Masanori Bun-ya

Characterization of a sterol carrier protein 2/3-oxoacyl-CoA thiolase from the cotton leafworm (Spodoptera littoralis): a lepidopteran mechanism closer to that in mammals than that in dipterans

Numerous invertebrate species belonging to several phyla cannot synthesize sterols de novo and rely on a dietary source of the compound. SCPx (sterol carrier protein 2/3-oxoacyl-CoA thiolase) is a protein involved in the trafficking of sterols and oxidation of branched-chain fatty acids. We have isolated SCPx protein from Spodoptera littoralis (cotton leafworm) and have subjected it to limited amino acid sequencing. A reverse-transcriptase PCR-based approach has been used to clone the cDNA (1.9 kb), which encodes a 57 kDa protein. Northern blotting detected two mRNA transcripts, one of 1.9 kb, encoding SCPx, and one of 0.95 kb, presumably encoding SCP2 (sterol carrier protein 2). The former mRNA was highly expressed in midgut and Malpighian tubules during the last larval instar. Furthermore, constitutive expression of the gene was detected in the prothoracic glands, which are the main tissue producing the insect moulting hormone. There was no significant change in the 1.9 kb mRNA in midgut throughout development, but slightly higher expression in the early stages. Conceptual translation of the cDNA and a database search revealed that the gene includes the SCP2 sequence and a putative peroxisomal targeting signal in the C-terminal region. Also a cysteine residue at the putative active site for the 3-oxoacyl-CoA thiolase is conserved. Southern blotting showed that SCPx is likely to be encoded by a single-copy gene. The mRNA expression pattern and the gene structure suggest that SCPx from S. littoralis (a lepidopteran) is evolutionarily closer to that of mammals than to that of dipterans.

New aspects of sterol carrier protein 2 (nonspecific lipid-transfer protein) in fusion proteins and in peroxisomes

Sterol carrier protein 2 (SCP2) is a 13-kDa peroxisomal protein, identical to nonspecific lipidtransfer protein, and stimulates various steps of cholesterol metabolism in vitro. Although the name is reminiscent of acyl carrier protein, which is involved in fatty acid synthesis, SCP2 does not bind to lipids specifically or stoichiometrically. This protein is expressed either as a small precursor or as a large fusion (termed SCPx) that carries at its C-terminal the complete sequence of SCP2. SCPx exhibits 3-oxoacyl-CoA thiolase activity, as well as sterol-carrier and lipid-transfer activities. The N- and C-terminal parts of SCPx are similar to the nematode protein P-44 and the yeast protein PXP-18, respectively. P-44, which has no SCP2 sequence, thiolytically cleaved the side chain of bile acid intermediate at a rate comparable to that of SCPx. This, together with the properties of other fusions with SCP2-like sequence, suggests that the SCP2 part of SCPx does not play a direct role in thiolase reaction. PXP-18, located predominantly inside peroxisomes, is similar to SCP2 in primary structure and lipid-transfer activity, and protects peroxisomal acyl-CoA oxidase from thermal denaturation. PXP-18 dimerized at a high temperature, formed an equimolar complex with the oxidase subunit, and released the active enzyme from the complex when the temperature went down. This article attempts to gain insight into the role of SPC2, and to present a model in which PXP-18, a member of the SCP2 family, functions as a molecular chaperone in peroxisomes.

Metabolic significance and expression of Caenorhabditis elegans type II 3-oxoacyl-CoA thiolase

The authors cloned the cDNA of the nematode Caenorhabditis elegans encoding a 44-kDa protein (P-44), which is similar to sterol carrier protein x (SCPx). Genomic DNA data and Northern blot analysis excluded the possibility of P-44 forming SCPx-like fusion protein. P-44 is required in the formation of bile acid in vitro from CoA esters of their enoyl-form intermediate in the presence of d-3-hydroxyacyl-CoA dehydratase/d-3-dehydrogenase bifunctional protein. Also, rat SCPx converts 24-hydroxy-form intermediate to bile acid under similar conditions. From this and other evidence, P-44 and SCPx were categorized as type II thiolase. The mRNA encoding P-44 was detected in every developmental stage of C. elegans: egg, larval stages, and adult. P-44, therefore, seems essential for the normal functioning of this organism.