Biomedical subjects

J D Kohles

Publications and source records attributed to J D Kohles.

4 recordsLinked to original sources

PubMed1999

The hololipoprotein complex of beta(2)-glycoprotein I and dicaproyl phosphatidylserine.

The ability of beta(2)-glycoprotein I (formerly referred to as apolipoprotein H) to act as an autoantigen for antibodies from patients with antiphospholipid syndrome is dependent upon its binding in vivo to anionic phospholipid surfaces or to surfaces in vitro which mimic their surface characteristics. The ability of the autoepitope(s) of beta(2)-glycoprotein I to be exposed by binding a short-chain (6-carbon), anionic phospholipid has not been explored. Here, we describe our studies of the hololipoprotein generated by reacting beta(2)-glycoprotein I with dicaproyl phosphatidylserine. The formation of the complex is accompanied by inhibition of beta(2)-glycoprotein I binding to phospholipid-coated polystyrene surfaces, with 50% reduction in binding occurring at about 10 mM. At concentrations >10 mM, dicaproyl phosphatidylserine also displaces beta(2)-glycoprotein I bound to anionic phospholipid surfaces. Physicochemical studies suggest that at DCPS concentrations >6 mM, the solutions are colloidal and that beta(2)-glycoprotein I forms a supramolecular complex with organized phospholipid structures. Using a standard human autoimmune anti-beta(2)-glycoprotein I plasma, as well as a series of six additional sera from patients with antiphospholipid syndrome, the complex did not generate a detectable epitope. We conclude that lipid binding, per se, is not sufficient for the presentation of the epitope(s) of beta(2)-glycoprotein I or its recognition by autoantibodies from patients with antiphospholipid syndrome.

Antiphospholipid Syndrome↗

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↗

PubMed1999

Inhibition of beta 2glycoprotein I binding to anionic phospholipids: a strategy for the development of antiphospholipid syndrome-specific drugs.

The binding of beta 2glycoprotein I (beta 2GPI) to anionic phospholipids (PL) leads to the presentation of one or more epitopes recognized by autoantibodies from patients with antiphospholipid syndrome (APS). The inhibition of beta 2GPI binding to PL mixtures coated on polystyrene microtiter wells (MTW) and to large, multilamellar PL vesicles (LMV) was examined. Inhibitors included phosphorylated monosaccharide metabolites, myo-inositol monophosphate (IMP), hexaphosphate (IHP) and hexasulfate (IHS), pyrophosphate (PPi), methyl bisphosphonate (MBP) and phenyl phosphonate, and a series of carboxylic and aromatic sulfonic acids. Inhibitors were incubated with beta 2GPI at 37 degrees C for 2 hr either with dimyristoylphosphatidic acid, 80%/dimyristoylphosphatidyl choline, 20% (DMPA/DMPC) coated on MTW or in a suspension of LMV. Phospholipid-bound beta 2GPI to PA/PC on MTW was detected using an immunoassay based on rabbit anti-beta 2GPI; free beta 2GPI (not bound to LMV) was detected by fluorescence spectroscopy. Inhibition was studied over the range 0.01-9.0 mumoles/10(-4)L (0.1-90 mM). Inhibition at maximum concentration in the MTW system ranged from 0.1% (for ADP) to > 94% (for IHP). IHP also provided the greatest inhibition in the LMV system (76%) and was also effective in displacing beta 2GPI already bound to PL surfaces (approximately 50% displaced at 0.25 mM). These data suggest that a strategy for development of therapeutic agents for APS may be based on the use of small cyclic, organic oligoanions such as inositol derivatives to act as ligands for lysine residues at the PL binding site of beta 2GPI.

Animals↗

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↗