Hillyer Lab News
Hillyer Lab News
Article showing that Drosophila prophenoloxidase is cleaved by chymotrypsin is published in the Journal Biochimie
Wednesday, March 19, 2014
As part of an international team lead by Professor Erjun Ling of the Chinese Academy of Sciences, Dr. Julián Hillyer co-authored a study showing that Drosophila prophenoloxidase is sequentially cleaved by a-chymotrypsin. This study was published in the journal Biochimie.
CITATION:
Lu, A., X. Li , J.F. Hillyer, B.T. Beerntsen, K. Söderhäll, and E. Ling. 2014. Recombinant Drosophila prophenoloxidase 1 is sequentially cleaved by α-chymotrypsin during in vitro activation. Biochimie. 102:154-165.
(Pubmed) (See it in Biochimie) (Email me for a pdf copy)
ARTICLE ABSTRACT:
Insect prophenoloxidase (PPO) is an essential innate immunity protein to induce pathogen into melanization. In Bombyx mori, pro-phenoloxidase-activating enzyme (PPAE) can directly cleave and activate PPO. However, PPO in Manduca sexta cannot be cleaved into active phenoloxidase (PO) by serine proteases unless cofactors are involved, which indicates that PPO activation is complicated. Here we use recombinant Drosophila melanogaster prophenoloxidase 1 (rPPO1) to study the mechanism of PPO activation induced by a typical serine protease, α-chymotrypsin. Small amounts of α-chymotrypsin cleave rPPO1 at the N- and C-terminus to produce a large fragment rPPO1(N1/C1) that needs further cleavage by α-chymotrypsin to produce a smaller fragment rPO1(60-kD) with PO activity. rPO1(60-kD) oxidizes dopamine without being affected by high temperature, or by having salt and Ethylene diamine tetraacetic acid (EDTA) in the solution. After incubation with dopamine, rPO1(60-kD) cannot be detected using reducing SDS-PAGE due to formation of a large complex. Trypsin, another typical serine protease, cleaves rPPO1 at the N- and C-terminus to produce a small fragment rPPO1(N'/C') without PO activity. Several rPPO1 mutants were created through over-expressing active fragments that have direct PO activity. They are easily cleaved by low amounts of α-chymotrypsin without increasing PO activity. Therefore, rPPO1 can be sequentially cleaved in at least three places by α-chymotrypsin to produce activated rPO1(60-kD).
(This post was modified on 6/4/2014 to include the full citation of the article)
“Biochimie is a multi-topical journal, publishing original work as well as review articles and mini-reviews in all areas of biology (enzymology, genetics, immunology, microbiology, structure of macromolecules, etc.), provided that the approach adopted stems from biochemistry, biophysics or molecular biology.”