EFFECTS OF POLYMANNOSYLATION OF RECOMBINANT CYSTATIN-C IN YEAST ON ITS STABILITY AND ACTIVITY

The effects of glycosylation on stability and activity of recombinant cystatin were investigated using two yeast expression systems. A great difference in the length of polymannosyl chains in addition to 2 mol of N-acetylglucosamine was observed in mouse cystatin C produced by Sa ccharomyces cerevisiae and Pichia pastoris transformants. Polymannosyl ated cystatin with degree of polymerization (DP) of 310 (Cyst310) was predominantly produced by S. cerevisiae as a heterogeneous glycoprotei n. In contrast, the DP of cystatin by P. pastoris was 90 (Cyst90). The se yeast transformants also produced a small amount of oligomannosylat ed cystatin with DP of 13 (Cyst13) as well as unglycosylated protein. Susceptibility of Cyst310 and Cyst90 to alpha-chymotrypsin dramaticall y decreased to <10%, while that of Cyst13 was 77% of the susceptibilit y of unglycosylated cystatin. Polymannosylation improved the heat stab ility of cystatin to an extent that no coagulation was observed under the conditions, which coagulated unglycosylated protein. Papain-inhibi ting activities of Cyst310, Cyst90, and Cyst13 were 18.5, 83.7, and 98 .3% of that of unglycosylated cystatin, respectively. The retentions o f inhibitory activity upon heating to 95 degrees C were 82.2 and 71.3% for Cyst310 and Cyst90, respectively, while those of Cyst13 and ungly cosylated cystatin were below 10%. The polymannosylation of cystatin b y P. pastoris is preferable to that by S. cerevisiae as the resulting protein is more stable and active in inhibiting papain.