Characterization of potato proteinase inhibitor II reactive site mutants

Abstract

Potato proteinase inhibitor II (PI-2) is composed of two sequence repeats. It contains two reactive site domains. We developed an improved protocol for the production of PI-2 using the yeast Pichia pastoris as the expression host. We then assessed the role of its two reactive sites in the inhibition of trypsin and chymotrypsin by mutating each of the two reactive sites in various ways. From these studies it appears that the second reactive site strongly inhibits both trypsin (K i 0.4 nm) and chymotrypsin (K i 0.9 nm), and is quite robust towards mutations at positions P2 or P1H . In contrast, the first reactive site inhibits only chymotrypsin (K i 2 nm), and this activity is very sensitive to mutations. Remarkably, replacing the reactive site amino acids of domain I with those of domain II did not result in inhibitory activities similar to domain II. The fitness for protein engineering of each domain is discussed.Keywords: mutational analysis; Pichia pastoris; serine proteinase inhibitor; Solanum tuberosum.Protease inhibitors are abundant proteins in the storage organs and seeds of plants [1]. In addition, their synthesis is induced to high levels in response to stress, infection and wounding [2]. Potato expresses many different proteinase inhibitors belonging to a wide range of inhibitor families. Members of the potato proteinase inhibitor II (PI-2) family have been shown to inhibit serine proteases, such as trypsin, chymotrypsin, subtilisin, oryzin and elastase [3,4]. Until now, they have been found only among the Solanaceae. Proteins and messenger RNAs have been identified in potato tubers [5,6], wounded tomato and tobacco leaves [4,7], eggplant fruits [8], green tomatoes [9] and ornamental tobacco flower stigma [10]. There is medical interest in the properties of PI-2. It has been shown to have anticarcinogenic properties, protecting mouse embryo fibroblasts from radiation-induced transformation [11]. Although the mechanism underlying this effect is poorly understood, it is correlated to the capacity of PI-2 to inhibit chymotrypsin-like enzymes [12].Plant protease inhibitors such as PI-2 have been proposed to function as part of the plant defense system [13]. The plant defense role is deduced from observations that proteinase inhibitors in leaves are synthesized in response to wounding [2,14,15] or viral infection [2,16]. In addition, direct evidence for a protective role has been obtained; it was shown that transgenic tobacco plants over-expressing potato PI-2 gained resistance against the tobacco hornworm Manduca sexta [17]. Also, when the PI-2 gene was introduced into rice under its own wound-inducible promoter, plants were protected from the major lepidopteran rice pest, Sesamia inferens [18]. PI-2 probably acts by inhibiting digestive proteases from the insects, thereby restricting the availability of amino acids [13].The anti-nutrient activity of PI-2 has been shown to be overcome by pest insects like Spodoptera exigua [19]. When these larvae are reared on tobacco leaves expressing potato PI-2, the spe...

Reverse zymography using fluorogenic substrates for protease inhibitor detection

“…They are known to be nonspecific protease inhibitors that can effectively inhibit both trypsin and chymotrypsin [19,23,24]. The lower sensitivity for detecting these inhibitors in trypsin-treated Z-Phe-Arg-MCA gel in comparison with Boc-Phe-Ser-Arg-MCA gel is in good agreement with kinetic data reported previously [13,14,25].…”

“…2). Under reducing conditions, the original PCI is known to have a molecular mass of 11 kDa as a monomer that is capable of binding to chymotrypsin or to trypsin [18,19]. Since the present technique preserved the inhibitory activity without heat treatment or reducing reagents, it is possible that the higher molecular mass forms observed in this case were due to aggregation of smaller inhibitor forms.…”

“…Previously, inhibitory capacities of aprotinin and PCI toward trypsin were reported by several investigators [18,19,23]. They are known to be nonspecific protease inhibitors that can effectively inhibit both trypsin and chymotrypsin [19,23,24].…”

Serine Protease Inhibitors Specifically Defend Solanum nigrum against Generalist Herbivores but Do Not Influence Plant Growth and Development

“…A characteristic feature of PI-II proteins is that they contain one to several sequence repeats, which code for one precursor. Proteolytic cleavage of these precursors at specific linker sites results in several single or multidomain PIs with different properties (Atkinson et al, 1993;Beekwilder et al, 2000;Horn et al, 2005;Tamhane et al, 2007), which could explain the absence of more than two bands when silencing genes encoding the two-domain proteins SPI2a and SPI2b. It is possible that other peptides with low activity or concentration were not detected or resolved with the method that we used and that there are in fact more than three SPI peptides missing in the (A) Mean 6 SE relative transcript abundance of SPI1, SPI2a, and SPI2b in leaves of wild-type plants and of transgenic lines expressing an irconstruct specific for SPI2a and SPI2b (irSPI2a+b) or a construct specific for SPI1, SPI2a, and SPI2b (irSPI1/2a+b) (n = 7).…”

Structural Basis of Inhibition Revealed by a 1:2 Complex of the Two-headed Tomato Inhibitor-II and Subtilisin Carlsberg

“…Secondary contacts not involving the reactive-site loop in Domain II of two-domain Pot II family inhibitors may be of critical importance to determining proteinase inhibition specificity, as suggested by the results of site-directed mutagenesis studies on PI-II from potato (66). In PI-II, the capacity for trypsin inhibition in Domain II could not be transferred to Domain I by mutation of the P1 residue, the P2-P1-P1Ј sequence, or even the entire stretch of sequence from P2 to P10Ј.…”