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    • Keywords

       

      Ecdysone; fat body; GPI anchor; haemolymph; hexamerin-binding protein; lepidopteran; CRD epitope

    • 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.

    • Author Affiliations

       

      Madhusudhan Budatha1 2 Thuirei Jacob Ningshen1 Aparna Dutta-Gupta1

      1. School of Life Sciences, University of Hyderabad, Hyderabad 500 046, India
      2. Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX, USA
    • Dates

       
  • Journal of Biosciences | News

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