Molecular cloning and functional properties of two early-stage encapsulation-relating proteins from the coleopteran insect, Tenebrio molitor larvae

Encapsulation is a major defensive reaction against foreign materials that are too large to be phagocytosed by individual hemocytes; however, the bioc hemical process of encapsulation is still obscure. To isolate and character ize the early-stage encapsulation-relating protein (ERP), we used the coleo pteran insect, Tenebrio molitor larvae, injecting three differing kinds of bead or inserting pieces of surgical suture into the abdomen of T. molitor larvae. The resulting proteins from the injected beads or the inserted piec es of surgical suture were recovered 10 min after injection or insertion. a nd were analyzed on SDS/PAGE under reducing conditions. Four different prot eins (86, 78, 56 and 48 kDa) were enriched compared with the crude hemolymp h. Among them, we purified 56-kDa and 48-kDa ERPs to homogeneity and raised polyclonal antibodies against each protein. Immunoblotting analysis showed that the affinity-purified antibodies of the 56-kDa and 48-kDa ERPs cross- reacted with the 48-kDa and 56-kDa ERPs, respectively. Analysis of the cDNA of 56-kDa ERP consisted of 579 amino acid residues and showed a novel glut amine-rich protein. Positive clones of the 48-kDa ERP showed the same DNA s equence as 56-kDa ERP. Interestingly. the chemically determined N-terminal amino acid sequence and the three partial amino acid sequences of the 48-kD a protein were found in the 56-kDa ERP, suggesting that the 48 kDa ERP was produced by the cleavage of Arg101-Gly102 of the 56-kDa ERP by a limited pr oteolysis, Western blotting analysis showed that these ERPs were detected e xclusively on membrane fractions of hemocytes. Also, when the early-stage e ncapsulated beads were coated with both the 56-kDa and 48-kDa ERP antibodie s and re-injected into larvae, no further encapsulation reaction was observ ed. However, when the early-stage encapsulated beads were incubated with 56 -kDa ERP antibody, 48-kDa ERP antibody or nonimmunized rabbit IgG and re-in jected into larvae, further encapsulation did occur.