scholarly journals The binding of 2,4,6-trinitrophenyl derivatives to the mouse myeloma immunoglobulin A protein MOPC 315

1978 ◽  
Vol 169 (1) ◽  
pp. 179-188 ◽  
Author(s):  
Steven K. Dower ◽  
Peter Gettins ◽  
Roland Jackson ◽  
Raymond A. Dwek ◽  
David Givol
Keyword(s):  
Heavy Chain ◽  
Light Chain ◽  
Chemical Shifts ◽  
Variable Region ◽  
Proton Nuclear Magnetic Resonance ◽  
Structural Basis ◽  
Tryptophan Residue ◽  
Difference Spectra ◽  
Fab Fragment ◽  
Mouse Myeloma

The binding of Tnp (2,4,6-trinitrophenyl) derivatives to the Fv fragment (variable region of heavy and light chains) of the mouse myeloma IgA protein MOPC 315 was investigated by 270MHz proton nuclear magnetic resonance. Two of the haptens, Tnp-glycine and Tnp-l-aspartate, are in fast exchange with the Fv fragment, and the changes in chemical shifts for both protein and hapten resonances were determined by titrations. For the tightly binding hapten ε-N-Tnp-α-N-acetyl-l-lysine, which is in slow exchange with the Fv fragment, the changes in chemical shifts for the hapten H3+H5 resonances were determined by cross-saturation. By using these data and the known structure of the combining site of protein MOPC 315 [Dwek, Wain-Hobson, Dower, Gettins, Sutton, Perkins & Givol (1977), Nature (London) 266, 31–37] the mode of binding of Tnp derivatives is deduced by ring-current calculations. The trinitrophenyl ring stacks with tryptophan-93L (light chain) in the ‘aromatic box’ formed by tryptophan-93L, tyrosine-34L and phenyl-alanine-34H (heavy chain). Further evidence for the stacking interaction with a tryptophan residue is provided by the similarity of the optical-difference spectra observed with Tnp-aminomethylphosphonate in the presence of either the Fab fragment (light chain and N-terminal half of heavy chain) of protein MOPC 315 or tryptophan. These data show that the modes of binding of all the Tnp derivatives are very similar, despite a 100-fold range in their affinities. It is also concluded that the modes of binding of Dnp (2,4-dinitrophenyl) and Tnp derivatives to protein MOPC 315 are very similar, and that the structural basis for this is that the aromatic box is large enought to allow the trinitrophenyl ring to stack with tryptophan-93L while still forming hydrogen bonds to asparagine-36L and tyrosine-34L.

scholarly journals The Structural Basis of Repertoire Shift in an Immune Response to Phosphocholine

2000 ◽  
Vol 191 (12) ◽  
pp. 2101-2112 ◽  
Author(s):  
McKay Brown ◽  
Maria A. Schumacher ◽  
Gregory D. Wiens ◽  
Richard G. Brennan ◽  
Marvin B. Rittenberg
Keyword(s):  
Immune Response ◽  
Light Chain ◽  
Indirect Effects ◽  
Somatic Mutations ◽  
Variable Region ◽  
Primary Response ◽  
Structural Basis ◽  
Antibody Repertoire ◽  
High Affinity

The immune response to phosphocholine (PC)–protein is characterized by a shift in antibody repertoire as the response progresses. This change in expressed gene combinations is accompanied by a shift in fine specificity toward the carrier, resulting in high affinity to PC–protein. The somatically mutated memory hybridoma, M3C65, possesses high affinity for PC–protein and the phenyl-hapten analogue, p-nitrophenyl phosphocholine (NPPC). Affinity measurements using related PC–phenyl analogues, including peptides of varying lengths, demonstrate that carrier determinants contribute to binding affinity and that somatic mutations alter this recognition. The crystal structure of an M3C65–NPPC complex at 2.35-Å resolution allows evaluation of the three light chain mutations that confer high-affinity binding to NPPC. Only one of the mutations involves a contact residue, whereas the other two have indirect effects on the shape of the combining site. Comparison of the M3C65 structure to that of T15, an antibody dominating the primary response, provides clear structural evidence for the role of carrier determinants in promoting repertoire shift. These two antibodies express unrelated variable region heavy and light chain genes and represent a classic example of the effect of repertoire shift on maturation of the immune response.

scholarly journals Structural basis for amino acid exchange by a human heteromeric amino acid transporter

2020 ◽  
Vol 117 (35) ◽  
pp. 21281-21287 ◽  
Author(s):  
Di Wu ◽  
Tamara N. Grund ◽  
Sonja Welsch ◽  
Deryck J. Mills ◽  
Max Michel ◽  
...  
Keyword(s):  
Amino Acid ◽  
Heavy Chain ◽  
Light Chain ◽  
Basic Amino Acid ◽  
Binding Pocket ◽  
Structural Basis ◽  
Amino Acid Transporters ◽  
Wide Range ◽  
Functional Complex ◽  
Protein Components

Heteromeric amino acid transporters (HATs) comprise a group of membrane proteins that belong to the solute carrier (SLC) superfamily. They are formed by two different protein components: a light chain subunit from an SLC7 family member and a heavy chain subunit from the SLC3 family. The light chain constitutes the transport subunit whereas the heavy chain mediates trafficking to the plasma membrane and maturation of the functional complex. Mutation, malfunction, and dysregulation of HATs are associated with a wide range of pathologies or represent the direct cause of inherited and acquired disorders. Here we report the cryogenic electron microscopy structure of the neutral and basic amino acid transport complex (b[0,+]AT1-rBAT) which reveals a heterotetrameric protein assembly composed of two heavy and light chain subunits, respectively. The previously uncharacterized interaction between two HAT units is mediated via dimerization of the heavy chain subunits and does not include participation of the light chain subunits. The b(0,+)AT1 transporter adopts a LeuT fold and is captured in an inward-facing conformation. We identify an amino-acid–binding pocket that is formed by transmembrane helices 1, 6, and 10 and conserved among SLC7 transporters.

Human Monoclonal Cryocrystalglobulinemia: First Complete Heavy and Light Chain Variable Region Nucleotide Sequences.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1381-1381
Author(s):  
Vittorio Perfetti ◽  
Valentina Navazza ◽  
Sofia Giorgietti ◽  
Loredana Marchese ◽  
Giovanni Palladini ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Synovial Fluid ◽  
Heavy Chain ◽  
Light Chain ◽  
Tertiary Structure ◽  
Variable Region ◽  
Inverse Pcr ◽  
Correct Identification ◽  
Type I ◽  
Vh Domain

Abstract Cryocrystalglobulinemia is characterized by a monoclonal immunoglobulin that is both cryoprecipitable and crystalforming (cryocrystalglobulin). It occours in humans as a rare complication of lymphoproliferative disorders. The monoclonal immunoglobulins may crystallize in serum and synovial fluid and are responsible for both microcrystalline synovial inflammation and occlusive vasculopathy in kidneys and skin. At present, there are no reports on sequence analysis and tertiary structure of human cryocrystalglobulins. In this study, we established the first complete light and heavy chain variable region sequences of a human cryocrystalglobulin (BEL) from a patient with severe microcrystalline arthropathy, cutaneous purpura and sacroiliitis. Serum cryoprecipitate was composed of unbound monoclonal IgGk at immunofixation (type I cryoglobulinemia) and revealed homogeneous crystals under light microscopy. Extra and intracellular crystals were also found in synovial fluid. Analysis of bone marrow aspirate showed 4% monoclonal plasma cell infiltration. Total RNA was extracted from Ficoll-separated bone marrow mononuclear cells and complete heavy (VH) and light chain (Vk) variable domain sequences of monoclonal Ig were obtained by means of an unbiased inverse-PCR strategy. Sequences were then matched in databases to identify rearranged germline V, D and J segments. The heavy chain sequence belonged to the g2 class and its variable region was most closely related (94% identity) to the VH3-30 germline gene (VH3 family) rearranged to D2-2 and JH4b segments, whereas the Vk region was derived (97% identity) from the germline VkL6 (VkIII) rearranged to Jk5. Partial protein sequence of serum cryocrystalprecipitate confirmed the correct identification of the monoclonal sequences. A previously reported murine model of cryocrystalglobulinemia suggested that acquisition of specific amino acids at critical positions within the VH domain (positions 6 and 23) could influence cryoprecipitation (Rengers J-U et al, Blood2000;95:3467–72). Analysis of BEL VH domain did not confirm these observations. Protein modelling and site-directed mutagenesis studies are undergoing to clarify the role of amino acid substitutions and interactions in immunoglobulin crystal-like aggregation and cryoprecipitation.

Complete variable region deletion in Aα heavy chain disease protein (roul). Correlation with light chain secretion

1993 ◽  
Vol 17 (6) ◽  
pp. 527-532 ◽  
Author(s):  
Michel Cogné ◽  
Pierre Aucouturier ◽  
André Brizard ◽  
Brigitte Dreyfus ◽  
Françoise Duarte ◽  
...  
Keyword(s):  
Heavy Chain ◽  
Light Chain ◽  
Variable Region ◽  
Disease Protein ◽  
Heavy Chain Disease

A Chimeric Streptokinase with Unexpected Fibrinolytic Selectivity

1996 ◽  
Vol 76 (03) ◽  
pp. 429-438 ◽  
Author(s):  
Joel Goldstein ◽  
Gary R Matsueda ◽  
Shyh-Yu Shaw
Keyword(s):  
Heavy Chain ◽  
Light Chain ◽  
Limited Proteolysis ◽  
Clot Lysis ◽  
Wild Type ◽  
Fab Fragment ◽  
Plasma Clot ◽  
N Terminus ◽  
Lag Period ◽  
Similar Kinetic

SummaryChimeric 59D8-SK was designed to confer fibrin-selectivity to streptokinase by fusion of the Fab fragment of anti-fibrin antibody 59D8 to the N-terminus of streptokinase (SK: Ile1-Lys414). It was expressed in a mouse hybridoma cell line and purified by affinity chromatography on a 59D8-antigen column. Chimeric 59D8-SK is a disulfide-linked heterodimer composed of an antibody light chain (Mr 27,000) and a N-glycosylated chimeric heavy chain (Mr 90,000). The fibrin targeting by 59D8 increased plasma clot lysis by 2-fold, but connecting 59D8 to SK has provided 59D8-SK several unique properties: (i) 59D8-SK activated human Glu-plasminogen with a significant lag period that coincided with limited proteolysis of 59D8-SK similar to that observed for wild-type SK. In a kinetic study, both gave very similar kinetic parameters for the activation of Glu-plasminogen even though 59D8-SK was N-glycosylated in its SK portion; (ii) 59D8-SK was relatively inactive in human plasma, compared to SK, but it became activated in the presence of clots; (iii) 59D8-SK lysed clots slowly but completely whereas SK lysed clots rapidly but incompletely. Even though the mechanism behind these new properties is not fully understood, they are characteristics of a second-generation plasminogen activator.

scholarly journals Amino acid-sequence variability at the N-terminal extra piece of mouse immunoglobulin light-chain precursors of the same and different subgroups

1976 ◽  
Vol 157 (1) ◽  
pp. 145-151 ◽  
Author(s):  
Y Burstein ◽  
I Schechter
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Light Chain ◽  
Variable Region ◽  
Amino Acid Residues ◽  
Free System ◽  
Methionine Residue ◽  
N Terminus ◽  
Mouse Myeloma

The proteins programmed in the wheat-germ cell-free system by the mRNA coding for the MOPC-63 mouse myeloma L (light) chain were labelled with six radioactive amino acids: [35S]methionine, [4,5-3H]leucine, [3,4-3H]proline, [3-3H]serine, [4,5-3H]isoleucine or [2,3-3H]alanine. Amino acid-sequence analyses showed that over 90% of the total cell-free product was one homogeneous protein, which corresponds to the MOPC-63 L-chain precursor. In this precursor an extra piece, 20 amino acid residues in length, precedes the N-terminus of the mature L chain. The extra piece contains one methionine residue at the N-terminus, six leucine residues, which are clustered in two triplets at positions 6, 7, 8 and 11, 12, 13, one proline residue at position 16, and one serine residue at position 18. The closely gathered leucine residues, as well as their abundance (30%), suggest that the extra-piece moiety is hydrophobic. In the precursors, the extra piece is coupled to the variable region of the L chain. Partial sequences of precursors of L chains of the same and different subgroups that were labelled with the above six radioactive amino acids indicate that the extra piece is part of the variable region. Thus the precursors of MOPC-63 and MOPC-321 L chains, which are of the same subgroup, have extra pieces of identical size (20 residues), and so far their partial sequences are also identical (see above). On the other hand, in the precursor of MOPC-41 L chain, which is of a different subgroup, the extra piece is 22 residues in length. Further, the sequence of the MOPC-41 extra piece differs in at least ten positions from sequences of the extra pieces of the precursors of MOPC-63 and MOPC-321 L chains.

scholarly journals Structural basis of polyethylene glycol recognition by antibody

2020 ◽  
Vol 27 (1) ◽  
Author(s):  
Cheng-Chung Lee ◽  
Yu-Cheng Su ◽  
Tzu-Ping Ko ◽  
Li-Ling Lin ◽  
Chih-Ya Yang ◽  
...  
Keyword(s):  
Polyethylene Glycol ◽  
Crown Ether ◽  
Heavy Chain ◽  
Light Chain ◽  
Underlying Mechanism ◽  
Binding Mode ◽  
Structural Basis ◽  
Side Chains ◽  
Arginine Residues ◽  
Aromatic Side Chains

Abstract Background Polyethylene glycol (PEG) is widely used in industry and medicine. Anti-PEG antibodies have been developed for characterizing PEGylated drugs and other applications. However, the underlying mechanism for specific PEG binding has not been elucidated. Methods The Fab of two cognate anti-PEG antibodies 3.3 and 2B5 were each crystallized in complex with PEG, and their structures were determined by X-ray diffraction. The PEG-Fab interactions in these two crystals were analyzed and compared with those in a PEG-containing crystal of an unrelated anti-hemagglutinin 32D6-Fab. The PEG-binding stoichiometry was examined by using analytical ultracentrifuge (AUC). Results A common PEG-binding mode to 3.3 and 2B5 is seen with an S-shaped core PEG fragment bound to two dyad-related Fab molecules. A nearby satellite binding site may accommodate parts of a longer PEG molecule. The core PEG fragment mainly interacts with the heavy-chain residues D31, W33, L102, Y103 and Y104, making extensive contacts with the aromatic side chains. At the center of each half-circle of the S-shaped PEG, a water molecule makes alternating hydrogen bonds to the ether oxygen atoms, in a similar configuration to that of a crown ether-bound lysine. Each satellite fragment is clamped between two arginine residues, R52 from the heavy chain and R29 from the light chain, and also interacts with several aromatic side chains. In contrast, the non-specifically bound PEG fragments in the 32D6-Fab crystal are located in the elbow region or at lattice contacts. The AUC data suggest that 3.3-Fab exists as a monomer in PEG-free solution but forms a dimer in the presence of PEG-550-MME, which is about the size of the S-shaped core PEG fragment. Conclusions The differing amino acids in 3.3 and 2B5 are not involved in PEG binding but engaged in dimer formation. In particular, the light-chain residue K53 of 2B5-Fab makes significant contacts with the other Fab in a dimer, whereas the corresponding N53 of 3.3-Fab does not. This difference in the protein-protein interaction between two Fab molecules in a dimer may explain the temperature dependence of 2B5 in PEG binding, as well as its inhibition by crown ether.

scholarly journals Genetic elements used for a murine lupus anti-DNA autoantibody are closely related to those for antibodies to exogenous antigens.

1985 ◽  
Vol 161 (4) ◽  
pp. 805-815 ◽  
Author(s):  
R Kofler ◽  
D J Noonan ◽  
D E Levy ◽  
M C Wilson ◽  
N P Møller ◽  
...  
Keyword(s):  
Heavy Chain ◽  
Light Chain ◽  
Variable Region ◽  
Leader Peptide ◽  
Murine Lupus ◽  
Heavy Chain Variable Region ◽  
Variable Gene ◽  
Vh Gene ◽  
Gene Origin ◽  
Common Variable

The mRNAs encoding heavy and light chains of a hybridoma-derived monoclonal IgM kappa anti-DNA autoantibody from lupus-prone MRL/Mp-lpr/lpr mice (Ighj) have been transcribed into cDNA copies and molecularly cloned, and their complete nucleotide sequences have been determined. The mRNA for the heavy chain variable region, including leader peptide and 5' untranslated region, is transcribed from a heavy chain variable region (VH) gene closely related (and possibly allelic) to VH genes of the C57BL/6 (Ighb) nitrophenyl antibody family. The deduced amino acid sequence corresponding to the light chain variable region of this autoantibody shows extensive similarities with non-autoantibody molecules of the V kappa 1 group, suggesting a common variable gene origin. The joining segments, constant regions, and 3' untranslated regions of both the heavy and light chain mRNAs are nearly identical to corresponding sequences of non-autoantibodies from normal mice. Our findings suggest that this anti-DNA autoantibody originated from the same germline repertoire as antibodies to exogenous antigens.

scholarly journals Clonal nature of the immune response to phosphorylcholine (PC). V. Cross-idiotypic specificity among heavy chains of murine anti-PC antibodies and PC-binding myeloma proteins.

1975 ◽  
Vol 141 (5) ◽  
pp. 1073-1083 ◽  
Author(s):  
J L Claflin ◽  
J M Davie
Keyword(s):  
Heavy Chain ◽  
Antigenic Determinant ◽  
Variable Region ◽  
Antigenic Determinants ◽  
Genetic Origin ◽  
Heavy Chains ◽  
Myeloma Proteins ◽  
Kappa Chain ◽  
Clonal Nature ◽  
Mouse Myeloma

Seven mouse myeloma proteins with specificity for phosphorylcholine (PC) were found to share a common antigenic determinant. This group of proteins contained members which differed in genetic origin, heavy chain class, kappa-chain subgroup, individual antigenic determinants and specificity for choline analogues. The cross-idiotypic determinant, VH-PC, was antigenically similar in each of the proteins and was associated with the variable portion of the heavy chain in the region of the antibody combining site. Further studies showed that an indistinguishable determinant was present on IgM anti-PC antibodies isolated from all strains of mice tested regardless of histocompatibility or heavy chain allotype. In view of the finding that this cross-idiotypic determinant was not found on antibodies or myeloma proteins which lacked specificity for PC, the data strongly suggest that a particular heavy chain variable region has been preserved in all mouse antibodies with specificity for PC.

scholarly journals Improvement of Fab expression by screening combinatorial synonymous signal sequence libraries

2019 ◽  
Vol 18 (1) ◽  
Author(s):  
Antti Kulmala ◽  
Tuomas Huovinen ◽  
Urpo Lamminmäki
Keyword(s):  
Codon Usage ◽  
Expression Profile ◽  
Heavy Chain ◽  
Light Chain ◽  
Signal Sequence ◽  
Secondary Structures ◽  
Antibody Fragments ◽  
Fab Fragment ◽  
Signal Sequences ◽  
Expression Levels

Abstract Background Antibody fragments can be expressed in Escherichia coli, where they are commonly directed to the periplasm via Sec pathway to enable disulphide bridge formations and correct folding. In order to transport antibody fragments to the periplasmic space via Sec pathway, they are equipped with N-terminal signal sequence. Periplasmic expression has many benefits but it’s also subjected to many hurdles like inefficient translocation across the inner membrane and insufficient capacity of the translocation system. One solution to overcome these hurdles is a modulation of codon usage of signal sequence which has proved to be an efficient way of tuning the translocation process. Modulation of codon usage of signal sequences has been successfully employed also in improving the expression levels of antibody fragments, but unfortunately the effect of codon usage on the expression has not been thoroughly analyzed. Results In the present study we established three synonymous PelB signal sequence libraries by modulating codon usage of light chain and heavy chain PelB signal sequences of a Fab fragment. Each region (n-region, hydrophobic region and c-region) of the PelB signal sequence in the both chains of the Fab fragment in a bicistronic expression vector was mutated separately. We then screened for clones with improved expression profile. The best source for improved clones was the n-region library but in general, improved clones were obtained from all of the three libraries. After screening, we analyzed the effects of codon usage and mRNA secondary structures of chosen clones on the expression levels of the Fab fragment. When it comes to codon usage based factors, it was discovered that especially codon usage of fifth leucine position of the light chain PelB affects the expression levels of Fab fragment. In addition, we observed that mRNA secondary structures in the translation initiation regions of the light and heavy chain have an effect on expression levels as well. Conclusions In conclusion, the established synonymous signal sequence libraries are good sources for discovering Fab fragments with improved expression profile and obtaining new codon usage related information.

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