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Mobilization of lipid stores in Manduca sexta: cDNA cloning and developmental expression of fat body triglyceride lipase, TGL.

ABSTRACT: Fatty acids stored as triglycerides (TG) in the fat body serve as precursor in multiple processes including energy production and synthesis of cellular components. Mobilization of fatty acids from TG depends on the action of lipases. The fat body triglyceride lipase from Manduca sexta, MsTGL, is the only insect lipase that has been purified and characterized, so far. A TGL cDNA from M. sexta fat body encoding a 649 amino acid protein was cloned and its identity confirmed by mass spectrometry and Edman sequencing data of the purified protein. The protein sequence has conserved domains and residues of potential importance for the function and regulation of TGL activity. The expression of TGL and the lipase activity of fat body homogenates were studied in several developmental stages of M. sexta. TG-hydrolase activity of fat body increased as larva grew to the last instar and, then, decreased to minimal levels during pupa stage. Lipase activity was progressively restored in adult insects and reached maximum values at this stage. The fat body lipase activity from adult insects, 1-2 day after emergence, was 9-fold higher than that from 2 to 3 days old 5th-instar larvae. A good correlation was found between the abundance of TGL protein and the lipase activity of fat body homogenates. This correlation and the expression pattern of TGL throughout development are consistent with the notion that TGL is the main fat body TG lipase of M. sexta.

SUBMITTER: Arrese EL

PROVIDER: S-EPMC2829384 | biostudies-literature | 2010 Feb

REPOSITORIES: biostudies-literature

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Mobilization of lipid stores in Manduca sexta: cDNA cloning and developmental expression of fat body triglyceride lipase, TGL.

Arrese Estela L EL Howard Alisha D AD Patel Rajesh T RT Rimoldi Omar J OJ Soulages Jose L JL

Insect biochemistry and molecular biology 20100107 2

Fatty acids stored as triglycerides (TG) in the fat body serve as precursor in multiple processes including energy production and synthesis of cellular components. Mobilization of fatty acids from TG depends on the action of lipases. The fat body triglyceride lipase from Manduca sexta, MsTGL, is the only insect lipase that has been purified and characterized, so far. A TGL cDNA from M. sexta fat body encoding a 649 amino acid protein was cloned and its identity confirmed by mass spectrometry and ...[more]

PMID: 20060045

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Project description:Analogous to blood coagulation and complement activation in mammals, some insect defense responses (e.g. prophenoloxidase (proPO) activation and Toll pathway initiation) are mediated by serine proteinase cascades and regulated by serpins in hemolymph. We recently isolated Manduca sexta serpin-6 from hemolymph of the bacteria-challenged larvae, which selectively inhibited proPO-activating proteinase-3 (PAP-3) (Wang, Y., and Jiang, H. (2004) Insect Biochem. Mol. Biol. 34, 387-395). To further characterize its structure and function, we cloned serpin-6 from an induced fat body cDNA library using a PCR-derived probe. M. sexta serpin-6 is 55% similar in amino acid sequence to Drosophila melanogaster serpin-5, an immune-responsive protein. We produced serpin-6 in an Escherichia coli expression system and purified the soluble protein by nickel affinity and hydrophobic interaction chromatography. The recombinant protein specifically inhibited PAP-3 and blocked proPO activation in vitro in a concentration-dependent manner. Matrix-assisted laser desorption ionization-time of flight mass spectrometry indicated that the cleavage site of serpin-6 is between Arg373 and Ser374. Serpin-6 is constitutively present in hemolymph of naive larvae, and its mRNA and protein levels significantly increase after a bacterial injection. The association rate constant of serpin-6 and PAP-3 is 2.6 x 10(4) m(-1) s(-1), indicating that serpin-6 may contribute to the inhibitory regulation of PAP-3 in the hemolymph. We also identified the covalent complex of serpin-6 and PAP-3 in induced hemolymph by immunoaffinity chromatography and mass spectrometry. Furthermore, immulectin-2, serine proteinase homologs, proPO, PO, attacin-2, and a complex of serpin-6 and hemolymph proteinase-8 were also detected in the proteins eluted from the immunoaffinity column using serpin-6 antibody. These results suggest that serpin-6 plays important roles in the regulation of immune proteinases in the hemolymph.

Dataset Information

Mobilization of lipid stores in Manduca sexta: cDNA cloning and developmental expression of fat body triglyceride lipase, TGL.

Publications

Mobilization of lipid stores in Manduca sexta: cDNA cloning and developmental expression of fat body triglyceride lipase, TGL.

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