Characterization of the protease processing sites in a multidomain proteinase inhibitor precursor from Nicotiana alata

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Title: Characterization of the protease processing sites in a multidomain proteinase inhibitor precursor from Nicotiana alata
Author(s): Heath, Robyn L.; Barton, Peter A.; Simpson, Richard J.; Reid, Gavin E.; Lim, Guan; Anderson, Marilyn A.
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.
Publication type: Journal article
Research centre: Swinburne University of Technology. School of Chemical Sciences
Source: European Journal of Biochemistry, Vol. 230, no. 1 (1995), pp. 250-257
Publication year: 1995
Keyword(s): Baculovirus precursor protein; Chymotrypsin inhibitor; EEKKND; Nicotiana alata; PI; Processing; Proteinase; Proteinase inhibitors; Protein precursors; Proteolytic enzymes; Solanaceae; Trypsin inhibitor
Publisher: Federation of European Biochemical Societies
ISSN: 0014-2956
Publisher URL: http://dx.doi.org/10.1111/j.1432-1033.1995.0250i.x
Additional information: The novel nucleotide sequence published here has been submitted to the GenBank data bank and is available under accession number U08219 (see: http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=nuccore&id=473590).
Peer reviewed