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Structure and Interactions of Human IgG-Fc
Linköping University, Department of Physics, Chemistry and Biology. Linköping University, The Institute of Technology.
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

This thesis involves structure and interaction studies of the Fc fragment of human IgG. For this purpose, hIgG-Fc of different subclasses were cloned and expressed in the eukaryotic host Pichia pastoris, where relevant protein modification at the post-translational level can be obtained.

Sometimes, changes in pH, temperature and salt concentration or addition of moderate amounts of denaturants to a protein solution are associated with the protein forming non-natively folded states, such as the molten globule or the A state. IgG and some parts thereof are capable of forming another, so called alternatively folded state, usually induced by acidification in the presence of anions. This state is in many aspects related to the molten globule and the A state but with distinguishing properties related mainly to chemical stability and formation of oligomeric structures. The first part of this thesis describes two different alternatively folded states of hIgG-Fc of subclass 4. One of them was induced by decreasing the pH of the protein solution. Observed structural changes were highly dependent on the concentration of sodium chloride. The alternatively folded protein showed drastic changes in its secondary structure compared to the native protein and significant tertiary structure was lost. Moreover, it displayed an apparently increased chemical stability and had surface exposed hydrophobic patches resulting in the formation of higher order assemblies. In addition, it was shown for the first time that thermal induction of an alternatively folded state is also possible, with similar, but not identical, properties as the acid-induced state. Heat incubation for 20 hours at neutral pH and at a physiological salt concentration further resulted in the formation of protein aggregates. The dye Congo red had affinity for these aggregates, and when viewed under polarized light, it showed green birefringence. They also displayed binding of Thioflavin T and had a typical fibril appearance in the transmission electron microscope. Hence, the formed aggregates share key properties with structures constituting amyloid.

The second part of this thesis is focused on interactions of the Fc-fragment with respect to both Fcγ-receptors on monocytes and the IgG autoantibody rheumatoid factor. Immune complexes and their binding to Fcγ-receptors are of pathogenic interest to rheumatoid arthritis. A surface mimic presenting full IgG molecules was designed as an in vitro immune complex model. Utilizing self-assembled monolayers composed of alkanethiolates with different chemical functionalities, the lateral IgG density could be tuned, enabling control of monocyte interaction with the surface. Importantly, the IgG molecules were homogeneously oriented to expose the Fc-fragment. The protein repellent properties of these  surfaces ensured that only differences in IgG concentration determined variations in cellular adhesion. In a separate study the specificities of IgG rheumatoid factor with respect to the different subclasses of hIgG-Fc were investigated, using sera from patients with early rheumatoid arthritis. Strikingly high IgG-RF reactivity against hIgG2-Fc was observed, together with raised levels against hIgG1-Fc and hIgG4-Fc. No reactivity against hIgG3-Fc was found.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press , 2011. , 71 p.
Series
Linköping Studies in Science and Technology. Dissertations, ISSN 0345-7524 ; 1361
National Category
Natural Sciences
Identifiers
URN: urn:nbn:se:liu:diva-65536ISBN: 978-91-7393-241-7 (print)OAI: oai:DiVA.org:liu-65536DiVA: diva2:396475
Public defence
2011-03-11, Planck, Fysikhuset, Campus Valla, Linköpings universitet, Linköping, 09:15 (Swedish)
Opponent
Supervisors
Available from: 2011-02-10 Created: 2011-02-10 Last updated: 2015-08-31Bibliographically approved
List of papers
1. Thermal Induction of an Alternatively Folded State in Human IgG-Fc
Open this publication in new window or tab >>Thermal Induction of an Alternatively Folded State in Human IgG-Fc
2011 (English)In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 50, no 6, 981-988 p.Article in journal (Refereed) Published
Abstract [en]

We report the formation of a non-native, folded state of human IgG4-Fc induced by a high temperature at neutral pH and at a physiological salt concentration. This structure is similar to the molten globule state in that it displays a high degree of secondary structure content and surface-exposed hydrophobic residues. However, it is highly resistant to chemical denaturation. The thermally induced state of human IgG4-Fc is thus associated with typical properties of the so-called alternatively folded state previously described for murine IgG, IgG-Fab, and individual antibody domains (V(L), V(H), C(H)1, and C(H)3) under acidic conditions in the presence of anions. Like some of these molecules, human IgG4-Fc in its alternative fold exists as a mixture of different oligomeric structures, dominated by an equilibrium between monomeric and heptameric species. Heating further induces the formation of fibrous structures in the micrometer range.

Place, publisher, year, edition, pages
American Chemical Society, 2011
National Category
Natural Sciences
Identifiers
urn:nbn:se:liu:diva-65532 (URN)10.1021/bi101549n (DOI)000287049500008 ()21261247 (PubMedID)
Available from: 2011-02-10 Created: 2011-02-10 Last updated: 2017-12-11Bibliographically approved
2. Human IgG-Fc Forms an Alternatively Folded State at Low pH
Open this publication in new window or tab >>Human IgG-Fc Forms an Alternatively Folded State at Low pH
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Anion-induced formation of so-called alternatively folded states has previously been described for murine IgG, IgG-Fab and several separate antibody domains, including CH3, at low pH. In this report, we confirm that this property can also be extended to the full Fc fragment of IgG. When incubating human IgG4-Fc at pH 2.0 in the presence of NaCl, the protein adopts a conformation that is characterized by a high degree of secondary structure but less well-defined tertiary structure compared to the native state. In contrast to both a classical molten globule and an acid-induced A state, however, the alternatively folded protein appears very stable toward chemical denaturation and unfolds in cooperative transitions. It further forms oligomeric assemblies that are primarily composed of dimers and dodecamers, and when incubated at physiologic temperatures with agitation, it displays prefibrillar structures that are both thioflavinophilic and congophilic.

National Category
Natural Sciences
Identifiers
urn:nbn:se:liu:diva-65533 (URN)
Available from: 2011-02-10 Created: 2011-02-10 Last updated: 2011-02-10Bibliographically approved
3. In Vitro Amyloid Fibril Formation of Human IgG-Fc
Open this publication in new window or tab >>In Vitro Amyloid Fibril Formation of Human IgG-Fc
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Both light and heavy chains of human antibodies are known to be associated with immunoglobulin related amyloidosis, but in vitro formation of amyloid fibrils has previously only been reported for light chain sequences. Here we show that fibrillation of the Fc fragment of human IgG of all subclasses can be induced by heating to at least 75°C at neutral pH and physiological salt concentration. The observed protein assemblies share key properties with those constituting amyloid, i.e. they are thioflavinophilic and congophilic and have a typical fibril appearance in the transmission electron microscope. This study of the amyloidogenic properties of human IgG-Fc, comprising the CH2 and CH3 domains of the IgG heavy chain, is important for increasing the understanding of which parts of IgG that could be involved in amyloid formation in vivo.

National Category
Natural Sciences
Identifiers
urn:nbn:se:liu:diva-65534 (URN)
Available from: 2011-02-10 Created: 2011-02-10 Last updated: 2011-02-10Bibliographically approved
4. Designed Surface with Tunable IgG Density as an in Vitro Model for Immune Complex Mediated Stimulation of Leukocytes
Open this publication in new window or tab >>Designed Surface with Tunable IgG Density as an in Vitro Model for Immune Complex Mediated Stimulation of Leukocytes
Show others...
2010 (English)In: Langmuir, ISSN 0743-7463, E-ISSN 1520-5827, Vol. 26, no 5, 3493-3497 p.Article in journal (Refereed) Published
Abstract [en]

We present the design of an in vitro for immune-complex-mediated stimulation of leukocytes and its functional characteristics with respect to monocyte adhesion. The model was based on orientation-controlled immobilization of a humanized IgG1 monoclonal antibody (rituximab) via its interaction with a biotinylated peptide epitope derived from the CD20 marker. The peptide was linked to neutravidin covalently attached to it mixed self-assembled monolayer of carboxyl- and methoxy-terminated oligo(ethylene glycol) alkane thiolates on gold. The surface adhesion propensity of human monocytes (cell line U917) was highly dependent on the lateral IgG density and indicated that there exists a distance between IgG-Fc on the surface where interactions with Fc gamma receptors are optimal. This well-defined platform allows for a careful control of the size and orientation of artificial IgG immune complexes, it is easily made compatible with, for example, cellular imaging, and it will become useful for in vitro studies on the importance of Fc gamma receptor interactions in chronic immune-mediated diseases.

National Category
Engineering and Technology
Identifiers
urn:nbn:se:liu:diva-54254 (URN)10.1021/la9030766 (DOI)000274636900076 ()
Available from: 2010-03-05 Created: 2010-03-05 Last updated: 2017-12-12Bibliographically approved
5. IgG Rheumatoid Factor Against the Four Human Fc-gamma Subclasses in Early Rheumatoid Arthritis (the Swedish TIRA Project)
Open this publication in new window or tab >>IgG Rheumatoid Factor Against the Four Human Fc-gamma Subclasses in Early Rheumatoid Arthritis (the Swedish TIRA Project)
Show others...
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Rheumatoid factor (RF), i.e. a family of autoantibodies against the Fc part of IgG, is an important seromarker of rheumatoid arthritis (RA). Traditional particle agglutination without disclosing the antibody isotype remains the predominating diagnostic method in clinical routine. Although IgG-RF attracts pathogenic interest, its detection remains technically challenging. The present study aimed at developing a set of tests identifying IgG-RFs directed against the four IgG subclasses. IgG-RF against either subclass of human IgG-Fc were analyzed with four novel enzyme-linked immunosorbent assays (ELISAs) utilizing four recombinant human Fc-gamma fragments (hIgG1-4) as sources of antigen. Sera from 40 patients with recent-onset RA (20 seropositive and 20 seronegative by IgM-RF and IgA-RF-isotype specific ELISA) were analyzed. Sera from 20 healthy blood donors served as reference. Among the IgM-/IgA-RF positive RA-sera, IgG-RF was found directed against hIgG1, hIgG4, and most notably, with strikingly high reactivity against hIgG2, but not hIgG3. Significant correlations were seen between IgG-RF against hIgG2-Fc and IgA-RF (r = 0.513) and IgM-RF (r = 0.736) levels. Further prospective studies are warranted to elucidate any correlation to disease course and outcome.

National Category
Natural Sciences
Identifiers
urn:nbn:se:liu:diva-65535 (URN)
Available from: 2011-02-10 Created: 2011-02-10 Last updated: 2015-08-31Bibliographically approved

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