Is hexamerin receptor a GPI-anchored protein in Achaea janata (Lepidoptera: Noctuidae)?

Abstract

The process of uptake of hexamerins during metamorphosis from insect haemolymph by fat body cells is reminiscent of receptor-mediated endocytosis. Previously, we had identified a hexamerin-binding protein (HBP) and reported for the first time that uptake of hexamerins is dependent on the phosphorylation of HBP partly by a tyrosine kinase, which is, in turn, activated by 20-hydroxyecdysone (20E). However, the exact nature of HBP and the mechanism of interaction are still unknown. Here we report the possibility of HBP being a GPI-anchored protein in the fat body of Achaea janata and its role in the tyrosine-kinase-mediated phosphorylation signalling. Digestion of fat body membrane preparation with bacterial phosphatidylinositol-specific phospholipase C (PI-PLC), and the subsequent recognition by antibodies specific for the cross-reacting determinant (CRD), revealed that HBP is glycosylphosphatidylinositol (GPI)-anchored protein and, further, that the hexamerin binding to HBP was inhibited after digestion. Hexamerin overlay assay (HOA) of co-immunoprecipitated in vitro phosphorylated HBP showed exclusive binding to ~120 kDa protein. Lectin-binding analysis of hexamerins revealed the presence of N-acetylgalactosamine (GalNAc) and N-acetylglucosamine (GluNAc), whereas HBP showed the presence of GalNac alone. Mild chemical deglycosylation studies and binding interaction in the presence of sugars revealed that glycan moieties are possibly not involved in the interaction between HBP and hexamerins. Taken together, these results suggest that HBP may be a GPI-anchored protein, and interaction and activation of HBP is through lipid-linked non-receptor src tyrosine kinases. However, additional studies are needed to prove that HBP is a GPI-anchored protein.

Insecticidal Effects of Hemocoelic Delivery of Bacillus thuringiensis Cry Toxins in Achaea janata Larvae

“…The hemolymph proteins were resolved by 10% SDS-PAGE and electro-blotted onto a nitrocellulose membrane using Trans-Blot apparatus (Bio-Rad) according to the procedure of Towbin et al (1979). Non-specific binding sites were blocked with 5% skimmed milk (w/v) in Tris-buffered saline (TBS) and then incubated with A. janata hexamerin polyclonal antibody (1:5,000 dilutions) (Budatha et al, 2011). Subsequently, the blots were incubated with ALP-conjugated goat anti-rabbit IgG and finally detected with NBT-BCIP substrate (Sigma-Aldrich).…”

Differential oxidative stress responses in castor semilooper, Achaea janata

“…janata larvae were obtained from Indian Institute of Oil Seeds Research (Hyderabad, India) and fed on castor leaves at 25±1°C and 70% relative humidity with a photoperiod of 12:12 hour light: dark cycle (Budatha M et al, 2011). The larvae were anesthetized on ice for 15 min and dissected in cold insect Ringer solution to isolate the tissues.…”