An Extended AE-Rich N-Terminal Trunk in Secreted Pineapple Cystatin Enhances Inhibition of Fruit Bromelain and Is Posttranslationally Removed during Ripening

Phytocystatins are potent inhibitors of cysteine proteases and have been shown to participate in senescence, seed and organ biogenesis, and plant defense. However, phytocystatins are generally poor inhibitors of the cysteine protease, bromelain, of pineapple (Ananas comosus). Here, we demonstrated that pineapple cystatin, AcCYS1, inhibited (>95%) stem and fruit bromelain. AcCYS1 is a unique cystatin in that it contains an extended N-terminal trunk (NTT) of 63 residues rich in alanine and glutamate. A signal peptide preceding the NTT is processed in vitro by microsomal membranes giving rise to a 27-kD species. AcCYS1 mRNA was present in roots and leaves but was most abundant in fruit. Using immunofluorescence and immunoelectron microscopy with an AcCYS1-specific antiserum, AcCYS1 was found in the apoplasm. Immunoblot analysis identified a 27-kD protein in fruit, roots, and leaves and a 15-kD species in mature ripe fruit. Ripe fruit extracts proteolytically removed the NTT of 27-kD AcCYS1 in vitro to produce the 15-kD species. Mass spectrometry analysis was used to map the primary cleavage site immediately after a conserved critical glycine-94. The AE-rich NTT was required to inhibit fruit and stem bromelain (>95%), whereas its removal decreased inhibition to 20% (fruit) and 80% (stem) and increased the dissociation equilibrium constant by 1.8-fold as determined by surface plasmon resonance assays. We propose that proteolytic removal of the NTT results in the decrease of the inhibitory potency of AcCYS1 against fruit bromelain during fruit ripening to increase tissue proteolysis, softening, and degradation.Phytocystatins are Cys protease inhibitors from plants that reside in the cystatin superfamily and contain a distinctive α-helix-forming sequence, LVI]-AGT]-RKE]-FY]-AS]-VI]-x-EDQV]-HYFQ]-N, in the main body (Margis et al., 1998). The most investigated phytocystatin is rice (Oryza sativa) oryzacystatin I (OC-I; Abe et al., 1987). Its three-dimensional structure (Nagata et al., 2000) resembles the structure of chicken egg white cystatin (Bode et al., 1988). These structural features of OC-I include a five-stranded antiparallel β-pleated sheet, which is wrapped around the α-helix. Two regions are predicted to reversibly bind to the active site of papain-like Cys proteases. They are the highly conserved QxVxG motif that is situated on a loop between the second and third β-strand and a conserved W on a loop between the fourth and fifth β-strand (Arai et al., 1991; Urwin et al., 1995). A conserved G immediately precedes the main body at the N terminus. The region preceding the conserved G is referred to as the N-terminal trunk (NTT) and has been shown to interact with Cys protease (Machleidt et al., 1989; Björk et al., 1995; Girard et al., 2007), but the role of the NTT in phytocystatins is less clear.Although the NTT of OC-I did not affect the inhibition of papain (Abe et al., 1988; Chen et al., 1992), the NTTs of other phytocystatins were subsequently shown to modulate the binding affinities to various enzymes (Urwin et al., 1995; Kiggundu et al., 2006). Some phytocystatins were predicted to possess an N-terminal signal peptide for transport into the lumen of the endoplasmic reticulum and/or a C-terminal extension, which may be involved in binding legumain-type Cys proteases (Lim et al., 1996; Womack et al., 2000; Martínez et al., 2005a, 2007; Abraham et al., 2006; Gianotti et al., 2006). Other phytocystatins, designated multicystatins, contain multiple copies of the main body (Kouzuma et al., 2000; Diop et al., 2004; Christova et al., 2006; Girard et al., 2007).Phytocystatins function in diverse biological processes, such as protein turnover during seed development and germination (Kuroda et al., 2001; Martínez et al., 2005c; Abraham et al., 2006; Kiyosaki et al., 2007; Valdés-Rodríguez et al., 2007), organogenesis (Corre-Menguy et al., 2002; Massonneau et al., 2005; Rivard et al., 2007), programmed cell death (Beers et al., 2000; Belenghi et al., 2003), fruit development (Ryan et al., 1998), and defense against a variety of pests and pathogens (Koiwa et al., 2000; Gholizadeh et al., 2005; Christova et al., 2006; Girard et al., 2007). Thus, phytocystatins inhibit both endogenous and exogenous Cys proteases. It is expected that cystatins have a high affinity for their endogenous cognate targets because they have coevolved functionally in the same cellular environment and the cystatin could control potentially damaging proteolytic activity (Otlewski et al., 2005). Similarly, exogenous targets require effective inhibitor-enzyme binding to confer resistance upon pathogen/herbivore attack (Kiggundu et al., 2006). The identification of natural targets of phytocystatins and the elucidation of their regulatory mechanisms are critical to improve our understanding of their roles in plants and for the development of practical applications (Urwin et al., 1997; Arai et al., 1998; Lilley et al., 2004).In pineapple (Ananas comosus), four major Cys proteases have been identified. They are the stem (Ritonja et al., 1989) and fruit bromelains (Yamada et al., 1976; Rowan et al., 1990) and unique ananain (Lee et al., 1997) and comosain (Rowan et al., 1990). Stem and fruit bromelains are encoded by distinct genes (Harrach et al., 1998; Jung et al., 2008) and share 68% sequence identity. They both contain signal peptides for entering the secretory pathway and propeptides for intramolecular inhibition and assisting protein folding. However, the primary species of bromelains that accumulate in plant cells have the propeptide removed (Yamada et al., 1976; Ritonja et al., 1989). Due to their broad substrate specificity and strong proteolytic activity, pineapple Cys proteases have become of considerable economical importance in the food and pharmaceutical industry (Rowan et al., 1990; Maurer, 2001). Fruit and stem bromelains are highly abundant and have been extensively studied (Vanhoof and Cooreman, 1997). Only kiwifruit (Actinidia deliciosa) cystatin has some inhibitory effect on stem bromelain (Rasaam and Laing, 2004). Here, we analyzed a ubiquitously expressed pineapple cystatin, AcCYS1, that we found to be secreted to the apoplast. AcCYS1 is unusual in that it contains an extended NTT of 63 residues that is rich in Ala and Glu. We showed that the NTT is important for complete inhibition of fruit and stem bromelain in the picomolar range and it is cleaved upon fruit ripening. Based on in vitro inhibition analysis against fruit and stem bromelain of three different species of AcCYS1, differing in the length of their NTT, we hypothesize that the cleavage of the NTT enhances the proteolytic activity of fruit bromelain during fruit ripening and senescence.