Insect lipoprotein biogenesis depends on an amphipathic β cluster in apolipophorin II/I and is stimulated by microsomal triglyceride transfer protein

Abstract

Lipoproteins transport lipids in the circulation of an evolutionally wide diversity of animals. The pathway for lipoprotein biogenesis has been revealed to a large extent in mammals only, in which apolipoprotein B (apoB) acquires lipids via the assistance of microsomal triglyceride transfer protein (MTP) and binds them by means of amphipathic protein structures. To investigate whether this is a common mechanism for lipoprotein biogenesis in animals, we studied the structural elements involved in the assembly of the insect lipoprotein, lipophorin. LOCATE sequence analysis predicted that the insect lipoprotein precursor, apolipophorin II/I (apoLp-II/I), contains clusters of amphipathic a-helices and b-strands, organized along the protein as N-a 1 -b-a 2 -C, reminiscent of a truncated form of apoB. Recombinant expression of a series of C-terminal truncation variants of Locusta migratoria apoLp-II/I in an insect cell (Sf9) expression system revealed that the formation of a buoyant high density lipoprotein requires the amphipathic b cluster. Coexpression of apoLp-II/I with the MTP homolog of Drosophila melanogaster affected insect lipoprotein biogenesis quantitatively as well as qualitatively, as the secretion of apoLp-II/I proteins was increased severalfold and the buoyant density of the secreted lipoprotein decreased concomitantly, indicative of augmented lipidation. Based on these findings, we propose that, despite specific modifications, the assembly of lipoproteins involves MTP as well as amphipathic structures in the apolipoprotein carrier, both in mammals and insects. Thus, lipoprotein biogenesis in animals appears to rely on structural elements that are of early metazoan origin. Lipoproteins function in the transport of lipids in the circulation of vertebrates as well as invertebrates. In mammals, the biosynthesis of these macromolecular proteinlipid complexes occurring in liver and intestine involves the intracellular transfer and subsequent stabilization of lipids onto a single protein component, the nonexchangeable apolipoprotein B (apoB) (for reviews, see Refs. 1-7).The major lipoprotein of insects, lipophorin, is stabilized by apolipopoteins derived from apolipophorin II/I (apoLp-II/I), a homolog of apoB (8). In contrast to apoB, apoLp-II/I is cleaved during lipoprotein biosynthesis, resulting in the presence of two apolipoproteins in the secreted insect lipoprotein, apoLp-I and apoLp-II (8-12; for reviews, see Refs. 13-17). Cleavage of apoLp-II/I is not essential for lipophorin biogenesis, as uncleaved apoLp-II/I is also able to form a lipoprotein (11). Lipophorin is produced in cells of the fat body, an organ that combines the functions of liver and adipose tissues in vertebrates. Lipophorin is secreted with a buoyant density in the high density or very high density lipoprotein range, dependent on the species (18)(19)(20)(21)(22). Accordingly, lipophorin contains far fewer associated lipids than apoB lipoproteins that are secreted as very low density lipoproteins or chylomicrons. Moreover, the...

Lipoprotein assembly and function in an evolutionary perspective

“…The a 1 cluster and a small N-terminal part of the b cluster of this tripartite organization constitute the LLT module (and could therefore, in accordance with apoB, be expanded to ba 1 , resulting in the tripartite structure N-ba 1 -b 1 -a 2 -C); recombinant expression experiments showed the b cluster to accommodate the apoLp-II/I lipid-binding capacity. After cleavage of apoLp-II/I, the b cluster is almost entirely situ- ated in apoLp-I, suggesting apoLp-I, and not apoLp-II, to bind the vast majority of lipids (82). This finding is consistent with HDLp dissociation experiments in which )98% of the total lipid in lipophorin remained associated with apoLp-I (83).…”

“…Based on the homology between the LLT modules of apoB and apoLp-II/I, the C-terminal sequences of both apolipoproteins can also share a common evolutionary origin. In this regard, it has been speculated that the b 2 and a 3 clusters in apoB would have arisen from duplication of the b 1 and a 2 clusters (82). At its C-terminal end, apoLp-II/I contains a von Willebrand factor D module (51); this module also appears to be present in many different insect Vtgs (84).…”

“…At its C-terminal end, apoLp-II/I contains a von Willebrand factor D module (51); this module also appears to be present in many different insect Vtgs (84). Although the function of this domain remains enigmatic, it does not appear to be involved in lipid binding (82).…”

“…(23)x provides significant support for the concept that an MTP-dependent mechanism for initial lipoprotein biosynthesis is also operative in the biogenesis of insect lipoproteins. Moreover, insect MTP was experimentally shown to stimulate insect lipoprotein biogenesis considerably, because coexpression of the Drosophila MTP homolog (dMTP) and recombinant fulllength locust (L. migratoria) apoLp-II/I cDNA in an insect cell (Sf9) expression system resulted in a several-fold increase in the secretion of apoLp-I and -II, as well as uncleaved apoLp-II/I (82). Concomitant with their secretion, dMTP significantly stimulated the lipidation of the apoLp-II/I proteins, because the secreted lipoprotein particles were recovered at a decreased buoyant density compared with control cells lacking the dMTP gene ( Figure 4).…”

Crossveinless d is a vitellogenin-like lipoprotein that binds BMPs and HSPGs, and is required for normal BMP signaling in the Drosophila wing

“…3D). This occurs despite the likely reduction in lipid binding with this construct; the DUF1943 domain contains two amphipathic -sheets (A and B) that make up a large part of the lipid-binding cavity (Smolenaars et al, 2007a), and similar deletions from apolipophorin largely eliminate lipid binding (Smolenaars et al, 2007b). Cv-d-V5(VWD) also co-precipitated with Dpp-FLAG but not with Gbb-FLAG (Fig.…”