Characterization of triacylglyceride lipase and mono- and diacylglycerol acyltransferases in Manduca sexta

Abstract

Triglyceride (TG) is the main form of lipid storage in the cell. The processes of hydrolysis and synthesis of TG play critical roles the lipid metabolism. Triglyceridelipase(TGL) is a major fat body lipase hydrolyzing TG in Manduca sexta. A WWE domain, presumably involved in protein-protein interactions, has been previously identified in the N-terminal region of TGL. So we searched for proteins partners that interact with the N-terminal region of TGL. Thirteen proteins were identified by mass spectrometry. The oxidoreductase lipoamide-dehydrogenase (LipDH) and the apolipoprotein components of the lipid transporter, HDLp, were among these proteins. LipDH is the common component of the mitochondrial ?-keto acid dehydrogenase complexes whereas HDLp occurs in the hemolymph. However, subcellular fractionation demonstrated these two proteins are relatively abundant in the soluble fraction of fat body adipocytes. TGL has critical thiol groups and studies with inhibitors suggested that LipDH, acting as a diaphorase could preserve the activity of TGL by controlling the redox state of thiol groups. The HDLp-TGL interaction affected the catalytic properties of TGL leading to a lower rate of hydrolysis of diacylglycerol (DG).

Monoacylglycerol acyltransferase (MGAT) and diacylglycerol acyltransferase (DGAT) are key enzymes of the synthesis of neutral glycerides. Very little is known about these acyltransferases in insects. We have cloned two predicted MGATs and a DGAT from M. sexta and compared their sequences with predicted MGAT and DGAT homologues from a number of insect species. The comparison suggested that insects may only have a single DGAT gene, DGAT1 and insects seem to have a single MGAT gene which is similar to the MGAT2 of vertebrates. Furthermore, the expression patterns of the acyltransferases suggest a significant role of the monoacylglycerol pathway in the production and mobilization of DG in M. sexta fat body.