Functional expression and enzymatic properties of two Sitophilus zeamais cysteine proteinases showing different autolytic processing profiles in vitro.
To characterize in more detail the cathepsin L-like cysteine proteinases from Sitophilus zeamais (SCPs) cloned in our previous study [Matsumoto et al. (1997) J. Biochem. 121, 464-476], we established a system for their functional expression and purification using a glutathione S-transferase (GST) fusion gene vector from Escherichia coli. The proenzyme forms of two representative SCPs, proSCPc1 and proSCPg3, were expressed as GST-fusion proteins and purified on a glutathione Sepharose column. GST-proSCPc1 undergoes autoproteolytic cleavage into the mature form efficiently at acidic pH, and exhibits significant proteolytic activity toward various substrates including hemoglobin and Z-Phe-Arg-MCA. The enzymatic characteristics of the activated form of SCPc1 are similar to those of mammalian cathepsin L, but its pH optimum for the hydrolysis of hemoglobin is significantly lower. The other proSCP, GST-proSCPg3, which has a shorter COOH-terminal domain than SCPc1, undergoes almost no autolytic processing and shows only very slight proteolytic activity, although the other enzymatic characteristics of GST-proSCPg3 are similar to those of GST-proSCPc1.
Matsumoto
I
I
Department of Applied Biological Chemistry, Faculty of Science, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657.
Abe
K
K
Arai
S
S
Emori
Y
Y
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D001517:Coleoptera / Q000201:enzymology*; D003546:Cysteine Endopeptidases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D018076:DNA, Complementary; D004792:Enzyme Precursors / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D005982:Glutathione Transferase / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D019476:Insect Proteins; D008969:Molecular Sequence Data; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000235:genetics; D013379:Substrate Specificity
D018076:DNA, Complementary; D004792:Enzyme Precursors; D019476:Insect Proteins; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase; D003546:Cysteine Endopeptidases; C112966:SCPc1 protein, Sitophilus zeamais; C112967:SCPg3 protein, Sitophilus zeamais