Structural characterization of antibodies against lipopolysaccharide antigens: Insights into primary antibody response

Antibody combining sites are constructed from limited set of germ-line gene segments, yet are capable of both recognizing a broad range of common epitopes and eliciting an adaptive response to newly encountered pathogens. Carbohydrate antigens generally do not draw T cell help and concomitant affin...

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Main Author: Haji-Ghassemi, Omid
Other Authors: Evans, S. V.
Format: Others
Language:English
en
Published: 2015
Subjects:
Online Access:http://hdl.handle.net/1828/6012
id ndltd-uvic.ca-oai-dspace.library.uvic.ca-1828-6012
record_format oai_dc
collection NDLTD
language English
en
format Others
sources NDLTD
topic Antibodies
Antigen
Carbohydrate Complex
Glycosylation
X-ray Crystallography
Autoimmunity
Lipid A
Lipopolysaccharide
Structure
Chlamydia
spellingShingle Antibodies
Antigen
Carbohydrate Complex
Glycosylation
X-ray Crystallography
Autoimmunity
Lipid A
Lipopolysaccharide
Structure
Chlamydia
Haji-Ghassemi, Omid
Structural characterization of antibodies against lipopolysaccharide antigens: Insights into primary antibody response
description Antibody combining sites are constructed from limited set of germ-line gene segments, yet are capable of both recognizing a broad range of common epitopes and eliciting an adaptive response to newly encountered pathogens. Carbohydrate antigens generally do not draw T cell help and concomitant affinity maturation in the humoral response. Therefore, anti-carbohydrate responses must rely more heavily on the primary germ-line gene repertoire. Antibodies are usually thought of as highly specific. It has been suggested that polyspecificity and cross-reactivity in germ-line antibodies is necessary to provide the protective mechanisms required to broaden the potential number of antigens recognized; however, the molecular mechanism underlying polyspecificity is poorly understood. To investigate the phenomena of specificity, cross-reactivity and polyspecificity in germ-line antibodies my thesis focuses first on the unique LPS inner core oligosaccharide of Chlamydiaceae, which contains variations within the conserved inner core trisaccharide Kdo(2→8)Kdo(2→4)Kdo (3-deoxy-D-manno-oct-2-ulosonic acid). Antibodies raised against this family-specific trisaccharide showed strong V-region restriction with two sets of heavy and light chain V genes accounting for almost all clones isolated. These groups were named after their prototypic clones as the ‘S25-2 type’ and the ‘S25-23 type’. In contrast to the cross-reactive S25-2 and related antibodies, the S25-23 family of antibodies were shown to be specific for the Chlamydiaceae-specific trisaccharide antigen with no cross-reactivity to Kdo mono or disaccharides or to the Kdo(2→4)Kdo(2→4)Kdo trisaccharide antigen. Interest in S25-23 was sparked by its rare high μM affinity and strict specificity for the family-specific trisaccharide antigen. The structures of the antigen binding fragments of four S25-23-type mAbs have been determined to high resolution in complex with the Chlamydiaceae-specific epitope, revealing the molecular basis for their binding behaviour. The germ-line-encoded paratopes of these antibodies differ significantly from previously characterized S25-2-type mAbs. Unlike the terminal Kdo recognition pocket that promotes cross-reactivity in S25-2-type antibodies, S25-26 and the closely related S25-23 utilize a groove composed of germ-line residues to recognize the length of the trisaccharide antigen. Further S25-23-type antibodies are glycosylated on the variable heavy chain. Analysis of the glycan reveals a heterogeneous mixture with a common root structure that contains an unusually high number of terminal αGal-Gal moieties. One of the unliganded structures in S25-26 shows significant order in the glycan with appropriate electron density for nine residues. The elucidation of the three-dimensional structure of a Gal(α1→3)Gal containing N-linked glycan on a mAb variable heavy chain has potential clinical interest, as it has been implicated in allergic responses in patients receiving therapeutic antibodies. The second focus of my thesis research is the lipid A moiety of LPS, which is involved in septic shock. Though the lipid A epitope appears to be cryptic during infection with Gram-negative bacteria, there have been several reported instances of lipid A specific antibodies isolated from human sera. While these antibodies are strictly selective for lipid A, there are reports of polyspecificity of some anti-lipid A antibodies for single stranded DNA. In such cases, the breakdown of negative selection through polyspecificity has been reported to result in the unfortunate consequences of autoimmune disease. This thesis reports the first crystal structures of antibodies in complex with lipid A and single stranded nucleic acids, elucidating their mechanism for polyspecificity. Perhaps more importantly, the structures may yield clues to the genesis of autoimmune diseases such as systemic lupus erythematosus, thyroiditis, and rheumatic autoimmune diseases. === Graduate === 2020-04-18 === 0487 === 0982
author2 Evans, S. V.
author_facet Evans, S. V.
Haji-Ghassemi, Omid
author Haji-Ghassemi, Omid
author_sort Haji-Ghassemi, Omid
title Structural characterization of antibodies against lipopolysaccharide antigens: Insights into primary antibody response
title_short Structural characterization of antibodies against lipopolysaccharide antigens: Insights into primary antibody response
title_full Structural characterization of antibodies against lipopolysaccharide antigens: Insights into primary antibody response
title_fullStr Structural characterization of antibodies against lipopolysaccharide antigens: Insights into primary antibody response
title_full_unstemmed Structural characterization of antibodies against lipopolysaccharide antigens: Insights into primary antibody response
title_sort structural characterization of antibodies against lipopolysaccharide antigens: insights into primary antibody response
publishDate 2015
url http://hdl.handle.net/1828/6012
work_keys_str_mv AT hajighassemiomid structuralcharacterizationofantibodiesagainstlipopolysaccharideantigensinsightsintoprimaryantibodyresponse
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spelling ndltd-uvic.ca-oai-dspace.library.uvic.ca-1828-60122019-03-23T05:36:27Z Structural characterization of antibodies against lipopolysaccharide antigens: Insights into primary antibody response Haji-Ghassemi, Omid Evans, S. V. Antibodies Antigen Carbohydrate Complex Glycosylation X-ray Crystallography Autoimmunity Lipid A Lipopolysaccharide Structure Chlamydia Antibody combining sites are constructed from limited set of germ-line gene segments, yet are capable of both recognizing a broad range of common epitopes and eliciting an adaptive response to newly encountered pathogens. Carbohydrate antigens generally do not draw T cell help and concomitant affinity maturation in the humoral response. Therefore, anti-carbohydrate responses must rely more heavily on the primary germ-line gene repertoire. Antibodies are usually thought of as highly specific. It has been suggested that polyspecificity and cross-reactivity in germ-line antibodies is necessary to provide the protective mechanisms required to broaden the potential number of antigens recognized; however, the molecular mechanism underlying polyspecificity is poorly understood. To investigate the phenomena of specificity, cross-reactivity and polyspecificity in germ-line antibodies my thesis focuses first on the unique LPS inner core oligosaccharide of Chlamydiaceae, which contains variations within the conserved inner core trisaccharide Kdo(2→8)Kdo(2→4)Kdo (3-deoxy-D-manno-oct-2-ulosonic acid). Antibodies raised against this family-specific trisaccharide showed strong V-region restriction with two sets of heavy and light chain V genes accounting for almost all clones isolated. These groups were named after their prototypic clones as the ‘S25-2 type’ and the ‘S25-23 type’. In contrast to the cross-reactive S25-2 and related antibodies, the S25-23 family of antibodies were shown to be specific for the Chlamydiaceae-specific trisaccharide antigen with no cross-reactivity to Kdo mono or disaccharides or to the Kdo(2→4)Kdo(2→4)Kdo trisaccharide antigen. Interest in S25-23 was sparked by its rare high μM affinity and strict specificity for the family-specific trisaccharide antigen. The structures of the antigen binding fragments of four S25-23-type mAbs have been determined to high resolution in complex with the Chlamydiaceae-specific epitope, revealing the molecular basis for their binding behaviour. The germ-line-encoded paratopes of these antibodies differ significantly from previously characterized S25-2-type mAbs. Unlike the terminal Kdo recognition pocket that promotes cross-reactivity in S25-2-type antibodies, S25-26 and the closely related S25-23 utilize a groove composed of germ-line residues to recognize the length of the trisaccharide antigen. Further S25-23-type antibodies are glycosylated on the variable heavy chain. Analysis of the glycan reveals a heterogeneous mixture with a common root structure that contains an unusually high number of terminal αGal-Gal moieties. One of the unliganded structures in S25-26 shows significant order in the glycan with appropriate electron density for nine residues. The elucidation of the three-dimensional structure of a Gal(α1→3)Gal containing N-linked glycan on a mAb variable heavy chain has potential clinical interest, as it has been implicated in allergic responses in patients receiving therapeutic antibodies. The second focus of my thesis research is the lipid A moiety of LPS, which is involved in septic shock. Though the lipid A epitope appears to be cryptic during infection with Gram-negative bacteria, there have been several reported instances of lipid A specific antibodies isolated from human sera. While these antibodies are strictly selective for lipid A, there are reports of polyspecificity of some anti-lipid A antibodies for single stranded DNA. In such cases, the breakdown of negative selection through polyspecificity has been reported to result in the unfortunate consequences of autoimmune disease. This thesis reports the first crystal structures of antibodies in complex with lipid A and single stranded nucleic acids, elucidating their mechanism for polyspecificity. Perhaps more importantly, the structures may yield clues to the genesis of autoimmune diseases such as systemic lupus erythematosus, thyroiditis, and rheumatic autoimmune diseases. Graduate 2020-04-18 0487 0982 2015-04-24T20:53:56Z 2016-04-17T11:22:06Z 2015 2015-04-24 Thesis http://hdl.handle.net/1828/6012 Haji-Ghassemi O, Muller-Loennies S, Saldova R, Muniyappa M, Brade L, Rudd PM, Harvey DJ, Kosma P, Brade H, Evans SV. (2014) Groove-type Recognition of Chlamydiaceae-specific Lipopolysaccharide Antigen by a Family of Antibodies Possessing an Unusual Variable Heavy Chain N-Linked Glycan. J Biol Chem. 289: 16644-16661. English en Available to the World Wide Web application/pdf