Characterization of the Protease Processing Sites in a Multidomain Proteinase Inhibitor Precursor from Nicotiana Alata

Abstract

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.

Evolution of proteinase inhibitor defenses in North American allopolyploid species of Nicotiana

“…This suggests that in No two domain expansion events occurred after speciation. The evolutionary advantages of repetitive TPI domain expansion have not been established, but it is reasonable to assume that repetitive domains provide plants with a more efficient use of transcription, translation, and cell-compartment targeting (Heath et al 1995).…”

The production of synthetic chemodisruptive peptides in planta disrupts the establishment of cyst nematodes

“…It is likely that processing occurs after the protein has passed through the endomembrane system (Atkinson et al ., 1993). Cleavage may occur adjacent to the linker or between the asparagine and aspartic acid residues, with subsequent trimming by nonspecific endogenous peptidases (Heath et al ., 1993). N‐terminal amino acid sequencing confirmed the presence of residues 3–16 (inclusive) of GFP for the smaller Western‐positive protein expressed by BY2 cells.…”

Expression analysis of proteinase inhibitor type-2 in response to southern root-knot nematode and its characterization in Nicotiana tabacum

“…Investigating the properties of IP-repeats and domains revealed that they had 5.2-5.7 kDa molecular weight. HEATH et al (1995) have shown that molecular weight of PI domains in N. alata is 6 kDa and sequence length is 53 amino acids. Domains had negative charge and only domain II was stable (Table 3).…”