Abstract
Most species of invertebrate animals cannot synthesise sterols de novo and many that feed on plants dealkylate phytosterols (mostly C29 and C28) yielding cholesterol (C27). The final step of this dealkylation pathway involves desmosterol reductase (DHCR24)-catalysed reduction of desmosterol to cholesterol. We now report the molecular characterisation in the silkworm, Bombyx mori, of such a desmosterol reductase involved in production of cholesterol from phytosterol, rather than in de novo synthesis of cholesterol. Phylogenomic analysis of putative desmosterol reductases revealed the occurrence of various clades that allowed for the identification of a strong reductase candidate gene in Bombyx mori (BGIBMGA 005735). Following PCR-based cloning of the cDNA (1.6 kb) and its heterologous expression in Saccharomyces cerevisae, the recombinant protein catalysed reduction of desmosterol to cholesterol in an NADH- and FAD- dependent reaction.Conceptual translation of the cDNA, that encodes a 58.9 kDa protein, and database searching, revealed that the enzyme belongs to an FAD-dependent oxidoreductase family. Western blotting revealed reductase protein expression exclusively in the microsomal subcellular fraction and primarily in the gut. The protein is peripherally associated with microsomal membranes. 2D-native gel and PAGE analysis revealed that the reductase is part of a large complex with molecular weight approximately 250kDa. The protein occurs in midgut microsomes at a fairly constant level throughout development in the last two instars, but is drastically reduced during the wandering stage in preparation for metamorphosis. Putative Broad Complex transcription factor-binding sites detectable upstream of the DHCR24 gene may play a role in this down-regulation.
Highlights
Most species of invertebrates are incapable of synthesis of sterols de novo and rely on a dietary source of these compounds [1]
The invertebrate phyla/species which have been demonstrated to be capable of phytosterol dealkylation, include phytophagous insect species [2,3], yellow fever mosquito, Aedes aegypti [4], an insect-parasitic [5] and certain free-living nematodes, such as Caenorhabditis elegans [6,7,8] and Turbatrix aceti [9], some Crustacea, Coelenterates, Molluscs [10,11,12], and a protozoan [13]
By comparison of the sterol composition of the nematodes with the host plants, good evidence exists for the occurrence of dealkylation in Ditylenchus dipsaci [14] and Rotylenchulus reniformis [15]
Summary
Most species of invertebrates are incapable of synthesis of sterols de novo and rely on a dietary source of these compounds [1]. Many of these species, such as plant pest insects and plant-parasitic nematodes, obtain primarily C29 and C28 sterols (e.g. sitosterol and campesterol) from plants. Such sterols, unprocessed, cannot satisfy the sterol requirement for normal growth and development of many of these invertebrates, which have a specific need for a C27 sterol, such as cholesterol [2,3]. Whereas the results of such an approach for Heterodera zeae [16], Meloidogyne incognita and M. arenaria [17] are suggestive of the existence of C-24 dealkylation in these species, they are inconclusive owing to the possibility of selective uptake of cholesterol from the diet [18,19]
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