Immunoblot analysis of chitinolytic enzymes in integument and molting fluid of the silkworm, Bombyx mori, and the tobacco hornworm, Manduca sexta

The occurrence of proteins related to chitinolytic enzymes in integument of the silkworm, Bombyx mori, and the tobacco hornworm, Manduca sexta, during the larval-pupal transformation was determined by immunoblot analysis using rabbit polyclonal antibodies for B. mori chitinase (EC 3.2.1.14) and β-N-acetylglucosaminidase (EC 3.2.1.30) as probes. Similar temporal patterns of appearance and immunoreactivities of chitinase-like and β-N-acetylglucosaminidase-like proteins were observed in the two species. Several chitinase-like proteins (MWapp 50–200 kDa) were resolved by denaturing electrophoresis. During the latter part of the fifth larval stadium, the major immunoreactive protein in B. mori integument and molting fluid had an apparent molecular mass of 88 kDa, which was previously observed (Koga et al., 1989). In M. sexta integument on days 6–8 of the fifth larval stadium and in molting fluid, the major immunoreactive protein had an apparent molecular mass of 97 kDa, which was 22 kDa larger than the 75 kDa chitinase detected in molting fluid (Koga et al., 1983). In integument on days 3–7 of the fifth larval stadium, another immunoreactive protein with an apparent molecular mass of 119 kDa was present. In contrast to multiple immunoreactive chitinase-like proteins, only a single major β-N-acetylglucosaminidase-immunoreactive protein was detected in integument and molting fluid from either species. The immunoreactive β-N-acetylglucosaminidase-like proteins had an apparent molecular mass of 67.5 kDa and a pI of 5.0, which are identical values to those of β-N-acetylglucosaminidases determined previously. The β-N-acetylglucosaminidases cleaved N-acetylchitooligosaccharides from the non-reducing end in an exo-fashion. The results of this study suggest that chitinases are synthesized in B. mori and M. sexta integuments as zymogens, which are activated by limited proteolysis whereas β-N-acetylglucosaminidases are not.