SPECTROSCOPIC AND LIPID-BINDING STUDIES ON THE AMINO AND CARBOXYL-TERMINAL FRAGMENTS OF LOCUSTA-MIGRATORIA APOLIPOPHORIN-III

The structural basis for the lipid binding capability of Locusta migratoria apolipophorin III (apoLp-III) was assessed by characterizing the amino and carboxyl terminal halves of the protein. The native molecule (similar to 20 kDa) was deglycosylated with endoglycosidase F (molecular mass of deglycosylated species similar to 18 kDa) and cleaved with endoproteinase Arg-C to yield two fragments with molecular masses of similar to 9 kDa each. The two fragments were purified by reversed-phase HPLC and identified by mass spectrometry, amino acid analysis and N-terminal sequencing as the amino terminal (N9) and carboxyl terminal (C9) halves. Due to the apparent discrepancy of the protease digestion pattern obtained compared to that expected from the deduced amino sequence of apoLp-III cDNA, we carried out partial amino acid sequencing of the fragments and cDNA sequencing for the entire protein. Circular dichroism spectroscopy of the N9 and C9 peptides revealed that both exist in buffer in a random coil state. However, addition of-trifluoroethanol, a helix-inducing agent, resulted in the formation of an alpha-helix, reflecting an innate propensity of the peptides to adopt a helical conformation. When cosonicated with dimyristoylphosphatidylcholine (DMPC) both peptides assumed an alpha-helical conformation, indicative of interaction with the phospholipid. In the presence of phospholipids, a 22 nm blue shift in Trp fluorescence emission was observed in the case of the C9 peptide, suggesting that the Trp residues are located in a more hydrophobic environment. Electron microscopy revealed that, compared to native apoLp-III, both peptides possessed a reduced ability to transform DMPC vesicles to disklike complexes. Further, both the N9 and C9 peptides were unable to interact with lipoprotein surfaces, as evidenced by their inability to prevent turbidity development due to aggregation of human low-density Lipoprotein induced by phospholipase C. These results show that the isolated amino and carboxyl terminal fragments of the protein, while able to interact with lipids, cannot mimic the functional capacity of the intact protein. Thus, we conclude that, aside from specific amphipathic alpha-helices, structural elements of the intact protein contribute to physiologically relevant lipid binding abilities of apoLp-III.