Ligand recycling by the insect LDL receptor homologue: a structural perspective

Abstract

Lipid transport potentially poses a problem to multicellular organisms, as lipids are hydrophobic and aggregate in an aqueous environment. In animals, specialized lipid-binding proteins, lipoproteins, are used to circumvent this problem. However, whereas in mammals, endocytic uptake of low density lipoprotein, LDL, mediated by LDL receptor (LDLR), results in their trafficking to lysosomes and subsequent degradation, the insect lipoprotein, HDLp, was shown to use a selective mechanism for transfer of its hydrophobic cargo. The recent identification of receptors of the LDLR family in insects has revealed that endocytic uptake of HDLp may constitute an additional mechanism at specific life stages, which would seem to conflict with the concept of HDLp acting as a reusable lipid shuttle. However, the lipoprotein endocytosed by the insect LDLR homologue, LpR, appeared to be recycled in a manner analogous to that of transferrin by the transferrin receptor. Such a pathway, in which the lipoprotein is ultimately resecreted, is highly comparable with the extracellular lipoprotein-mediated selective delivery of lipids, and clearly is of physiological relevance in insects although the precise function of the LpR-mediated lipoprotein recycling process awaits disclosure. The data presented in this thesis highlight both the parallels and the differences in structure of the mammalian and insect lipoprotein receptors LDLR and LpR, leading to the intriguing difference in functioning, namely ligand degradation versus ligand recycling. Using a flow cytometric assay, it was demonstrated that, in contrast to the complex of LDLR and LDL, the complex of LpR and HDLp is resistant to pertinent conditions prevailing in the early endosome, such as low pH and a decrease in Ca2+ concentration, suggesting that HDLp and LpR remain in complex during intracellular trafficking. An important issue remains, however, how HDLp is released from the receptor when it is recycled back to the plasma membrane. Due to its expression pattern, it was hypothesized that LpR-mediated recycling of HDLp contributes to intracellular lipid delivery without degradation of its apolipoprotein matrix, in agreement with the shuttle function of HDLp in the insect blood. Similar to the iron delivery by transferrin, this would imply a mechanism in which endocytosed HDLp unloads (part of) its lipid cargo intracellularly, resulting in a lower affinity of HDLp for LpR. However, contrary to our expectations, binding studies using a partially delipidated HDLp particle revealed that LpR displayed a 2.4-fold higher affinity for delipidated HDLp than for wt HDLp. The complex stability and endocytic fate were similar to that previously described for HDLp. Therefore, we propose that LpR specifically endocytoses circulatory lipid-poor HDLp to be reloaded during its intracellular route, resulting in a decreased affinity of the particle for LpR. The proposed mechanism allows the uptake of lipids from the strongly diminished reserves in the fat body and their release into the circulation for delivery to other tissues.