the reason for the observed differences in antibody fine specificity.

Amino Acid Sequence↗
PubMed1993

Variable region genes of anti-HIV human monoclonal antibodies: non-restricted use of the V gene repertoire and extensive somatic mutation.

The extent of the expressed human V gene repertoire for the most part has been derived from fetal cDNA libraries, autoantibodies, and myeloma proteins. In order to continue to explore the utilization of the VH and VL gene repertoire in response to exogenous viral antigens, the heavy and light chain cDNAs from four human anti-HIV monoclonal antibodies were PCR amplified from human-mouse heterohybridomas, cloned, and nucleotide sequence analysis performed. Of the monoclonals analyzed, three were directed against gp120 and one reacted with gp41. Three of the antibodies were of the IgG1 lambda isotype and one was an IgG1 kappa. Three of the four heavy chains were derived from VHI gene segments and one VHII was observed. D segments showed evidence of D-D joining and three JH4 and one JH5 gene were utilized. Two V lambda II lambda chains and one from the V lambda III gene family were observed and the single kappa chain sequenced was from the V kappa III family. DNA sequence comparison with known germline gene segments identified putative precursor V gene segments for one of the heavy chains and two light chains. Comparison of the expressed amino acid sequences with the predicted germline sequences indicated that changes were clustered in the CDRs and FR3 regions of the V gene segments. We reported previously the nucleotide sequences of five human monoclonal antibodies from HIV-infected individuals, three of which utilized VHIV, one VHV and one a VHI gene segment and also found extensive evidence of somatic mutation. Collectively, our results indicate that an antigen driven response is functioning following HIV infection and, surprisingly, to date we have not encountered a VHIII gene segment. Since VHIII is the largest human VH gene family, it may well be that this under-representation has both functional and clinical implications.

Amino Acid Sequence↗