Role of phospholipids in the protein stability of an insect lipoprotein, lipophorin from Rhodnius prolixus

Lipophorin (Lp) is the major lipoprotein in insect hemolymph. The structural organization proposed for Lp is basically the same as that suggested for vertebrate lipoproteins, consisting of a hydrophobic core containing neutral lipids, stabilized in the aqueous environment by surrounding polar moieties of protein and phospholipids at the particle surface. After complete removal of phospholipids from Lp by phospholipase A2, the particle remains soluble [Gondim, K.C., Atella, G.C., Kawooya, J.K., & Masuda, H. (1992) Arch. Insect Biochem. Physiol. 20, 303-314]. However, studies on the roles of phospholipid on the structural stability of Lp are still lacking. In the present work, we have studied the structure and stability of dephospholipidated lipophorin (d-Lp). Trypsinolysis of d-Lp indicated no exposure of new cleavage sites on the protein when compared to Lp. However, an enhanced rate of proteolysis of the apoproteins (especially apolipophorin II) was observed in d-Lp. Circular dichroism analysis indicated that the secondary structure of Lp was not significantly affected by phospholipid removal. Furthermore, the exposure of tryptophan residues to the aqueous solvent in d-Lp was the same as in Lp, as indicated by intrinsic fluorescence emission spectra and fluorescence quenching experiments. Interestingly, d-Lp was more resistant to denaturation by guanidine hydrochloride than Lp. d-Lp was also found to be less sensitive than Lp to structural changes induced by hydrostatic pressure. Taken together, these results indicate that, although changes in its structural organization were subtle, dephospholipidated lipophorin may have additional protein-protein and/or protein-neutral lipid interactions that are responsible for the observed increase in stability. Therefore, phospholipids are not only not essential for Lp stability, but their presence in the particle seems to result in a less stable structure in the aqueous environment.