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.

Figure 1. Nucleotide and deduced amino acid sequence of TGL from M. sexta

cDNA nucleotide (62–2044) is shown above the deduced amino acid sequences (1–649). Amino acid residues are aligned with the first nucleotide of each codon. The stop codon TGA is marked by an asterisk. The amino acid sequences underlined represent matched peptides obtained from the MALDI-Tof analysis of the tryptic digest of the gel excised protein. The amino acid microsequence obtained by LC/MS/MS and Edman degradation from MsTGL is in bold. The consensus lipase catalytic motif is highlighted in gray. The cDNA sequence of MsTGL has been deposited in GenBank (Accession number FJ807781).

Figure 2. Size of MsTGL transcript

Northern blot of Manduca sexta RNA extracted from the fat body of 2-day adult male insects. PolyA-RNA was probed with a 0.6kb radiolabeled anti-sense RNA probe.

Figure 3. Multiple alignment of the three conserved regions identified in MsTGL deduced amino acid sequence and the partial deduced sequences of five representative insects

Culex quinquefasciatus (Cq, NM_135341.1), Drosophila melanogaster (Dm, XM_001654068.1), Apis mellifera (Am, XM_392149.3), Nasonia vitripennis (Nv, XM_001602690.1), and Tribolium castaneous (Tc, XM_001812847.1). A- Alignment of the N-terminal WWE domains (Pfam: PF02825); B- Alignment of the central regions containing the lipase consensus sequence GHSLG; C- Alignment of the C-terminal domains that are homologous to the DDHD domain (Pfam: PF02862). A central sequence of approximately 80 residues was omitted (~) in the alignment from brevity. Identities in all sequences are indicated with asterisks; fully conservative substitutions are denoted with two dots. Partially conserved substitutions are indicated with a single dot and sequence gaps with dashes.

Figure 4. Sketch of TGL structure showing key conserved residues and structural regions

The figure shows the locations of conserved Cys residues and phosphorylation sites present in insect TGLs. It also shows the predicted catalytic triad comprising the serine residue within the GHSLG sequence (³⁶⁶S) and the conserved aspartic (⁴⁵⁷D) and histidine (⁵⁹⁰H) located in the DDHD domain.

Figure 5. Fat body lipase activity during development

The infranatant fractions (200μg protein) of fat body homogenates from larva (L), pupa (P) and adult (A) M. sexta were examined for lipase activity against an emulsion of [³H-triolein] and Triton X-100 as indicated in Methods. Panel A shows the lipase activities normalized by protein content (nmol FFA/min/mg protein); Panel B shows the lipase activities per fat body (nmol FFA/min.fat body). Data that are expressed as means ± SE (n = 4–8). Significant differences (P<0.001) were estimated between A-24 and A-48 and all other fat body TG lipase activities. The lipase activities during the pupal stages were significantly lower (P<0.05 or lower) than those observed at all other stages with the exception of 2^nd-instar. Abbreviations: HC, head capsules; W, wanderer; P, pupa; and A, adult (males).

Figure 6. Changes in TGL protein level during development

A- TGL protein levels were determined by western blotting in the infranatants of larval fat body homogenates (40μg of protein). The infranatants of adult fat body homogenates were first ran through a single step of Q-Sepharose chromatography and then subjected to western blotting using 20μg of protein. The proteins were separated on SDS-PAGE, transferred to nitrocellulose, and analyzed using anti-lipase antibody; Pooled samples, 4 to 8 insects per developmental stage, were used for western blotting B- The figure shows the average lipase activities corresponding to the developmental stages analyzed by western blotting.