Drosophila CTLA-2-like protein (D/CTLA-2) inhibits cysteine proteinase 1 (CP1), a cathepsin L-like enzyme.
In this study, we present a propeptide-like cysteine proteinase inhibitor, Drosophila CTLA-2-like protein (D/CTLA-2), a CG10460 (crammer) gene product, with an amino acid sequence significantly similar to the proregion of Drosophila cysteine proteinase 1 (CP1). Recombinant D/CTLA-2, expressed in E. coli, strongly inhibited Bombyx cysteine proteinase (BCP) with a Ki value of 4.7 nM. It also inhibited cathepsins L and H with Ki values of 3.9 (human liver) and 0.43 (rabbit liver) nM, and 7.8 nM (human liver), respectively. Recombinant D/CTLA-2 exhibited low but significant inhibitory activities to cathepsin B with Ki values of 15 nM (human liver) and 110 nM (rat liver), but hardly inhibited papain. We attempted to purify cysteine proteinases inhibited by D/CTLA-2 from total bodies of adult Drosophila. Recombinant D/CTLA-2 significantly inhibited CP1 with a Ki value of 12 nM, indicating that CP1, a cognate enzyme of D/CTLA-2, is a target enzyme of the inhibitor in Drosophila cells. These results indicate that D/CTLA-2 is a selective inhibitor of cathepsin L-like cysteine proteinases similar to other propeptide-like cysteine proteinase inhibitors such as Bombyx cysteine proteinase inhibitors (BCPI) and cytotoxic T-lymphocyte antigen-2 (CTLA-2). D/CTLA-2 was expressed over the whole life cycle of Drosophila. Strong expression was observed in the garland cells and prothoracic gland in the late stages of embryonic development. These results suggest that D/CTLA-2, implicated in intra- and extra-cellular digestive processes, functions in these tissues by suppressing uncontrolled enzymatic activities of CP1.
Deshapriya
Rathnayaka M C
RM
Department of Veterinary Sciences, Faculty of Agriculture,Yamaguchi University, Japan.
Takeuchi
Akiyo
A
Shirao
Khoji
K
Isa
Kenji
K
Watabe
Shoji
S
Murakami
Ryutaro
R
Tsujimura
Hidenobu
H
Yamamoto
Yoshimi
Y
D000595:Amino Acid Sequence; D000818:Animals; D000943:Antigens, Differentiation; D001483:Base Sequence; D015153:Blotting, Western; D002403:Cathepsins / Q000037:antagonists & inhibitors; D003546:Cysteine Endopeptidases; D017931:DNA Primers; D004330:Drosophila / Q000235:genetics* / Q000378:metabolism; D029721:Drosophila Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics* / Q000378:metabolism*; D004926:Escherichia coli; D006801:Humans; D017403:In Situ Hybridization; D008099:Liver / Q000378:metabolism; D008969:Molecular Sequence Data; D011817:Rabbits; D011994:Recombinant Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D013602:T-Lymphocytes, Cytotoxic
D000943:Antigens, Differentiation; D017931:DNA Primers; D029721:Drosophila Proteins; D011994:Recombinant Proteins; C526367:cer protein, Drosophila; D002403:Cathepsins; D003546:Cysteine Endopeptidases; C106944:Cysteine proteinase-1, Drosophila