Effect of phospholipase C and apolipophorin III on the structure and stability of lipophorin subspecies

Four distinct subspecies of the insect hemolymph lipoprotein, lipophorin, that range in diacylglycerol (DAG) content from approximately 100 to 1000 molecules per particle, were treated with phospholipase C. Lipid analysis demonstrated that both phosphatidylcholine and phosphatidylethanolamine were hydrolyzed to DAG. Phospholipase C was used to remove 74-82% of the phospholipid of different lipophorins and these were analyzed for aggregation. Low density lipophorin (LDLp), the largest subspecies, with a diameter of ~23 nm, developed turbidity (monitored by sample absorbance at 340 nm) suggesting the formation of lipoprotein aggregates. High density lipophorin-adult (HDLp-A) and high density lipophorin-wanderer 1 (HDLp-W1) also displayed an increase in A₃₄₀ when incubated with phospholipase C, although the maximal increase observed was considerably less than that for LDLp on a per particle basis. Phospholipase C caused only a minimal increase in A₃₄₀ in a fourth subspecies, high density lipophorin-wanderer 2 (HDLp- W2), which contains an even lower amount of DAG. Electron microscopy was used to evaluate changes in particle morphology as a result of phospholipid depletion. HDLp-W2 and HDLp-W1 showed signs of progressive aggregation and particle fusion. A similar aggregation/fusion was seen in the case of high density lipophorin adult (HDLp-A) while LDLp samples contained multiple aggregation/fusion foci and resultant very large particles. In the presence of exogenous apolipophorin III (apoLp-III), phospholipase C-induced lipophorin aggregation/fusion was prevented. Electron microscopy of LDLp and HDLp-A samples revealed that apoLp-III-stabilized, phospholipase C-treated particles had a morphology similar to that of control particles. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of HDLp-W1, HDLp-A, and LDLp after incubation with phospholipase C and apoLp-III demonstrated the association of apoLp-III with these lipoproteins. Scanning densitometry of the stained gels showed that phospholipase C-treated, apoLp-III-stabilized lipophorin samples acquired 3-5 apoLp-III molecules/particle as a result of phospholipase C-catalyzed phospholipid conversion to DAG. Thus, these experiments establish a correlation between the generation of DAG and the binding of apoLp-III to lipophorin particles. Furthermore, they provide direct evidence that association of apoLp-III with DAG-enriched lipophorins functions to stabilize particle structure.