OVEREXPRESSION OF HUMAN PROCARBOXYPEPTIDASE A2 IN PICHIA-PASTORIS ANDDETAILED CHARACTERIZATION OF ITS ACTIVATION PATHWAY

The cDNA of human procarboxypeptidase A2 has been overexpressed in the methylotrophic yeast Pichia pastoris and secreted into the culture me dium by means of the alpha-mating factor signal sequence, yielding a m ajor protein of identical size and N-terminal sequence as the wild-typ e form, Two other forms containing the proenzyme have also been overex pressed: one of them resulted from an incomplete processing of the sig nal peptide, whereas the other was a glycosylated derivative. Recombin ant procarboxypeptidase A2 was purified to homogeneity, and it was sho wn that its mature active form displays functional properties similar to those of the enzyme directly isolated from human pancreas, The over all yield was similar to 250 mg of proenzyme or 180 mg of mature enzym e/liter of cell culture. The proteolysis-promoted activation process o f the recombinant proenzyme has been studied in detail. During maturat ion by trypsin, the increase in activity of the enzyme is a rapid and monotonic event, which reflects the rate of the proteolytic release of the inhibitory pro-segment and the weaker nature of its interactions with the enzyme moiety compared with procarboxypeptidases of the A1 ty pe, Three main forms of the pro-segment (96, 94, and 92 amino acids), with no inhibitory capability in the severed state, and a single matur e carboxypeptidase A2 are produced during this process. No further pro teolysis of these pro-segments by the generated carboxypeptidase A2 oc curs, in contrast with observations made in other procarboxypeptidases (A1 and B), This differential behavior is a result of the extreme spe cificity of carboxypeptidase A2.