CHARACTERIZATION OF THE PROTEASE PROCESSING SITES IN A MULTIDOMAIN PROTEINASE-INHIBITOR PRECURSOR FROM NICOTIANA-ALATA

A gene encoding a 40.3-kDa serine proteinase inhibitor (PI) precursor is expressed at high levels in the stigma of the ornamental tobacco, N icotiana alata. The precursor is processed proteolytically in vivo to release five homologous proteinase inhibitors of approximately 6 kDa, as well as two flanking peptides. The five PIs have been purified from stigmas and identified by N-terminal sequencing, electrospray mass sp ectrometry and inhibition activity against chymotrypsin or trypsin. On e of the PIs inhibits chymotrypsin and the other four are most active on trypsin. Cleavage occurs in a linker region (EEKKND) that is repeat ed six times in the precursor molecule. In the plant, the initial clea vage probably occurs between asparagine and the aspartate residues and ragged ends are formed by subsequent trimming. In vitro, the protease -sensitive linker region is selectively cleaved by the endoproteinases Asp-N, Glu-C and Lys-C to release fully active approximately 6-kDa PI s that are resistant to further proteolytic digestion. The precursor, produced by a recombinant baculovirus, inhibits chymotrypsin more effe ctively than trypsin. The stoichiometry of 2.6 trypsin molecules/1 pre cursor molecule indicates that processing is required to activate or e xpose all of the four trypsin inhibitory sites.