A serine protease in the midgut of the silkworm, Bombyx mori: protein sequencing, identification of cDNA, demonstration of its synthesis as zymogen form and activation during midgut remodeling.
We identified a serine protease with a molecular mass of 37kDa in the midgut of the silkworm, Bombyx mori. The activity of this protease (37-kDa protease: p37k) appears after pupation, when the metamorphic remodeling of the midgut is under progress. The sequence analysis of the purified protease and its cDNA revealed that p37k is a trypsin-type serine protease, which is highly similar to serine proteases of other insects, including CG4386 of Drosophila melanogaster. In our molecular phylogenetic analysis, these proteases are grouped together with CG4386-like serine proteases of other insects to form an isolated cluster. The p37k protein and its putative orthologs present in this cluster have two unique sequence motifs, CxxCxC and FIDWLxxLLG, in the N-terminal side of the catalytic region. The gene for p37k is expressed in the midgut on day 2 of the silk-spinning larva, and the p37k polypeptide becomes detectable with a specific antibody at this stage of the midgut. On the other hand, p37k activity is not detectable until pupation, indicating that p37k is present in the larval midgut as an inactive precursor, which then is activated after pupation. A recombinant p37k produced using a baculovirus system is also inactive in its intact form. However, the recombinant p37k can be converted to an active protease when incubated in the homogenate of the midgut, suggesting that some unidentified midgut factor(s) are involved in the activation of p37k.
Kaji
Kentaro
K
Department of Biological Sciences, Tokyo Metropolitan University, Hachioji, Tokyo, Japan.
Tomino
Shiro
S
Asano
Tsunaki
T
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D012831:Bombyx / Q000145:classification / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development*; D018076:DNA, Complementary / Q000235:genetics / Q000378:metabolism; D004064:Digestive System / Q000737:chemistry / Q000201:enzymology / Q000254:growth & development; D004789:Enzyme Activation; D019476:Insect Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D007814:Larva / Q000737:chemistry / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D008969:Molecular Sequence Data; D010802:Phylogeny; D012697:Serine Endopeptidases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
D018076:DNA, Complementary; D019476:Insect Proteins; D012697:Serine Endopeptidases