A zymogen form of masquerade-like serine proteinase homologue is cleaved during pro-phenoloxidase activation by Ca2+ in coleopteran and Tenebrio molitor larvae.
To elucidate the biochemical activation mechanism of the insect pro-phenoloxidase (pro-PO) system, we purified a 45-kDa protein to homogeneity from the hemolymph of Tenebrio molitor (mealworm) larvae, and cloned its cDNA. The overall structure of the 45-kDa protein is similar to Drosophila masquerade serine proteinase homologue, which is an essential component in Drosophila muscle development. This Tenebrio masquerade-like serine proteinase homologue (Tm-mas) contains a trypsin-like serine proteinase domain in the C-terminal region, except for the substitution of Ser to Gly at the active site triad, and a disulfide-knotted domain at the amino-terminal region. When the purified 45-kDa Tm-mas was incubated with CM-Toyopearl eluate solution containing pro-PO and other pro-PO activating factors, the resulting phenoloxidase (PO) activity was shown to be independent of Ca2+. This suggests that the purified 45-kDa Tm-mas is an activated form of pro-PO activating factor. The55-kDa zymogen form of Tm-mas was detected in the hemolymph when PO activity was not evident. However, when Tenebrio hemolymph was incubated with Ca2+, a 79-kDa Tenebrio pro-PO and the 55-kDa zymogen Tm-mas converted to 76-kDa PO and 45-kDa Tm-mas, respectively, with detectable PO activity. Furthermore, when Tenebrio hemolymph was incubated with Ca2+ and beta-1,3-glucan, the conversion of pro-PO to PO and the 55-kDa zymogen Tm-mas to the 45-kDa protein, was faster than in the presence of Ca2+ only. These results suggest that the cleavage of the 55-kDa zymogen of Tm-mas by a limited proteolysis is necessary for PO activity, and the Tm-mas is a pro-PO activating cofactor.
Lee
Kum Young
KY
College of Pharmacy, Pusan National University, Jangjeon Dong, Korea.
Zhang
Rong
R
Kim
Moon Suk
MS
Park
Ji Won
JW
Park
Ho Young
HY
Kawabata
Shun-ichiro
S
Lee
Bok Luel
BL
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002118:Calcium / Q000494:pharmacology*; D020134:Catalytic Domain; D004156:Catechol Oxidase / Q000378:metabolism*; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004330:Drosophila; D029721:Drosophila Proteins; D010450:Endopeptidases / Q000378:metabolism*; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000378:metabolism*; D006434:Hemocytes / Q000378:metabolism; D006458:Hemolymph / Q000737:chemistry; D019476:Insect Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D007814:Larva / Q000187:drug effects* / Q000201:enzymology; D008969:Molecular Sequence Data; D011994:Recombinant Proteins; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases; D013712:Tenebrio / Q000201:enzymology*
D018076:DNA, Complementary; D029721:Drosophila Proteins; D004792:Enzyme Precursors; D019476:Insect Proteins; D011994:Recombinant Proteins; C038451:pro-phenoloxidase; D004156:Catechol Oxidase; D010450:Endopeptidases; C091914:mas protein, Drosophila; D012697:Serine Endopeptidases; D002118:Calcium