FUNCTIONAL EXPRESSION AND ENZYMATIC-PROPERTIES OF 2 SITOPHILUS-ZEAMAIS CYSTEINE PROTEINASES SHOWING DIFFERENT AUTOLYTIC PROCESSING PROFILESIN-VITRO

To characterize in more detail the cathepsin L-like cysteine proteinas es from Sitophilus zeamais (SCPs) cloned in our previous study [Matsum oto et al, (1997) J, Biochem, 121, 464-476], we established a system f or their functional expression and purification using a glutathione S- transferase (GST) fusion gene vector from Escherichia coil, The proenz yme forms of two representative SCPs, proSCPc1 and proSCPg3, were expr essed as GST-fusion proteins and purified on a glutathione Sepharose c olumn, GST-proSCPc1 undergoes autoproteolytic cleavage into the mature form efficiently at acidic pH, and exhibits significant proteolytic a ctivity toward various substrates including hemoglobin and Z-Phe-Arg-M CA. The enzymatic characteristics of the activated form of SCPc1 are s imilar to those of mammalian cathepsin L, but its pH optimum for the h ydrolysis of hemoglobin is significantly lower, The other proSCP, GST- proSCPg3, which has a shorter COOH-terminal domain than SCPc1, undergo es almost no autolytic processing and shows only very slight proteolyt ic activity, although the other enzymatic characteristics of GST-proSC Pg3 are similar to those of GST-proSCPc1.