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Molecular cloning of a multidomain cysteine protease and protease inhibitor precursor gene from the tobacco hornworm (Manduca sexta) and functional expression of the cathepsin F-like cysteine protease domain.
Insect Biochem Mol Biol. 2010 Dec; 40(12):835-46.IB

Abstract

A Manduca sexta (tobacco hornworm) cysteine protease inhibitor, MsCPI, purified from larval hemolymph has an apparent molecular mass of 11.5 kDa, whereas the size of the mRNA is very large (∼9 kilobases). MsCPI cDNA consists of a 9,273 nucleotides that encode a polypeptide of 2,676 amino acids, which includes nine tandemly repeated MsCPI domains, four cystatin-like domains and one procathepsin F-like domain. The procathepsin F-like domain protein was expressed in Escherichia coli and processed to its active mature form by incubation with pepsin. The mature enzyme hydrolyzed Z-Leu-Arg-MCA, Z-Phe-Arg-MCA and Boc-Val-Leu-Lys-MCA rapidly, whereas hydrolysis of Suc-Leu-Tyr-MCA and Z-Arg-Arg-MCA was very slow. The protease was strongly inhibited by MsCPI, egg-white cystatin and sunflower cystatin with K(i) values in the nanomolar range. When the MsCPI tandem protein linked to two MsCPI domains was treated with proteases, it was degraded by the cathepsin F-like protease. However, tryptic digestion converted the MsCPI tandem protein to an active inhibitory form. These data support the hypothesis that the mature MsCPI protein is produced from the MsCPI precursor protein by trypsin-like proteases. The resulting mature MsCPI protein probably plays a role in the regulation of the activity of endogenous cysteine proteases.

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Authors+Show Affiliations

Miyaji T
Laboratory of Food Molecular Functionality, College of Agriculture, Ibaraki University, 3-21-1, Chuo, Ami-machi, Inashiki-gun, Ibaraki 300-0393, Japan.
Murayama S
No affiliation info available
Kouzuma Y
No affiliation info available
Kimura N
No affiliation info available
Kanost MR
No affiliation info available
Kramer KJ
No affiliation info available
Yonekura M
No affiliation info available

MeSH

Amino Acid SequenceAnimalsBase SequenceCathepsin FCloning, MolecularCystatinsCysteine ProteasesCysteine Proteinase InhibitorsDNA, ComplementaryGenes, InsectHemolymphInsect ProteinsLarvaManducaMolecular Sequence DataProtein Precursors

Pub Type(s)

Journal Article
Research Support, U.S. Gov't, Non-P.H.S.

Language

eng

PubMed ID

20727410

Citation

Miyaji, Takayuki, et al. "Molecular Cloning of a Multidomain Cysteine Protease and Protease Inhibitor Precursor Gene From the Tobacco Hornworm (Manduca Sexta) and Functional Expression of the Cathepsin F-like Cysteine Protease Domain." Insect Biochemistry and Molecular Biology, vol. 40, no. 12, 2010, pp. 835-46.
Miyaji T, Murayama S, Kouzuma Y, et al. Molecular cloning of a multidomain cysteine protease and protease inhibitor precursor gene from the tobacco hornworm (Manduca sexta) and functional expression of the cathepsin F-like cysteine protease domain. Insect Biochem Mol Biol. 2010;40(12):835-46.
Miyaji, T., Murayama, S., Kouzuma, Y., Kimura, N., Kanost, M. R., Kramer, K. J., & Yonekura, M. (2010). Molecular cloning of a multidomain cysteine protease and protease inhibitor precursor gene from the tobacco hornworm (Manduca sexta) and functional expression of the cathepsin F-like cysteine protease domain. Insect Biochemistry and Molecular Biology, 40(12), 835-46. https://doi.org/10.1016/j.ibmb.2010.08.003
Miyaji T, et al. Molecular Cloning of a Multidomain Cysteine Protease and Protease Inhibitor Precursor Gene From the Tobacco Hornworm (Manduca Sexta) and Functional Expression of the Cathepsin F-like Cysteine Protease Domain. Insect Biochem Mol Biol. 2010;40(12):835-46. PubMed PMID: 20727410.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Molecular cloning of a multidomain cysteine protease and protease inhibitor precursor gene from the tobacco hornworm (Manduca sexta) and functional expression of the cathepsin F-like cysteine protease domain. AU - Miyaji,Takayuki, AU - Murayama,Satoshi, AU - Kouzuma,Yoshiaki, AU - Kimura,Nobutada, AU - Kanost,Michael R, AU - Kramer,Karl J, AU - Yonekura,Masami, Y1 - 2010/08/18/ PY - 2010/06/03/received PY - 2010/08/10/revised PY - 2010/08/10/accepted PY - 2010/8/24/entrez PY - 2010/8/24/pubmed PY - 2011/2/25/medline SP - 835 EP - 46 JF - Insect biochemistry and molecular biology JO - Insect Biochem Mol Biol VL - 40 IS - 12 N2 - A Manduca sexta (tobacco hornworm) cysteine protease inhibitor, MsCPI, purified from larval hemolymph has an apparent molecular mass of 11.5 kDa, whereas the size of the mRNA is very large (∼9 kilobases). MsCPI cDNA consists of a 9,273 nucleotides that encode a polypeptide of 2,676 amino acids, which includes nine tandemly repeated MsCPI domains, four cystatin-like domains and one procathepsin F-like domain. The procathepsin F-like domain protein was expressed in Escherichia coli and processed to its active mature form by incubation with pepsin. The mature enzyme hydrolyzed Z-Leu-Arg-MCA, Z-Phe-Arg-MCA and Boc-Val-Leu-Lys-MCA rapidly, whereas hydrolysis of Suc-Leu-Tyr-MCA and Z-Arg-Arg-MCA was very slow. The protease was strongly inhibited by MsCPI, egg-white cystatin and sunflower cystatin with K(i) values in the nanomolar range. When the MsCPI tandem protein linked to two MsCPI domains was treated with proteases, it was degraded by the cathepsin F-like protease. However, tryptic digestion converted the MsCPI tandem protein to an active inhibitory form. These data support the hypothesis that the mature MsCPI protein is produced from the MsCPI precursor protein by trypsin-like proteases. The resulting mature MsCPI protein probably plays a role in the regulation of the activity of endogenous cysteine proteases. SN - 1879-0240 UR - https://www.unboundmedicine.com/medline/citation/20727410/Molecular_cloning_of_a_multidomain_cysteine_protease_and_protease_inhibitor_precursor_gene_from_the_tobacco_hornworm__Manduca_sexta__and_functional_expression_of_the_cathepsin_F_like_cysteine_protease_domain_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S0965-1748(10)00171-2 DB - PRIME DP - Unbound Medicine ER -