A new easter-type serine protease cleaves a masquerade-like protein during prophenoloxidase activation in Holotrichia diomphalia larvae.
The prophenoloxidase (proPO) activation pathway, like the vertebrate complement system, consists of a protease cascade and functions as a non-self-recognition system in these animals. Determining the molecular mechanism by which pattern recognition molecules differentiate non-self from self and transduce signals that stimulate defense responses is a key for understanding the ways in which innate immune systems are regulated. However, the proPO system is poorly defined at the molecular level. The proPO-activating system of the insect Holotrichia diomphalia comprises several components, some of which have been cloned and characterized, such as the novel 27-kDa proPO-activating factor-III (PPAF-III) from the plasma of H. diomphalia larvae and two prophenoloxidases. The PPAF-III gene encodes an easter-type serine protease zymogen consisting of 351 amino acid residues with a mass of 40 kDa. The purified 27-kDa PPAF-III specifically cleaved a 55-kDa proPPAF-II to generate a 45-kDa PPAF-II with or without Ca2+ present. Furthermore, two Holotrichia prophenoloxidases (proPO-I and -II) have been characterized, and their structural changes during activation were examined by in vitro reconstitution experiments. When the proPOs were incubated with PPAF-I, the 79-kDa proPOs were converted to 76-kDa proPOs, which did not exhibit any phenoloxidase (PO) activity. However, when the proPOs were incubated simultaneously with PPAF-I, proPPAF-II, and PPAF-III in the presence of Ca2+, a 60-kDa protein (PO-1) with PO activity was detected in addition to the 76-kDa proPO-II protein. These results indicate that the conversion of Holotrichia proPOs to enzymatically active phenoloxidase is accomplished by PPAF-I, PAF-II, and PPAF-III through a two-step limited proteolysis in the presence of Ca2+.
Kim
Moon Suk
MS
College of Pharmacy, Pusan National University, Jangjeon Dong, Kumjeong Ku, Busan, 609-735, Korea.
Baek
Min Ji
MJ
Lee
Mi Hee
MH
Park
Ji Won
JW
Lee
So Young
SY
Soderhall
Kenneth
K
Lee
Bok Luel
BL
D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism; D020134:Catalytic Domain; D004156:Catechol Oxidase / Q000378:metabolism*; D003001:Cloning, Molecular; D001517:Coleoptera; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000378:metabolism*; D006458:Hemolymph / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid
D004792:Enzyme Precursors; C038451:pro-phenoloxidase; D004156:Catechol Oxidase; D002118:Calcium