Lipophorin: The Lipid Shuttle.

Insects need to transport lipids through the aqueous medium of the hemolymph to the organs in demand, after they are absorbed by the intestine or mobilized from the lipid-producing organs. Lipophorin is a lipoprotein present in insect hemolymph, and is responsible for this function. A single gene encodes an apolipoprotein that is cleaved to generate apolipophorin I and II. These are the essential protein constituents of lipophorin. In some physiological conditions, a third apolipoprotein of different origin may be present. In most insects, lipophorin transports mainly diacylglycerol and hydrocarbons, in addition to phospholipids. The fat body synthesizes and secretes lipophorin into the hemolymph, and several signals, such as nutritional, endocrine, or external agents, can regulate this process. However, the main characteristic of lipophorin is the fact that it acts as a reusable shuttle, distributing lipids between organs without being endocytosed or degraded in this process. Lipophorin interacts with tissues through specific receptors of the LDL receptor superfamily, although more recent results have shown that other proteins may also be involved. In this chapter, we describe the lipophorin structure in terms of proteins and lipids, in addition to reviewing what is known about lipoprotein synthesis and regulation. In addition, we reviewed the results investigating lipophorin's function in the movement of lipids between organs and the function of lipophorin receptors in this process.

Lipophorin receptor is required for the accumulations of cuticular hydrocarbons and ovarian neutral lipids in Locusta migratoria.

Lipophorin is the most abundant lipoprotein particle in insect hemolymph. Lipophorin receptor (LPR) is a glycoprotein that binds to the lipophorin and mediates cellular uptake and metabolism of lipids by endocytosis. However, the roles of LPR in uptake of lipids in the integument and ovary remain unknown in the migratory locust (Locusta migratoria). In present study, we characterized the molecular properties and biological roles of LmLPR in L. migratoria. The LmLPR transcript level was high in the first 2 days of the adults after eclosion, then gradually declined. LmLPR was predominately expressed in fat body, ovary and integument. Using immuno-detection methods, we revealed that LmLPR was mainly localized in the membrane of oenocytes, epidermal cells, fat body cells and follicular cells. RNAi-mediated silencing of LmLPR led to a slight decrease of the cuticle hydrocarbon contents but with little effect on the cuticular permeability. However, the neutral lipid content was significantly decreased in the ovary after RNAi against LmLPR, which led to a retarded ovarian development. Taken together, our results indicated that LmLPR is involved in the uptake and accumulation of lipids in the ovary and plays a crucial role in ovarian development in L. migratoria. Therefore, LmLPR could be a promising RNAi target for insect pest management by disrupting insect ovarian development.

Lipophorin receptors regulate mushroom body development and complex behaviors in Drosophila.

BACKGROUND: Drosophila melanogaster lipophorin receptors (LpRs), LpR1 and LpR2, are members of the LDLR family known to mediate lipid uptake in a range of organisms from Drosophila to humans. The vertebrate orthologs of LpRs, ApoER2 and VLDL-R, function as receptors of a glycoprotein involved in development of the central nervous system, Reelin, which is not present in flies. ApoER2 and VLDL-R are associated with the development and function of the hippocampus and cerebral cortex, important association areas in the mammalian brain, as well as with neurodevelopmental and neurodegenerative disorders linked to those regions. It is currently unknown whether LpRs play similar roles in the Drosophila brain. RESULTS: We report that LpR-deficient flies exhibit impaired olfactory memory and sleep patterns, which seem to reflect anatomical defects found in a critical brain association area, the mushroom bodies (MB). Moreover, cultured MB neurons respond to mammalian Reelin by increasing the complexity of their neurite arborization. This effect depends on LpRs and Dab, the Drosophila ortholog of the Reelin signaling adaptor protein Dab1. In vitro, two of the long isoforms of LpRs allow the internalization of Reelin, suggesting that Drosophila LpRs interact with human Reelin to induce downstream cellular events. CONCLUSIONS: These findings demonstrate that LpRs contribute to MB development and function, supporting the existence of a LpR-dependent signaling in Drosophila, and advance our understanding of the molecular factors functioning in neural systems to generate complex behaviors in this model. Our results further emphasize the importance of Drosophila as a model to investigate the alterations in specific genes contributing to neural disorders.

Lipophorin receptor regulates the cuticular hydrocarbon accumulation and adult fecundity of the pea aphid Acyrthosiphon pisum.

Cuticular hydrocarbons form a barrier that protects terrestrial insects from water loss via the epicuticle. Lipophorin loads and transports lipids, including hydrocarbons, from one tissue to another. In some insects, the lipophorin receptor (LpR), which binds to lipophorin and accepts its lipid cargo, is essential for female fecundity because it mediates the incorporation of lipophorin by developing oocytes. However, it is unclear whether LpR is involved in the accumulation of cuticular hydrocarbons and its precise role in aphid reproduction remains unknown. We herein present the results of our molecular characterization, phylogenetic analysis, and functional annotation of the pea aphid (Acyrthosiphon pisum) LpR gene (ApLpR). This gene was transcribed throughout the A. pisum life cycle, but especially during the embryonic stage and in the abdominal cuticle. Furthermore, we optimized the RHA interference (RNAi) parameters by determining the ideal dose and duration for gene silencing in the pea aphid. We observed that the RNAi-based ApLpR suppression significantly decreased the internal and cuticular hydrocarbon contents as well as adult fecundity. Additionally, a deficiency in cuticular hydrocarbons increased the susceptibility of aphids to desiccation stress, with decreased survival rates under simulated drought conditions. Moreover, ApLpR expression levels significantly increased in response to the desiccation treatment. These results confirm that ApLpR is involved in transporting hydrocarbons and protecting aphids from desiccation stress. Furthermore, this gene is vital for aphid reproduction. Therefore, the ApLpR gene of A. pisum may be a novel RNAi target relevant for insect pest management.

Site-specific O-glycosylation of members of the low-density lipoprotein receptor superfamily enhances ligand interactions.

The low-density lipoprotein receptor (LDLR) and related receptors are important for the transport of diverse biomolecules across cell membranes and barriers. Their functions are especially relevant for cholesterol homeostasis and diseases, including neurodegenerative and kidney disorders. Members of the LDLR-related protein family share LDLR class A (LA) repeats providing binding properties for lipoproteins and other biomolecules. We previously demonstrated that short linker regions between these LA repeats contain conserved O-glycan sites. Moreover, we found that O-glycan modifications at these sites are selectively controlled by the GalNAc-transferase isoform, GalNAc-T11. However, the effects of GalNAc-T11-mediated O-glycosylation on LDLR and related receptor localization and function are unknown. Here, we characterized O-glycosylation of LDLR-related proteins and identified conserved O-glycosylation sites in the LA linker regions of VLDLR, LRP1, and LRP2 (Megalin) from both cell lines and rat organs. Using a panel of gene-edited isogenic cell line models, we demonstrate that GalNAc-T11-mediated LDLR and VLDLR O-glycosylation is not required for transport and cell-surface expression and stability of these receptors but markedly enhances LDL and VLDL binding and uptake. Direct ELISA-based binding assays with truncated LDLR constructs revealed that O-glycosylation increased affinity for LDL by ∼5-fold. The molecular basis for this observation is currently unknown, but these findings open up new avenues for exploring the roles of LDLR-related proteins in disease.

Lipophorin receptor regulates Nilaparvata lugens fecundity by promoting lipid accumulation and vitellogenin biosynthesis.

Insect lipophorin receptor (LpR) belongs to the low-density lipoprotein receptor (LDLR) superfamily and plays an essential role in fecundity by mediating the incorporation of lipophorin into developing oocytes. Here we report the identification and characterization of a full-length cDNA encoding a putative LpR from the brown planthopper, Nilaparvata lugens. The deduced amino acid sequence of NlLpR possesses the conserved structural motifs of LDLR family members, and displays a high degree of similarity with sequences from other insect LpRs. NlLpR is transcribed throughout oogenesis with its maximum level on day 7 after adult female emergence. NlLpR is highly expressed in the fat body and ovary, with relative low levels in the head, epidermis and midgut. Knockdown of NlLpR using double-stranded RNA (dsRNA) led to decreased triacylglycerol (TAG) content, retarded development of ovaries and decreased fecundity. Further functional analyses revealed that NlLpR works through nutritional signaling pathway-dependent activation of S6 kinase to regulate vitellogenin (Vg) biosynthesis during vitellogenesis and oocyte development. Disrupting of ecdysone receptor (EcR) expression and 20-hydroxyecdysone (20E) topical application demonstrated that NlLpR is regulated by ecdysone at transcript level. These results suggest that LpR is essential for Vg synthesis in the fat body and lipid uptake by developing oocytes, thus playing a critical role in insect reproduction.

Recent progress in molecular genetic studies on the carotenoid transport system using cocoon-color mutants of the silkworm.

The existence of tissue-specific delivery for certain carotenoids is supported by genetic evidence from the silkworm Bombyx mori and the identification of cocoon color mutant genes, such as Yellow blood (Y), Yellow cocoon (C), and Flesh cocoon (F). Mutants with white cocoons are defective in one of the steps involved in transporting carotenoids from the midgut lumen to the middle silk gland via the hemolymph lipoprotein, lipophorin, and the different colored cocoons are caused by the accumulation of specific carotenoids into the middle silk gland. The Y gene encodes carotenoid-binding protein (CBP), which is expected to function as the cytosolic transporter of carotenoids across the enterocyte and epithelium of the middle silk gland. The C and F genes encode the C locus-associated membrane protein, which is homologous to a mammalian high-density lipoprotein receptor-2 (Cameo2) and scavenger receptor class B member 15 (SCRB15), respectively; these membrane proteins are expected to function as non-internalizing lipophorin receptors in the middle silk gland. Cameo2 and SCRB15 belong to the cluster determinant 36 (CD36) family, with Cameo2 exhibiting specificity not only for lutein, but also for zeaxanthin and astaxanthin, while SCRB15 seems to have specificity toward carotene substrates such as α-carotene and ß-carotene. These findings suggest that Cameo2 and SCRB15 can discriminate the chemical structure of lutein and ß-carotene from circulating lipophorin during uptake. These data provide the first evidence that CD36 family proteins can discriminate individual carotenoid molecules in lipophorin.

Yeast two-hybrid screen reveals novel protein interactions of the cytoplasmic tail of lipophorin receptor in silkworm brain.

The silkworm, Bombyx mori lipophorin receptor (BmLpR), is expressed as splice variants. The alternative splicing of its primary gene transcripts yields four isoforms namely, LpR1 through 4. Among these isoforms, the LpR4 is unique, expressed only in the brain and CNS and with a unique amino acid tail sequence in its cytoplasmic domain. We carried out yeast two-hybrid screens to identify effector proteins that interact specifically with the cytoplasmic tail of LpR4 from a cDNA library derived from silkworm brain. The validity of the screen was confirmed by immunoblotting and further by co-immunoprecipitation. We have identified 11 novel proteins that are capable of interacting with the cytoplasmic domain of LpR4 in the silkworm brain. Most of these newly identified target proteins have known functions in lipid signalling, protein kinase pathways, cell motility, and organization of cytoskeleton, neurotransmission, and neuroprotection. These findings, for the first time, demonstrate a molecular link between LpR4 and the interacting proteins that might be involved in the regulation of signalling pathways in silkworm brain.

Interaction of lipophorin with Rhodnius prolixus oocytes: biochemical properties and the importance of blood feeding

Lipophorin (Lp) is the main haemolymphatic lipoprotein in insects and transports lipids between different organs. In adult females, lipophorin delivers lipids to growing oocytes. In this study, the interaction of this lipoprotein with the ovaries of Rhodnius prolixus was characterised using an oocyte membrane preparation and purified radiolabelled Lp (125I-Lp). Lp-specific binding to the oocyte membrane reached equilibrium after 40-60 min and when 125I-Lp was incubated with increasing amounts of membrane protein, corresponding increases in Lp binding were observed. The specific binding of Lp to the membrane preparation was a saturable process, with a Kdof 7.1 ± 0.9 x 10-8M and a maximal binding capacity of 430 ± 40 ng 125I-Lp/µg of membrane protein. The binding was calcium independent and pH sensitive, reaching its maximum at pH 5.2-5.7. Suramin inhibited the binding interaction between Lp and the oocyte membranes, which was completely abolished at 0.5 mM suramin. The oocyte membrane preparation from R. prolixus also showed binding to Lp from Manduca sexta. When Lp was fluorescently labelled and injected into vitellogenic females, the level of Lp-oocyte binding was much higher in females that were fed whole blood than in those fed blood plasma.

Molecular cloning and partial characterization of an ovarian receptor with seven ligand binding repeats, an orthologue of low-density lipoprotein receptor, in the cutthroat trout (Oncorhynchus clarki).

Teleost fish eggs contain a substantial yolk mass consisting of lipids and proteins that provides essential nutrients for embryonic and larval development. The polar lipid and protein components of the yolk are delivered to oocytes by circulating vitellogenins, however the source(s) of the neutral lipid remains unknown. We cloned a cDNA encoding an orthologue of low-density-lipoprotein receptor (LDLR) from the ovary of cutthroat trout, Oncorhynchus clarki (ct-Ldlr). Predominant expression of ct-ldlr mRNA was observed in the ovary and moderate expression was detected in intestine, gill and brain. The relative abundance of ct-ldlr transcripts was highest in early pre-vitellogenic ovaries and significantly decreased during vitellogenesis, followed by a slight increase during final maturation and in post-ovulatory follicles. In situ hybridization revealed an intense and evenly distributed localization of ct-ldlr transcripts in the ooplasm of pre-vitellogenic oocytes and these signals disappeared in vitellogenic follicles. Collectively, these results suggest that the Ldlr is involved in deposition of yolk lipids in cutthroat trout oocytes. The ct-ldlr transcripts also were detected in theca and granulosa cells, suggesting that this receptor may be involved in cholesterol uptake for ovarian steroidogenesis. This is the first report on partial characterization of an ldlr orthologue in any fish species.