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, Nicotiana 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 spectrometry and inhibition activity against chymotrypsin or trypsin. One of the PIs inhibits chymotrypsin and the other four are most active on trypsin. Cleavage occurs in a linker region (EEKKND) that is repeated six times in the precursor molecule. In the plant, the initial cleavage 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 PIs that are resistant to further proteolytic digestion. The precursor, produced by a recombinant baculovirus, inhibits chymotrypsin more effectively than trypsin. The stoichiometry of 2.6 trypsin molecules/1 precursor molecule indicates that processing is required to activate or expose all of the four trypsin inhibitory sites.
Heath
R L
RL
Plant Cell Biology Research Centre, School of Botany, University of Melbourne, Victoria, Australia.
Barton
P A
PA
Simpson
R J
RJ
Reid
G E
GE
Lim
G
G
Anderson
M A
MA
D000595:Amino Acid Sequence; D016367:Baculoviridae / Q000235:genetics; D010450:Endopeptidases / Q000494:pharmacology; D008969:Molecular Sequence Data; D010947:Plants, Toxic; D011498:Protein Precursors / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000096:biosynthesis; D015842:Serine Proteinase Inhibitors / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification*; D014026:Nicotiana / Q000737:chemistry*
D011498:Protein Precursors; D011994:Recombinant Proteins; D015842:Serine Proteinase Inhibitors; D010450:Endopeptidases