scholarly journals Variable domain-linked oligosaccharides of a human monoclonal IgG: structure and influence on antigen binding

1999 ◽  
Vol 338 (2) ◽  
pp. 529-538 ◽  
Author(s):  
Haike LEIBIGER ◽  
Daniel WÜSTNER ◽  
Rolf-Dietrich STIGLER ◽  
Uwe MARX
Keyword(s):  
Consensus Sequence ◽  
Variable Region ◽  
Glycosylation Site ◽  
Exchange Chromatography ◽  
Variable Domain ◽  
Dimensional Structure ◽  
Computer Assisted ◽  
Antigen Binding ◽  
Fab Fragment ◽  
Human Igg

The variable-domain-attached oligosaccharide side chains of a human IgG produced by a human–human–mouse heterohybridoma were analysed. In addition to the conserved N-glycosylation site at Asn-297, an N-glycosylation consensus sequence (Asn-Asn-Ser) is located at position 75 in the variable region of its heavy chain. The antibody was cleaved into its antigen-binding (Fab) and crystallizing fragments. The oligosaccharides of the Fab fragment were released by digestion with various endo- and exoglycosidases and analysed by anion-exchange chromatography and fluorophore-assisted carbohydrate electrophoresis. The predominant components were disialyl- bi-antennary and tetra-sialyl tetra-antennary complex carbohydrates. Of note is the presence in this human IgG of oligosaccharides containing N-glycolylneuraminic acid and N-acetylneuraminic acid in the ratio of 94:6. Furthermore, we determined N-acetylgalactosamine in the Fab fragment of this antibody, suggesting the presence of O-linked carbohydrates. A three-dimensional structure of the glycosylated variable (Fv) fragment was suggested using computer-assisted modelling. In addition, the influence of the Fv-associated oligosaccharides of the CBGA1 antibody on antigen binding was tested in several ELISA systems. Deglycosylation resulted in a decreased antigen-binding activity.

scholarly journals Crystal structure of a glycosylated Fab from an IgM cryoglobulin with properties of a natural proteolytic antibody

2006 ◽  
Vol 395 (3) ◽  
pp. 473-481 ◽  
Author(s):  
Paul A. Ramsland ◽  
Simon S. Terzyan ◽  
Gwendolyn Cloud ◽  
Christina R. Bourne ◽  
William Farrugia ◽  
...  
Keyword(s):  
Serine Proteases ◽  
Three Dimensional ◽  
Catalytic Triad ◽  
Dimensional Structure ◽  
Gel Point ◽  
Antigen Binding ◽  
Antigen Binding Site ◽  
Fab Fragment ◽  
Peripheral Tissues ◽  
Fragment Antigen Binding

The 2.6 Å (1 Å=0.1 nm) resolution structure has been determined for the glycosylated Fab (fragment antigen binding) of an IgM (Yvo) obtained from a subject with Waldenström's macroglobulinaemia. Dynamic light scattering was used to estimate the gel point and monitor the formation of an ordered hydroscopic gel of Yvo IgM upon cooling. If a cryoglobulin forms gels in peripheral tissues and organs, the associated swelling and damage to microvasculature can result in considerable morbidity and mortality. The three-dimensional structure of the branched N-linked oligosaccharide associated with the CH1 domain (first constant domain of heavy chain) is reported. The carbohydrate may act to shield part of the lateral surface of the CH1 domain and crowd the junction between the CH1 and CH2 domains, thereby limiting the segmental flexibility of the Fab arms in intact Yvo IgM, especially at low temperatures. Recently, Yvo IgM was shown to have the properties of a naturally occurring proteolytic antibody [Paul, Karle, Planque, Taguchi, Salas, Nishiyama, Handy, Hunter, Edmundson and Hanson (2004) J. Biol. Chem. 279, 39611–39619; Planque, Bangale, Song, Karle, Taguchi, Poindexter, Bick, Edmundson, Nishiyama and Paul (2004) J. Biol Chem. 279, 14024–14032]. The Yvo protein displayed the ability to cleave, by a nucleophilic mechanism, the amide bonds of a variety of serine protease substrates and the gp120 coat protein of HIV. An atypical serine, arginine and glutamate motif is located in the middle of the Yvo antigen-binding site and displays an overall geometry that mimics the classical serine, histidine and aspartate catalytic triad of serine proteases. Our present findings indicate that pre-existing or natural antibodies can utilize at least one novel strategy for the cleavage of peptide bonds.

scholarly journals The mycoplasma surface proteins MIB and MIP promote the dissociation of the antibody-antigen interaction

2021 ◽  
Vol 7 (10) ◽  
pp. eabf2403
Author(s):  
Pierre Nottelet ◽  
Laure Bataille ◽  
Geraldine Gourgues ◽  
Robin Anger ◽  
Carole Lartigue ◽  
...  
Keyword(s):  
Surface Proteins ◽  
Mycoplasma Species ◽  
Antigen Binding ◽  
Antigen Binding Site ◽  
Fab Fragment ◽  
Cryo Electron Microscopy ◽  
Antigen Complex ◽  
Antigen Interaction ◽  
Immunoglobulin Binding

Mycoplasma immunoglobulin binding (MIB) and mycoplasma immunoglobulin protease (MIP) are surface proteins found in the majority of mycoplasma species, acting sequentially to capture antibodies and cleave off their VH domains. Cryo–electron microscopy structures show how MIB and MIP bind to a Fab fragment in a “hug of death” mechanism. As a result, the orientation of the VL and VH domains is twisted out of alignment, disrupting the antigen binding site. We also show that MIB-MIP has the ability to promote the dissociation of the antibody-antigen complex. This system is functional in cells and protects mycoplasmas from antibody-mediated agglutination. These results highlight the key role of the MIB-MIP system in immunity evasion by mycoplasmas through an unprecedented mechanism, and open exciting perspectives to use these proteins as potential tools in the antibody field.

scholarly journals Structural analysis of mutations in the Drosophila beta 2-tubulin isoform reveals regions in the beta-tubulin molecular required for general and for tissue-specific microtubule functions.

Genetics ◽  
1995 ◽  
Vol 139 (1) ◽  
pp. 267-286 ◽  
Author(s):  
J D Fackenthal ◽  
J A Hutchens ◽  
F R Turner ◽  
E C Raff
Keyword(s):  
Amino Acid ◽  
Three Dimensional ◽  
Variable Region ◽  
Internal Variable ◽  
Microtubule Assembly ◽  
Dimensional Structure ◽  
Wild Type ◽  
Beta Tubulin ◽  
Assembly Kinetics ◽  
Beta 2

Abstract We have determined the lesions in a number of mutant alleles of beta Tub85D, the gene that encodes the testis-specific beta 2-tubulin isoform in Drosophila melanogaster. Mutations responsible for different classes of functional phenotypes are distributed throughout the beta 2-tubulin molecule. There is a telling correlation between the degree of phylogenetic conservation of the altered residues and the number of different microtubule categories disrupted by the lesions. The majority of lesions occur at positions that are evolutionarily highly conserved in all beta-tubulins; these lesions disrupt general functions common to multiple classes of microtubules. However, a single allele B2t6 contains an amino acid substitution within an internal cluster of variable amino acids that has been identified as an isotype-defining domain in vertebrate beta-tubulins. Correspondingly, B2t6 disrupts only a subset of microtubule functions, resulting in misspecification of the morphology of the doublet microtubules of the sperm tail axoneme. We previously demonstrated that beta 3, a developmentally regulated Drosophila beta-tubulin isoform, confers the same restricted morphological phenotype in a dominant way when it is coexpressed in the testis with wild-type beta 2-tubulin. We show here by complementation analysis that beta 3 and the B2t6 product disrupt a common aspect of microtubule assembly. We therefore conclude that the amino acid sequence of the beta 2-tubulin internal variable region is required for generation of correct axoneme morphology but not for general microtubule functions. As we have previously reported, the beta 2-tubulin carboxy terminal isotype-defining domain is required for suprastructural organization of the axoneme. We demonstrate here that the beta 2 variant lacking the carboxy terminus and the B2t6 variant complement each other for mild-to-moderate meiotic defects but do not complement for proper axonemal morphology. Our results are consistent with the hypothesis drawn from comparisons of vertebrate beta-tubulins that the two isotype-defining domains interact in a three-dimensional structure in wild-type beta-tubulins. We propose that the integrity of this structure in the Drosophila testis beta 2-tubulin isoform is required for proper axoneme assembly but not necessarily for general microtubule functions. On the basis of our observations we present a model for regulation of axoneme microtubule morphology as a function of tubulin assembly kinetics.

scholarly journals Refined three-dimensional structure of the Fab fragment of a murine IgGl,λ antibody

1994 ◽  
Vol 50 (5) ◽  
pp. 768-777 ◽  
Author(s):  
T. Bizebard ◽  
R. Daniels ◽  
R. Kahn ◽  
B. Golinelli-Pimpaneau ◽  
J. J. Skehel ◽  
...  
Keyword(s):  
Three Dimensional ◽  
Dimensional Structure ◽  
Fab Fragment ◽  
Three Dimensional Structure

THE THREE-DIMENSIONAL STRUCTURE OF THE ANTIGEN BINDING SITE OF McPC 603 PROTEIN

1974 ◽  
pp. 7-14 ◽  
Author(s):  
E.A. Padlan ◽  
D.M. Segal ◽  
G.H. Cohen ◽  
D.R. Davies ◽  
S. Rudikoff ◽  
...  
Keyword(s):  
Binding Site ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Antigen Binding ◽  
Antigen Binding Site ◽  
Three Dimensional Structure

scholarly journals Three-dimensional structure of actin filaments and of an actin gel made with actin-binding protein.

1983 ◽  
Vol 96 (5) ◽  
pp. 1400-1413 ◽  
Author(s):  
R Niederman ◽  
P C Amrein ◽  
J Hartwig
Keyword(s):  
Actin Filaments ◽  
Binding Protein ◽  
Three Dimensional ◽  
Actin Binding ◽  
Dimensional Structure ◽  
Molar Ratio ◽  
Computer Assisted ◽  
Three Dimensional Structure ◽  
Actin Binding Protein ◽  
Critical Point Drying

Purified muscle actin and mixtures of actin and actin-binding protein were examined in the transmission electron microscope after fixation, critical point drying, and rotary shadowing. The three-dimensional structure of the protein assemblies was analyzed by a computer-assisted graphic analysis applicable to generalized filament networks. This analysis yielded information concerning the frequency of filament intersections, the filament length between these intersections, the angle at which filaments branch at these intersections, and the concentration of filaments within a defined volume. Purified actin at a concentration of 1 mg/ml assembled into a uniform mass of long filaments which overlap at random angles between 0 degrees and 90 degrees. Actin in the presence of macrophage actin-binding protein assembled into short, straight filaments, organized in a perpendicular branching network. The distance between branch points was inversely related to the molar ratio of actin-binding protein to actin. This distance was what would be predicted if actin filaments grew at right angles off of nucleation sites on the two ends of actin-binding protein dimers, and then annealed. The results suggest that actin in combination with actin-binding protein self-assembles to form a three-dimensional network resembling the peripheral cytoskeleton of motile cells.

Differential binding of heavy chain variable domain 3 antigen binding fragments to protein a chromatography resins

2015 ◽  
Vol 1409 ◽  
pp. 60-69 ◽  
Author(s):  
Julia Bach ◽  
Nathaniel Lewis ◽  
Kathy Maggiora ◽  
Alison J. Gillespie ◽  
Lisa Connell-Crowley
Keyword(s):  
Heavy Chain ◽  
Protein A ◽  
Variable Domain ◽  
Antigen Binding ◽  
Differential Binding ◽  
Domain 3
Sign in / Sign up

Export Citation Format

Share Document