scholarly journals Evidence supporting somatic assembly of the DNA segments (minigenes), coding for the framework, and complementarity-determining segments of immunoglobulin variable regions.

1979 ◽  
Vol 149 (6) ◽  
pp. 1299-1313 ◽  
Author(s):  
E A Kabat ◽  
T T Wu ◽  
H Bilofsky
Keyword(s):  
Adult Mouse ◽  
Germ Line ◽  
Variable Region ◽  
Single Copy ◽  
Variable Regions ◽  
X Ray ◽  
V Genes ◽  
V Region ◽  
Complementarity Determining Regions ◽  
The One

Two sets of apparently conflicting data on the genes coding for the variable region are being accumulated. One suggests that the sets of nucleotides coding for the framework segments of immunoglobulin light and heavy (VL and VH) chains assort independently and are therefore germ-line minigenes which, together with sets of nucleotides coding for the complementarity-determining regions (CDR) or segments assemble to form complete variable (V)-region genes (15, 16, 33). The other, based on the findings with clones from 12-d-old embryo and adult mouse coding for V-regions, infer that the first three frameworks and the three complementarity-determining segments are already assembled as germ-line V-genes (17-21). It is now generally accepted that the J segment, which in the one instance sequenced (21) is made up of nucleotides coding for framework (FR)4 plus two residues of CDR3, is a minigene. An examination of sequences of human, mouse, and rabbit V-regions, assuming the latter hypothesis, indicates that individual framework sets would have to be present in many copies. The FR2 segment found in one human, 20 mice, and 13 rabbits would have to be present in at least 10/14 copies in the NZB, and 5/6 in the BALB/c mouse, and 12/13 in the rabbit. The X-ray crystallographic data show this region to be a loop, projecting out from the V-domain, capable of accommodating many substiutions and 12 and 8 alternative sequences for this FR2 segment have been found in mouse and rabbit VK chains with substitutions possible at 13 of the 15 positions. These alternative sequences occur much less frequently than the preserved FR2 segment. Thus, there is no basis in the protein structure to account for evolutionary stability of this FR2 segment if it occurs in so many copies in germ-line genes coding for residues 1-96, but its stability is easily explained if it were coded for by a separate germ-line minigene present as a single copy; the alternative forms could then have arisen by duplication and mutation of this minigene. Somatic assembly of the minigene segments for the three framework and three complementarity-determining segments during differentiation would account completely for our assortment data from which FR4 was inferred to be a minigene.

scholarly journals Disjoint combinations profiling (DCP): a new method for the prediction of antibody CDR conformation from sequence

2014 ◽  
Author(s):  
Dimitris Nikoloudis ◽  
Jim E. Pitts ◽  
José W. Saldanha
Keyword(s):  
Protein Engineering ◽  
Prediction Accuracy ◽  
Variable Region ◽  
New Method ◽  
Engineering Applications ◽  
Variable Regions ◽  
Blind Prediction ◽  
Novel Method ◽  
Complementarity Determining Regions ◽  
Sequence Rules

The accurate prediction of the conformation of Complementarity-Determining Regions (CDRs) is important in modelling antibodies for protein engineering applications. Specifically, the Canonical paradigm has proved successful in predicting the CDR conformation in antibody variable regions. It relies on canonical templates which detail allowed residues at key positions in the variable region framework or in the CDR itself for 5 of the 6 CDRs. While no templates have as yet been defined for the hypervariable CDR-H3, instead, reliable sequence rules have been devised for predicting the base of the CDR-H3 loop. Here a new method termed Disjoint Combinations Profiling (DCP) is presented, which contributes a considerable advance in the prediction of CDR conformations. This novel method is explained and compared with canonical templates and sequence rules in a 3-way blind prediction. DCP achieved 93% accuracy over 951 blind predictions and showed an improvement in cumulative accuracy compared to predictions with canonical templates or sequence-rules. In addition to its overall improvement in prediction accuracy, it is suggested that DCP is open to better implementations in the future and that it can improve as more antibody structures are deposited in the databank. In contrast, it is argued that canonical templates and sequence rules may have reached their peak.

scholarly journals Disjoint combinations profiling (DCP): a new method for the prediction of antibody CDR conformation from sequence.

2014 ◽  
Author(s):  
Dimitris Nikoloudis ◽  
Jim E. Pitts ◽  
José W. Saldanha
Keyword(s):  
Protein Engineering ◽  
Prediction Accuracy ◽  
Variable Region ◽  
New Method ◽  
Engineering Applications ◽  
Variable Regions ◽  
Blind Prediction ◽  
Novel Method ◽  
Complementarity Determining Regions ◽  
Sequence Rules

The accurate prediction of the conformation of Complementarity-Determining Regions (CDRs) is important in modelling antibodies for protein engineering applications. Specifically, the Canonical paradigm has proved successful in predicting the CDR conformation in antibody variable regions. It relies on canonical templates which detail allowed residues at key positions in the variable region framework or in the CDR itself for 5 of the 6 CDRs. While no templates have as yet been defined for the hypervariable CDR-H3, instead, reliable sequence rules have been devised for predicting the base of the CDR-H3 loop. Here a new method termed Disjoint Combinations Profiling (DCP) is presented, which contributes a considerable advance in the prediction of CDR conformations. This novel method is explained and compared with canonical templates and sequence rules in a 3-way blind prediction. DCP achieved 93% accuracy over 951 blind predictions and showed an improvement in cumulative accuracy compared to predictions with canonical templates or sequence-rules. In addition to its overall improvement in prediction accuracy, it is suggested that DCP is open to better implementations in the future and that it can improve as more antibody structures are deposited in the databank. In contrast, it is argued that canonical templates and sequence rules may have reached their peak.

scholarly journals Highly sensitive and unbiased approach for elucidating antibody repertoires

2016 ◽  
Vol 113 (28) ◽  
pp. 7846-7851 ◽  
Author(s):  
Sherry G. Lin ◽  
Zhaoqing Ba ◽  
Zhou Du ◽  
Yu Zhang ◽  
Jiazhi Hu ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Germ Line ◽  
Variable Region ◽  
Cell Receptor ◽  
Affinity Maturation ◽  
Antigen Binding ◽  
Variable Regions ◽  
Antibody Repertoires ◽  
B Lineage

Developing B lymphocytes undergo V(D)J recombination to assemble germ-line V, D, and J gene segments into exons that encode the antigen-binding variable region of Ig heavy (H) and light (L) chains. IgH and IgL chains associate to form the B-cell receptor (BCR), which, upon antigen binding, activates B cells to secrete BCR as an antibody. Each of the huge number of clonally independent B cells expresses a unique set of IgH and IgL variable regions. The ability of V(D)J recombination to generate vast primary B-cell repertoires results from a combinatorial assortment of large numbers of different V, D, and J segments, coupled with diversification of the junctions between them to generate the complementary determining region 3 (CDR3) for antigen contact. Approaches to evaluate in depth the content of primary antibody repertoires and, ultimately, to study how they are further molded by secondary mutation and affinity maturation processes are of great importance to the B-cell development, vaccine, and antibody fields. We now describe an unbiased, sensitive, and readily accessible assay, referred to as high-throughput genome-wide translocation sequencing-adapted repertoire sequencing (HTGTS-Rep-seq), to quantify antibody repertoires. HTGTS-Rep-seq quantitatively identifies the vast majority of IgH and IgL V(D)J exons, including their unique CDR3 sequences, from progenitor and mature mouse B lineage cells via the use of specific J primers. HTGTS-Rep-seq also accurately quantifies DJH intermediates and V(D)J exons in either productive or nonproductive configurations. HTGTS-Rep-seq should be useful for studies of human samples, including clonal B-cell expansions, and also for following antibody affinity maturation processes.

scholarly journals Disjoint combinations profiling (DCP): a new method for the prediction of antibody CDR conformation from sequence.

2014 ◽  
Author(s):  
Dimitris Nikoloudis ◽  
Jim E. Pitts ◽  
José W. Saldanha
Keyword(s):  
Protein Engineering ◽  
Prediction Accuracy ◽  
Variable Region ◽  
New Method ◽  
Engineering Applications ◽  
Variable Regions ◽  
Blind Prediction ◽  
Novel Method ◽  
Complementarity Determining Regions ◽  
Sequence Rules

The accurate prediction of the conformation of Complementarity-Determining Regions (CDRs) is important in modelling antibodies for protein engineering applications. Specifically, the Canonical paradigm has proved successful in predicting the CDR conformation in antibody variable regions. It relies on canonical templates which detail allowed residues at key positions in the variable region framework or in the CDR itself for 5 of the 6 CDRs. While no templates have as yet been defined for the hypervariable CDR-H3, instead, reliable sequence rules have been devised for predicting the base of the CDR-H3 loop. Here a new method termed Disjoint Combinations Profiling (DCP) is presented, which contributes a considerable advance in the prediction of CDR conformations. This novel method is explained and compared with canonical templates and sequence rules in a 3-way blind prediction. DCP achieved 93% accuracy over 951 blind predictions and showed an improvement in cumulative accuracy compared to predictions with canonical templates or sequence-rules. In addition to its overall improvement in prediction accuracy, it is suggested that DCP is open to better implementations in the future and that it can improve as more antibody structures are deposited in the databank. In contrast, it is argued that canonical templates and sequence rules may have reached their peak.

scholarly journals Disjoint combinations profiling (DCP): a new method for the prediction of antibody CDR conformation from sequence

2014 ◽  
Author(s):  
Dimitris Nikoloudis ◽  
Jim E. Pitts ◽  
José W. Saldanha
Keyword(s):  
Protein Engineering ◽  
Prediction Accuracy ◽  
Variable Region ◽  
New Method ◽  
Engineering Applications ◽  
Variable Regions ◽  
Blind Prediction ◽  
Novel Method ◽  
Complementarity Determining Regions ◽  
Sequence Rules

The accurate prediction of the conformation of Complementarity-Determining Regions (CDRs) is important in modelling antibodies for protein engineering applications. Specifically, the Canonical paradigm has proved successful in predicting the CDR conformation in antibody variable regions. It relies on canonical templates which detail allowed residues at key positions in the variable region framework or in the CDR itself for 5 of the 6 CDRs. While no templates have as yet been defined for the hypervariable CDR-H3, instead, reliable sequence rules have been devised for predicting the base of the CDR-H3 loop. Here a new method termed Disjoint Combinations Profiling (DCP) is presented, which contributes a considerable advance in the prediction of CDR conformations. This novel method is explained and compared with canonical templates and sequence rules in a 3-way blind prediction. DCP achieved 93% accuracy over 951 blind predictions and showed an improvement in cumulative accuracy compared to predictions with canonical templates or sequence-rules. In addition to its overall improvement in prediction accuracy, it is suggested that DCP is open to better implementations in the future and that it can improve as more antibody structures are deposited in the databank. In contrast, it is argued that canonical templates and sequence rules may have reached their peak.

scholarly journals Sequence intrinsic somatic mutation mechanisms contribute to affinity maturation of VRC01-class HIV-1 broadly neutralizing antibodies

2017 ◽  
Vol 114 (32) ◽  
pp. 8614-8619 ◽  
Author(s):  
Joyce K. Hwang ◽  
Chong Wang ◽  
Zhou Du ◽  
Robin M. Meyers ◽  
Thomas B. Kepler ◽  
...  
Keyword(s):  
B Cells ◽  
Neutralizing Antibodies ◽  
Somatic Hypermutation ◽  
Human Subjects ◽  
Variable Region ◽  
Affinity Maturation ◽  
Broadly Neutralizing Antibodies ◽  
Variable Regions ◽  
Complementarity Determining Regions ◽  
Hiv 1

Variable regions of Ig chains provide the antigen recognition portion of B-cell receptors and derivative antibodies. Ig heavy-chain variable region exons are assembled developmentally from V, D, J gene segments. Each variable region contains three antigen-contacting complementarity-determining regions (CDRs), with CDR1 and CDR2 encoded by the V segment and CDR3 encoded by the V(D)J junction region. Antigen-stimulated germinal center (GC) B cells undergo somatic hypermutation (SHM) of V(D)J exons followed by selection for SHMs that increase antigen-binding affinity. Some HIV-1–infected human subjects develop broadly neutralizing antibodies (bnAbs), such as the potent VRC01-class bnAbs, that neutralize diverse HIV-1 strains. Mature VRC01-class bnAbs, including VRC-PG04, accumulate very high SHM levels, a property that hinders development of vaccine strategies to elicit them. Because many VRC01-class bnAb SHMs are not required for broad neutralization, high overall SHM may be required to achieve certain functional SHMs. To elucidate such requirements, we used a V(D)J passenger allele system to assay, in mouse GC B cells, sequence-intrinsic SHM-targeting rates of nucleotides across substrates representing maturation stages of human VRC-PG04. We identify rate-limiting SHM positions for VRC-PG04 maturation, as well as SHM hotspots and intrinsically frequent deletions associated with SHM. We find that mature VRC-PG04 has low SHM capability due to hotspot saturation but also demonstrate that generation of new SHM hotspots and saturation of existing hotspot regions (e.g., CDR3) does not majorly influence intrinsic SHM in unmutated portions of VRC-PG04 progenitor sequences. We discuss implications of our findings for bnAb affinity maturation mechanisms.

scholarly journals Generation of antibody diversity in the immune response of BALB/c mice to influenza virus hemagglutinin. I. Significant variation in repertoire expression between individual mice

1983 ◽  
Vol 157 (2) ◽  
pp. 687-704 ◽  
Author(s):  
LM Staudt ◽  
W Gerhard
Keyword(s):  
Immune Response ◽  
Influenza Virus ◽  
Influenza A ◽  
Germ Line ◽  
Cross Reactivity ◽  
Influenza Viruses ◽  
Antibody Diversity ◽  
V Genes ◽  
V Region ◽  
Individual Adult

The paratypic and idiotypic diversity of the BALB/c antibody response to the hemagglutinin (HA) of the influenza A/PR/8/34 virus (PR8) was investigated using a panel of 125 anti-HA hybridoma antibodies derived from 14 BALB/c mice. The paratypic diversity, as assessed by a fine specificity analysis using 51 related influenza viruses, was extensive: 104 distinct paratopes were observed. In three instances, antibodies with indistinguishable paratopes were isolated from two individual mice. A minimum estimate of the size of the adult BALB/c anti-HA paratypic repertoire, calculated from these data, is 1,500. The generation of this diverse repertoire was studied by screening the anti-HA hybridoma panel for the presence of idiotypes (Id) that are markers for variable (V) region sequences derived from related germ line V genes. Three cross-reactive Id (IdX) that are markers for the V(k)21C, V(k)21B, and V(k)21A, D, E, or F L chain subgroups were found, respectively on 16, 1, and 10 anti-HA hybridoma antibodies derived from seven individual BALB/c mice. Thus, the V(k)21 IdX(+) hybridomas constitute 22 percent of the anti-HA hybridoma panel. The V(k)21 IdX are also present on 8.6 percent of K-bearing immunoglobulin in normal BALB/c serum. This suggests that the V(k)21 group is used preferentially in the BALB/c anti-HA immune response. The generation of the anti-HA repertoire was further studied using large panels of anti-HA hybridomas derived from two individual adult BALB/c mice. Anti-idiotypic antisera were raised in rabbits against individual hybridomas from each mouse. One anti-Id serum defined a family of four idiotypically and paratypically related, but not identical, antibodies from mouse 36, which represented 31 percent of the hybridoma antibodies isolated from this mouse. None of the 112 anti-HA hybridoma antibodies derived from 13 other individual mice showed idiotypic cross-reactivity. Furthermore, this Id could not be detected in anti-PR8 antisera from 75 individual BALB/c mice. Another anti-Id serum defined a family of 27 idiotypically related antibodies from mouse 37, which represented 50 percent of the hybridoma antibodies isolated from this mouse. Only 1 of the 71 hybridoma antibodies isolated from 13 other individuals was idiotypically cross-reactive. These results demonstrate that individual adult BALB/c mice express paratypically and idiotypically distinct antibody repertoires to the HA of influenza virus PR8. Based on these observations, we suggest that somatic mutation plays an important role in the generation of the adult anti-HA repertoire. Mechanisms that could account for differences in repertoire expression among individual mice are discussed.

The Functional Role of Acquired N-Linked Glycosylation Sites On Follicular Lymphoma B Cell Antigen Receptors.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2704-2704
Author(s):  
Dunja Schneider ◽  
Hendrik Veelken ◽  
Hassan Jumaa
Keyword(s):  
Immune System ◽  
B Cells ◽  
B Cell ◽  
Innate Immune System ◽  
Germ Line ◽  
Variable Region ◽  
Innate Immune ◽  
Variable Regions ◽  
Glycosylation Sites

Abstract Abstract 2704 Follicular lymphoma (FL) is an indolent B-cell lymphoma characterized by apoptosis resistance due to overexpression of Bcl-2 as a consequence of the t(14;18) translocation, ongoing somatic hypermutation (SHM), and expression of B-cell receptors (BCR) with glycosylation of the antigen binding sites. Translocation and concomitant Bcl-2 overexpression can be found in healthy human blood B cells and is insufficient to drive lymphoma outgrowth in mouse models. Since most FL cells still express a surface B cell receptor (BCR) despite the disruption of one immunoglobulin heavy chain allele by the t(14;18) translocation, expression of an antigen receptor seems to be indispensable for FL development. Around 80% of FLs possess asparagine (N)-linked glycosylation sites (amino acid sequence: N-X-S/T) in their BCR variable regions that are not encoded in germ-line but are acquired through SHM. In contrast to germ-line-encoded glycosylation sites in the constant BCR region, where normal processing of the glycans results in termination on branched sugars like sialic acid, the variable region glycosylation sites carry mannose-terminating sugars. Recently, it has been shown that C-type lectins bind to and stimulate FL cells. Such lectins are normally expressed on cells of the innate immune system, e.g. dendritic cells (DCs), which also reside in close interaction with the transformed B cells in germinal centers. Importantly, previous studies point to an outstanding role of the tumor microenvironment in survival and proliferation of the FL cells. In this study, we demonstrate that the variable region glycosylation in FL BCRs contribute to stimulation of the cells as well as adhesion to cells of the innate immune system. The BCR from six FL and the appropriate glycosylation-defective controls in which the N-linked glycosylation sequons are removed by replacing the asparagine (N) residues with glutamine (Q) residues were expressed in the tko cellular reconstitution system. In tko cells, the BCR signaling cascade can be rendered functional at will through a tamoxifen-dependent mutant of the signal transducer SLP-65 (Meixlsperger et al., Immunity 2007; Dühren von Minden et al., Nature 2012). Tko cells expressing FL BCRs and their glycosylation-defective controls were tested for binding of a recombinant DC-SIGN/Fc fusion protein by flow cytometry. The mannosylated FL-derived BCR but not glycosylation-mutated receptors bound DC-SIGN. Stepwise mutation of individual glycosylation sites demonstrated variable contribution to the strength of lectin binding. Despite this specific binding to mannosylated FL BCRs, DC-SIGN/Fc failed to induce significant calcium mobilization of transduced tko cells. Crosslinking with anti-IgM, in contrast, led to a readily detectable BCR-mediated signal, thereby demonstrating functionality of the transduced BCR. To study the role of mannosylated FL receptors in interaction with their environment, we co-cultured cells expressing FL receptors containing or lacking N-linked glycans in the variable regions together with macrophages. Western blot analyses with a pan-phosphotyrosine antibody demonstrated higher global tyrosine phosphorylation in the lysates of cells expressing glycosylated receptors, thereby indicating a specific role for mannosylated V-regions in FL stimulation. Glycan-mediated interactions fulfill multiple important functions in the mammalian immune system including pathogen recognition and cell adhesion or trafficking. DC-SIGN serves as receptor for the uptake of mannosylated pathogens and contributes to cell-cell interaction by binding to the heavily glycosylated ICAM-2/3 (intracellular adhesion molecules-2/3). In the case of FL, it is therefore conceivable that DC-SIGN expressed on follicular DCs binds to the heavily mannosylated FL BCRs and serves thereby as adhesion molecule to keep the FL B cells within the follicular structure. We tested this hypothesis using live cell imaging on a DC sublayer and detected slightly slower movement and shorter tracks of cells expressing glycosylated FL BCRs as compared to control cells. Together, our results ascribe a role of the acquired glycosylation sites in FL BCRs for B-cell/DC interaction, thereby keeping the cells in the appropriate environment in a process that involves active signal transduction rather than triggering a classical antigen-induced BCR stimulation. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Essentially Leading Antibody Production: An Investigation of Amino Acids, Myeloma, and Natural V-Region Signal Peptides in Producing Pertuzumab and Trastuzumab Variants

2020 ◽  
Vol 11 ◽  
Author(s):  
Wei-Li Ling ◽  
Chinh Tran-To Su ◽  
Wai-Heng Lua ◽  
Jun-Jie Poh ◽  
Yuen-Ling Ng ◽  
...  
Keyword(s):  
Amino Acids ◽  
Culture Media ◽  
Essential Amino Acids ◽  
Equation Model ◽  
Signal Peptides ◽  
Variable Regions ◽  
Amino Acid Availability ◽  
V Genes ◽  
V Region ◽  
Large Repertoire

Boosting the production of recombinant therapeutic antibodies is crucial in both academic and industry settings. In this work, we investigated the usage of varying signal peptides by antibody V-genes and their roles in recombinant transient production, systematically comparing myeloma and the native signal peptides of both heavy and light chains in 168 antibody permutation variants. We found that amino acids count and types (essential or non-essential) were important factors in a logistic regression equation model for predicting transient co-transfection protein production rates. Deeper analysis revealed that the culture media were often incomplete and that the supplementation of essential amino acids can improve the recombinant protein yield. While these findings are derived from transient HEK293 expression, they also provide insights to the usage of the large repertoire of antibody signal peptides, where by varying the number of specific amino acids in the signal peptides attached to the variable regions, bottlenecks in amino acid availability can be mitigated.

scholarly journals Sequence similarities among kappa IIIb chains of monoclonal human IgM kappa autoantibodies.

1984 ◽  
Vol 160 (3) ◽  
pp. 893-904 ◽  
Author(s):  
B Pons-Estel ◽  
F Goñi ◽  
A Solomon ◽  
B Frangione
Keyword(s):  
Amino Acid ◽  
Germ Line ◽  
Sequence Data ◽  
Selection Process ◽  
Variable Region ◽  
Light Chains ◽  
Partial Amino Acid Sequence ◽  
Kappa Chain ◽  
V Region ◽  
Sequence Similarities

Light chains of the serologically and chemically defined V region sub-subgroup kappa IIIb are preferentially associated with several types of human IgM kappa (monoclonal) autoantibodies and are remarkably homologous in primary structure, as evidenced by partial amino acid sequence data. To establish the extent of homology among such proteins, we have determined the complete variable region (V) sequence of the light chains of four monoclonal IgM kappa autoantibodies, of which two (GAR and GOT) are rheumatoid factors (RFs), the third (SON) has anti-apo beta lipoprotein specificity, and the fourth (PIE) binds specifically to intermediate filaments. The region encoded by the V kappa segment gene (positions 1-95) in all four light (L) chains is virtually identical in sequence, differing by only one residue in the FR3 of protein SON and in the first CDR of protein GOT. Further, the CDR3 of kappa chain SON contains an additional residue (prolyl) located at the carboxyl-terminus of the V segment. The region encoded by the J gene (positions 96-108) is identical after position 96 for the two RFs GAR and GOT (J kappa 2), but different in proteins SON (J kappa 4) and PIE (J kappa 1). The amino acid residue at position 96, located in CDR3 at the site of combinatoriaL joining of the V kappa and J kappa gene segments and involved as a contacting residue in the hapten binding site, is different in all four light chains. These results demonstrate the extensive homology in sequence among light chains of IgM kappa autoantibodies and indicate that a particular V kappa germ line gene, kappa IIIb, is expressed as a phylogenetic response to certain self antigens or as part of a selection process by which these autoimmune responses are regulated.

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