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M Galazka

Publications and source records attributed to M Galazka.

6 recordsLinked to original sources
PubMed1999

Modification of beta 2glycoprotein I by glutardialdehyde. Conformational changes and aggregation accompany exposure of the cryptic autoepitope.

Autoantibodies from patients with antiphospholipid syndrome (APS) recognize an epitope on beta 2glycoprotein I (beta 2 GPI) only when native beta 2 GPI is adsorbed on surfaces composed of anionic phospholipids or oxidized polystyrene. beta 2 GPI was modified with the crosslinking agent, glutardialdehyde (GDA), which induced exposure of the anti-beta 2 GPI epitope at GDA:beta 2 GPI mol ratios in the range of 500-2000. A second crosslinking agent, dimethyl-suberimidate (DMS), did not expose the epitope, which may be a consequence of its having less tendency than GDA to form intermolecular links. SDS-PAGE experiments demonstrate that GDA does promote extensive intermolecular crosslinking of beta 2 GPI, and DMS does not. Formaldehyde also reacts with the lysine residues of beta 2 GPI, but does not expose the epitope. The circular dichroism spectra of native and modified beta 2 GPI confirm that GDA induces changes in conformation that are qualitatively different from those caused by formaldehyde. These data provide evidence that binding of lysine residues is not a sufficient condition for exposure of the autoepitope, and also support the likelihood that anti-beta 2 GPI antibodies bind only to aggregates of the protein. Thus, by synthesizing an active holoantigen of beta 2 GPI, conditions were defined that are necessary for binding of human autoantibodies.

Autoantibodies↗
PubMed1998

A stable, multi-subunit complex of beta2glycoprotein I.

Beta2glycoprotein I (beta2GPI) is a 54-kDa plasma protein which is recognized as an autoantigen for antibodies from patients with antiphospholipid syndrome (APS). SDS-PAGE (under reducing conditions) of beta2GPI from three sources indicates that the 54-kDa beta2GPI band is accompanied by a band corresponding to an 8-kDa protein. In the absence of detergent and reducing agents (native PAGE), beta2GPI demonstrated a large complex (molecular mass approximately 320 kDa) which is dissociable by boiling in 6-8 M urea, yielding several lower molecular mass bands, one of which corresponds to the 8-kDa protein observed in SDS-PAGE. Sera from five healthy adults demonstrated native beta2GPI migration equivalent to the commercially purified protein. Atomic force microscopy (AFM) images of native beta2GPI show aggregrates of particles each having a diameter of 30-35 nm. This is consistent with a globular unit the size of which would be substantially larger than that expected for a 54-kDa protein. These experiments suggest that the 54-kDa beta2GPI monomer subunits exist as a multimeric complex with the 8-kDa protein.

Adult↗
PubMed1995

Binding of beta 2 glycoprotein I to activated polystyrene and its recognition by human IgG autoantibodies.

Beta 2 glycoprotein I (apolipoprotein H, beta 2GPI) is involved in the formation of epitope(s) recognized by clinically relevant autoantibodies from patients with antiphospholipid syndrome. We studied the binding of beta 2GPI to chemically activated polystyrene in a microtitre plate format. Adsorption isotherms (at 37 degrees C) were generated for beta 2GPI on activated polystyrene and on unactivated polystyrene, with both human serum antibodies and rabbit polyclonal IgG antibodies as probes, and horseradish peroxidase (HRP)-tagged anti-IgG to detect binding. Additionally, beta 2GPI was biotinylated and isotherms were developed by using HRP-streptavidin as the recognition sequence. Human serum autoantibodies, which did not precipitate beta 2GPI in solution, yielded a characteristic chemisorption isotherm on activated polystyrene but did not recognize beta 2GPI bound to untreated polystyrene. The rabbit IgG, which did precipitate beta 2GPI in solution, detected beta 2GPI bound to both activated polystyrene and, to a lesser extent, to untreated polystyrene. The binding of beta 2GPI to untreated polystyrene was confirmed by the use of biotinylated beta 2GPI. To assess the prevalence of IgG anti-beta 2GPI autoantibodies, we surveyed 113 sera submitted to our laboratory for anticardiolipin antibody (aCL) testing. Only nine (8%) had anti-beta 2GPI activity greater than two standard deviations above the mean for those sera in which aCL activity was within normal limits. We conclude that epitope presentation of beta 2GPI for human autoantibody binding is dependent on surface properties of the polystyrene, and that beta 2GPI autoantibodies are found only in a subpopulation of sera positive for aCL.

Adsorption↗