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

Abstract

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.

Protein Disulfide Isomerase-2 of Arabidopsis Mediates Protein Folding and Localizes to Both the Secretory Pathway and Nucleus, Where It Interacts with Maternal Effect Embryo Arrest Factor

“…Samples were mixed with an equal volume of 2× loading buffer and electrophoresed on a 10% resolving, 5% stacking SDS-PAGE gel (described above). After electrophoresis for 1 h at 60 V, then 2.0 h at 80 V in 1 × Tris-Glycine buffer, pH 8.3, the gel was vacuum dried at 80°C for 1 h and exposed to X-ray film O/N (Neuteboom et al, 2009).…”

X‐ray crystallography and NMR studies of domain‐swapped canecystatin‐1

“…To investigate this further, we applied chemical shift mapping, using amino acid-specific calculations of the combined chemical shift perturbation Dd comb as implemented in AUREMOL [32]. Backbone atoms ( 1 HN, 15 NH, 13 Ca, and 13 CO) were used for this calculation. As shown in Fig.…”

“…However, in this case, the tetrameric arrangement is retained in solution, and the interactions that occur in the handshake are believed to contribute to its stability. Furthermore, the stability of the stefin B tetramer in solution is believed to be [32] of canecystatin-1 derived from backbone atoms ( 1 HN, 15 NH, 13 Ca, and 13 CO). Residues with values above the cut-off r 0 (black line) have significant resonance shifts as a consequence of the dimerization.…”

Overexpression of the protease inhibitor BoCPI-1 in broccoli delays chlorophyll loss after harvest and causes down-regulation of cysteine protease gene expression

“…Expression is usually limited to specific organs or specific phases of development, such as fruit maturation and ripening (Ryan et al, 1998;Neuteboom et al, 2009), senescence (Huang et al, 2001;Sugawara et al, 2002;Tajima et al, 2011), PCD (Solomon et al, 1999), or defence against abiotic stresses (Gaddour et al, 2001;Van der Vyver et al, 2003;Diop et al, 2004;Zhang et al, 2008). Eight putative phytocystatins have been isolated from Brassica oleracea (cabbage) (Rawlings, 2010).…”