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Title: Purification of a cysteine protease inhibitor from larval hemolymph of the Tobacco Hornworm (Manduca sexta) and functional expression of the recombinant protein.

Author
item MIYAJI, TAKAYUKI - IBARAKI UNIVERSITY, JAPAN
item KOUZUMA, YOSHIAKI - IBARAKI UNIVERSITY, JAPAN
item YAGUCHI, JUN - IBARAKI UNIVERSITY, JAPAN
item YATSUMOTO, RIKA - IBARAKI UNIVERSITY, JAPAN
item KANOST, MICHAEL - KANSAS STATE UNIV
item KRAMER, KARL - 5430-05-30 RETIRED
item YONEKURA, MASAMI - IBARAKI UNIVERSITY, JAPAN
item Beeman, Richard

Submitted to: Journal of Insect Biochemistry and Molecular Biology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/1/2007
Publication Date: 6/2/2007
Citation: Miyaji, T., Kouzuma, Y., Yaguchi, J., Yatsumoto, R., Kanost, M.R., Kramer, K.J., Yonekura, M., Beeman, R.W. 2007. Purification of a cysteine protease inhibitor from larval hemolymph of the Tobacco Hornworm (Manduca sexta) and functional expression of the recombinant protein. Journal of Insect Biochemistry and Molecular Biology 37: 960-968.

Interpretive Summary: The role of many enzyme inhibitors in insect growth and development is poorly understood. Together with collaborators at Ibaraki University in Japan and at Kansas State University, we purified a novel enzyme inhibitor from insect blood and several of its properties were determined. One very unique property of this inhibitor is that it is initially synthesized as a very large and complex precursor protein containing multiple inhibitory and other subunits that are processed into single inhibitory and other types of molecules. The results contribute to our basic understanding of insect biochemistry and physiology, and identify a new type of insect metabolism that is a potential target of insect growth regulators.

Technical Abstract: A cysteine protease inhibitor (CPI) with an apparent molecular mass of 11.5 kDa was purified from larval hemolymph of the tobacco hornworm (Manduca sexta) by gel filtration of Sephadex G-50 followed by hydrophobic and ion-exchange column chromatographies. The purified cysteine proteinase inhibitor, denoted as MsCPI, strongly inhibited the plant cysteine protease, papain, with a Ki value of 5.5 X 10-9 M. Nucleotide sequence analysis of a partial cDNA encoding MsCPI indicated that MsCPI consists of 105 amino acid residues in a sequence that is similar to sarcocystatin A from Sarcophaga peregrine. However, northern blotting and PCR analyses using the specific primers of MsCPI suggested that the mRNA encoding MsCPI had a size of more than 12 kilobases, which included at least six tandemly repeated MsCPI segments. MsCPI was expressed in E. coli and the recombinant protein effectively inhibited cysteine proteases from plants as well as from animals such as cathepsins B (Ki, 6.8 nM), H (3.0 nM), and L (0.87 nM). There was no inhibition exhibited toward trypsin, chymotrypsin, subtilisin, pepsin, or themolysin.