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To assess the effects of perturbing the surface of low density lipoprotein (LDL) on the conformation of apoB-100, LDL (d 1.030-1.050 g/ml) isolated from normal subjects were treated with phospholipase A2 (PL-A2) for 0.5 to 15 min. The resulting P-LDL and concurrent control LDL (C-LDL) incubated without PL-A2 were isolated by gel permeation chromatography. Approximately 50% of LDL-phosphatidylcholine was hydrolyzed in 2 min and approximately 85% in 5 min. Lysophosphatidylcholine compounds (LPC) and free fatty acids (FFA) accumulated during lipolysis but most of the LPC and all of FFA could be removed by adding FFA-free albumin to the lipolysis mixtures. Immunoreactivities of P-LDL and C-LDL were evaluated in competitive radioimmunoassays, using a library of anti-human LDL monoclonal antibodies directed against the major regions of apoB-100 (the T4, T3, and T2 thrombin fragments). One epitope defined by monoclonal antibody 465B6C3 and localized near the carboxyl end of the apoB-100 molecule became less immunoreactive (ED 50s increased); three other epitopes on the T2 fragment near the LDL receptor recognition site and four epitopes localized towards the middle (T3) and amino terminal (T4) regions did not change. Altered immunoreactivities were not related to LPC and FFA contents. Thus, the conformation of apoB-100 was selectively altered by phospholipolysis. The interactions of P-LDL with cultured fibroblasts were grossly altered: P-LDL were bound nonspecifically to fibroblasts of both normal and homozygous familial hypercholesterolemic subjects and P-LDL were not degraded. LPC and FFA retained in LDL did not explain these alterations, nor did changes of epitope expression near the LDL receptor recognition site. It is likely that the apoB-100 aberrant cell interaction is due to loss of surface phospholipids and "uncovering" of core lipids that react nonspecifically with cell surface components.
Kleinman et al. (Wed,) studied this question.