Molecular cloning and characterization of the cathepsin B-like proteinase from the cotton boll worm, Helicoverpa armigera

An enzyme purified from the ovaries of Helicoverpa armigera, as an active form with molecular mass of 30 kDa on SDS‐PAGE, was identified as a cysteine proteinase because it could be inhibited by E‐64, a specific inhibitor of cysteine proteinase, and required reducing conditions for activity. This en...

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Bibliographic Details
Published in:Insect molecular biology 2002-12, Vol.11 (6), p.567-575
Main Authors: Zhao, X.-F., Wang, J.-X., Xu, X.-L., Schmid, R., Wieczorek, H.
Format: Article
Language:English
Subjects:
Adipose Tissue - enzymology
Amino Acid Sequence
Animals
cathepsin B
Cathepsin B - genetics
Cathepsin B - isolation & purification
Cathepsin B - metabolism
characterization
Cloning, Molecular
DNA Primers
Electrophoresis, Polyacrylamide Gel
Endopeptidases - genetics
Endopeptidases - isolation & purification
Endopeptidases - metabolism
Female
Helicoverpa armigera
In Situ Hybridization
Male
molecular cloning
Molecular Sequence Data
Moths - classification
Moths - enzymology
Moths - genetics
Phylogeny
Sequence Alignment
Sequence Homology, Amino Acid
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Summary:An enzyme purified from the ovaries of Helicoverpa armigera, as an active form with molecular mass of 30 kDa on SDS‐PAGE, was identified as a cysteine proteinase because it could be inhibited by E‐64, a specific inhibitor of cysteine proteinase, and required reducing conditions for activity. This enzyme was further identified as a cathepsin B‐like cysteine proteinase by partial amino acid sequencing. A cDNA encoding this proteinase was cloned from H. armigera, using degenerate primers and RACE techniques. Results of Northern blots indicated that the mRNA encoding the proteinase was transcribed in the ovaries, the fat bodies of female and male adults, pupae and in the larvae. No mRNA was detected from the larval epidermis or from the midgut. Hence, transcription of the cathepsin B‐like cysteine proteinase from H. armigera was tissue‐specific, but not gender‐ or developmental stage‐specific. However, proteolytic activities were only detected from ovaries, and adult female and male fat bodies. No activity was observed from pupal and larval fat bodies, from the larval epidermis or from the midgut. Only one form of mRNA of ≈ 1100 bases was detected, and in situ hybridization showed that the transcripts were distributed in the adult female fat bodies, follicular cells and the oocytes. Since the proteinase expressed in ovaries was able to degrade vitellin in vitro, it may be involved in the degradation of vitellin during embryonic development.
ISSN:0962-1075
1365-2583
DOI:10.1046/j.1365-2583.2002.00366.x