Biomedical subjects

C Wingren

Publications and source records attributed to C Wingren.

9 recordsLinked to original sources

PubMed2000

Crystal structure of a gammadelta T cell receptor ligand T22: a truncated MHC-like fold.

Murine T10 and T22 are highly related nonclassical major histocompatibility complex (MHC) class Ib proteins that bind to certain gammadelta T cell receptors (TCRs) in the absence of other components. The crystal structure of T22b at 3.1 angstroms reveals similarities to MHC class I molecules, but one side of the normal peptide-binding groove is severely truncated, which allows direct access to the beta-sheet floor. Potential gammadelta TCR-binding sites can be inferred from functional mapping of T10 and T22 point mutants and allelic variants. Thus, T22 represents an unusual variant of the MHC-like fold and indicates that gammadelta and alphabeta TCRs interact differently with their respective MHC ligands.

Alleles↗

PubMed2000

Recombining germline-derived CDR sequences for creating diverse single-framework antibody libraries.

We constructed a single-chain Fv antibody library that permits human complementarity-determining region (CDR) gene fragments of any germline to be incorporated combinatorially into the appropriate positions of the variable-region frameworks VH-DP47 and VL-DPL3. A library of 2 x 109 independent transformants was screened against haptens, peptides, carbohydrates, and proteins, and the selected antibody fragments exhibited dissociation constants in the subnanomolar range. The antibody genes in this library were built on a single master framework into which diverse CDRs were allowed to recombine. These CDRs were sampled from in vivo-processed gene sequences, thus potentially optimizing the levels of correctly folded and functional molecules, and resulting in a molecule exhibiting a lower computed immunogenicity compared to naive immunoglobulins. Using the modularized assembly process to incorporate foreign sequences into an immunoglobulin scaffold, it is possible to vary as many as six CDRs at the same time, creating genetic and functional variation in antibody molecules.

Germ-Line Mutation↗

PubMed1997

Conformational isomerism of IgG antibodies.

The purpose of this study was to determine why apparently homogeneous IgG antibodies were, in some cases, fractionated into at least two components by liquid-liquid partition chromatography (LLPC) in an aqueous two-phase system. Four mouse monoclonal IgG antibodies, two against albumin, one against IgG and one against thyroxine, were shown to adopt different conformational isomeric forms. The four antibodies existed in an equilibrium between two or three conformational forms, the proportion of which could also be estimated by LLPC. Since LLPC detects mainly conformational differences within the antigen-binding sites of IgG antibodies, it could be concluded that the conformational forms differed with respect to their combining sites. Moreover, the isomeric forms of an antibody directed against a protein antigen, formed antigen-antibody complexes with almost identical surface properties. In contrast, complexes with different surface properties were formed when the hapten or hapten conjugated to BSA was bound. Thus, both the conformational isomers could bind antigen, at least when the antigen was a small hapten or a hapten conjugated to a carrier protein. Our results suggest that six out of 57 monoclonal IgG antibodies exist in equilibrium between at least two conformational forms and the biological significance of this isomerism is discussed.

Animals↗

PubMed1997

Surface properties of antigen-antibody complexes.

In this paper, the authors show that liquid-liquid partition chromatography in an aqueous two-phase system offers unique possibilities of comparing the overall surface properties of intact antibodies in solution before and after binding of antigen. The authors demonstrate that the surface properties of antigen-antibody complexes are dependent on the variable regions of the antibodies, the nature of the antigen and/or possible conformational changes induced by antigen binding. Thus, each antigen-IgG antibody pair formed one type of complex with respect to the exposed dominant surface. The antigen-binding sites of IgG antibodies were exposed and dominant even after binding of hapten or hapten-carrier. In contrast, the antibody-combining sites were concealed upon protein binding and the exposed surfaces of the protein-antibody complexes were related mainly to those of the antigen. IgA1, IgA2, IgE and IgM formed, in comparison to the IgG, hapten-antibody complexes which exhibited surface properties that could be related to both the antigen-binding sites and Fc parts of the antibodies. Moreover, the results indicated that antigen-induced conformational changes occurred in either IgA1, IgA2, IgE, or IgM, but not in IgG1, -2, -3 and -4, making the surfaces of their heavy chain constant regions more similar.

Animals↗

PubMed1996

Enzyme conformational alterations detected by partition column chromatography.

In this paper, we demonstrate the ability of liquid-liquid partition chromatography (LLPC) to detect conformational alterations occurring in well-characterized enzymes. The conformational changes induced in dehydrogenases such as alcohol dehydrogenase (ADH), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), lactate dehydrogenases (LDH) and malate dehydrogenase (MDH) upon binding of ligand(s) were detectable by LLPC. The ligand-dependent equilibrium between two forms of citrate synthase (CS), glutamate-oxaloacetate transaminase (GOT), hexokinase (HK) and 3-phosphoglycerate kinase (PGK) could also be demonstrated. Furthermore, different conformational forms of some of the apoenzymes could also be detected and separated by LLPC. The results obtained here are discussed in relation to those obtained by other methods.

Chromatography, Liquid↗

PubMed1996

Comparison of surface properties of human IgA, IgE, IgG and IgM antibodies with identical and different specificities.

In this paper, the authors report the use of liquid-liquid partition chromatography (LLPC) in an aqueous polyethylene glycol (PEG)/dextran two-phase system to compare the surface properties (partition properties) of human antibodies and fragments thereof. The surface properties of all the monoclonal antibodies of different classes and subclasses investigated were within the same broad range as that observed for the polyclonal antibodies and no relationship was found between the exposed surfaces of the immunoglobulins (Ig) and their heavy chain isotype. Moreover, Fc fragments from various IgG1, 2 and 4 myeloma proteins were found to exhibit similar surface properties. Employing chimeric antibodies with identical variable regions the authors found that intact IgG1, 2 and 4 displayed identical surface properties, while the corresponding IgA1, IgA2, IgG3, IgE and IgM antibodies differed both from each other and from the IgGs. The surface properties of chimeric IgG3 could be made similar to those of the IgG1, 2 and 4 chimers by partially reducing the length of the hinge section, but new differences in surface properties appeared when their hinges were of similar length. Thus, LLPC can be used to detect differences or similarities in the surface properties of the antigen-binding regions as well as the Fc part in the various isotypes. This can shed light on biological activities such as antigen binding and effector function.

Antibody Specificity↗

PubMed1995

A new approach to examine conformational changes occurring upon binding of ligand by biomolecules.

Liquid-liquid partition chromatography in an aqueous poly(ethylene glycol)/dextran two-phase system (LLPC) is shown to be a quick and sensitive method for detecting conformational changes occurring upon binding of ligands by biospecific molecules. Two groups of well-characterized proteins, enzymes and monoclonal antibodies, were employed. As an example, LLPC demonstrated that isoforms of lactate dehydrogenase as well as of hexokinase existed in a ligand-dependent equilibrium between two forms and that conformational changes occurred when monoclonal antibodies bound haptens. We also demonstrate that the method could be used to detect and separate subfractions in preparations of unliganded proteins that appeared to be homogeneous when analysed by other techniques.

Antibodies↗

PubMed1995

Antigen-binding sites dominate the surface properties of IgG antibodies.

A new technique, liquid-liquid partition chromatography in an aqueous polyethylene glycol-dextran two-phase system, was used to detect differences in surface properties of antibodies with different antigen-binding sites. Employing well-characterized monoclonal IgG antibodies and Fab and Fc fragments thereof as well as chimeric IgG antibodies we found a remarkable relationship between structure of the antibody combining site and chromatographic behaviour. The surface properties of the IgG antibodies were dominated by those of its antigen-binding regions. In addition, our results indicated that the constant parts of the IgGs form similar scaffoldings, on to which CDRs of variable shapes and sizes are interspaced and constitute the major dominant differences in exposed surface properties.

Animals↗

PubMed1993

Liquid-liquid partition chromatography as a method to examine surface properties of antibodies and antigen-antibody complexes.

We demonstrate liquid-liquid partition chromatography in aqueous two-phase systems (LLPC) as a simple method for examining the surface properties of immunoglobulins and antigen-antibody complexes in solution. LLPC separates molecules with respect to the properties of the exposed surfaces. As an example, the method may be used to detect changes in the conformation of IgG following chemical modification like acylation or iodination. We have studied the partitioning of antibodies and antigen-antibody complexes, modelled by rabbit antibodies against three human serum proteins, in aqueous polyethylene glycol/dextran two-phase systems at pH 7. Analysis of both polyclonal and monoclonal antibodies against various antigens suggested that the partition properties of immunoglobulins are related mainly to their antigen specificity and not to subclass-specific structures. Furthermore, experiments indicated that changes in the surface properties of antigen and/or antibody following complexation may be detected. Thus, LLPC may prove to be a new way of studying the relation between antibody structure and function in solution.

Acylation↗