Microheterogeneity of beta-2 glycoprotein I: implications for binding to anionic phospholipids

Considerable interest is currently focused on the interactions of beta-2 gl ycoprotein I(beta(2)GPI) and anti-phospholipid antibodies with anionic phos pholipids in an attempt to understand the association between these antibod ies and clinical diseases such as thrombosis. The interactions of beta(2)GP I and anionic phospholipids have only been characterized partially, and the physiological role of this glycoprotein remains uncertain. In this study w e have explored in detail the physical and phospholipid-binding characteris tics of a number of beta(2)GPI preparations. We have found (i) that perchlo ric acid-purification methods are damaging to beta(2)GPI during purificatio n, (ii) that the dissociation constants of the various preparations for pho sphatidylserine vary between 0.1-2 mu M and are considerably weaker than pr eviously reported, (iii) that considerable differences in affinity of the v arious beta(2)GPI preparations for anionic phospholipids are obtained when comparing anionic phospholipids immobilized to a solid-phase versus phospho lipid assembled in unilamellar vesicles, (iv) that the integrity of the fif th domain of beta(2)GPI is important for binding immobilized anionic phosph olipid but not especially important in binding vesicular anionic phospholip id, and (v) that beta(2)GPI preparations with differing isoelectric species content bind anionic phospholipids differently, suggesting that varying gl ycosylation and/or protein polymorphisms impact upon phospholipid binding. These results highlight the importance of assessing the determinants of the interaction of beta(2)GPI with anionic phospholipids assembled in unilamel lar vesicles.