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.
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.
Miyaji
Takayuki
T
Laboratory of Food Molecular Functionality, College of Agriculture, Ibaraki University, 3-21-1, Chuo, Ami-machi, Inashiki-gun, Ibaraki 300-0393, Japan.
Murayama
Satoshi
S
Kouzuma
Yoshiaki
Y
Kimura
Nobutada
N
Kanost
Michael R
MR
Kramer
Karl J
KJ
Yonekura
Masami
M
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D056646:Cathepsin F / Q000737:chemistry / Q000235:genetics; D003001:Cloning, Molecular; D015891:Cystatins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D057056:Cysteine Proteases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D015853:Cysteine Proteinase Inhibitors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D018076:DNA, Complementary / Q000235:genetics; D017344:Genes, Insect; D006458:Hemolymph / Q000737:chemistry; D019476:Insect Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D007814:Larva / Q000201:enzymology / Q000235:genetics; D018402:Manduca / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D011498:Protein Precursors / Q000235:genetics
D015891:Cystatins; D015853:Cysteine Proteinase Inhibitors; D018076:DNA, Complementary; D019476:Insect Proteins; D011498:Protein Precursors; D057056:Cysteine Proteases; D056646:Cathepsin F