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First published online July 31, 2009; 10.1104/pp.109.142232 Plant Physiology 151:515-527 (2009) © 2009 American Society of Plant Biologists OPEN ACCESS ARTICLE
An Extended AE-Rich N-Terminal Trunk in Secreted Pineapple Cystatin Enhances Inhibition of Fruit Bromelain and Is Posttranslationally Removed during Ripening1,[W],[OA]Department of Molecular Biosciences and Bioengineering, University of Hawaii, Honolulu, Hawaii 96822
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  -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 -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
Phytocystatins function in diverse biological processes, such as protein turnover during seed development and germination (Kuroda et al., 2001
In pineapple (Ananas comosus), four major Cys proteases have been identified. They are the stem (Ritonja et al., 1989
AcCYS1 Contains a Signal Peptide and an Extended NTT Rich in Ala and Glu
A full-length cDNA encoding AcCYS1 was isolated after reverse northern hybridization analysis (Neuteboom et al., 2002
The unusual AE-rich NTT is unique to pineapple cystatin as determined by an extensive search of the databases. Among the presumably complete cystatins, the main body of cowpea (Vigna unguiculata) CPI-1 (Diop et al., 2004 ; Martínez et al., 2005b) was most similar to AcCYS1. However, CPI-1 has only six amino acids between the predicted signal peptide processing site and the main body (Fig. 1). CPI-1 also has a C-terminal extension of 71 residues that is lacking in AcCYS1. Some phytocystatins (WC4, wheat [Triticum aestivum]; HvCPI-2 barley [Hordeum vulgare]) have weak similarity around Met-51 to AcCYS1 and are much shorter. The NTT of sweet potato (Ipomoea batatas) cystatin, SPC15 (To et al., 1999), is 27 residues shorter than the NTT of AcCYS1. In addition, SPC15 has only five Glu and two Ala residues relative to the 17 Glu and 16 Ala residues in the NTT of AcCYS1 (Fig. 1).
The presence of a putative signal peptide and extended, AE-rich NTT in AcCYS1 prompted us to study the function of these regions in further detail. The full-length cDNA (designated SPPC for signal peptide pineapple cystatin; Fig. 2A) contains the signal peptide and complete AE-rich NTT, whereas a shorter cDNA (MPC, for medium pineapple cystatin; Fig. 2A), lacks the signal peptide coding region and a portion of the AE-rich NTT. MPC has an N-terminal Met at position 51. The SPPC cDNA contains two translational start codons, one for the signal peptide and a second downstream coinciding with the start of MPC (Met-51). These cDNAs were transcribed and translated in vitro in the presence of canine microsomal membranes. Both SPPC and MPC arise from translation of SPPC mRNA (Fig. 2, B and C) in the cell-free system, whereas only MPC is translated from the truncated MPC mRNA. Microsomes cleaved the SPPC to a shorter polypeptide corresponding to the size of AcCYS1 containing the complete AE-rich NTT (designated as AEPC), but MPC was not affected (Fig. 2B). The decrease in size of SPPC is consistent with the predicted processing of the 33-amino-acid N terminus (Figs. 1 and 2A), although the precise cleavage site cannot be determined in this assay. Posttranslational addition of microsomes to SPPC also resulted in the specific processing of SPPC (Fig. 2C). This experiment demonstrated that microsomes processed the predicted signal peptide that functioned as a genuine constituent of AcCYS1.
We determined AcCYS1 mRNA levels throughout the plant and at different stages of development by RNA gel-blot hybridization analysis (Fig. 3 ). An AcCYS1 transcript of approximately 600 nucleotides was most abundant in fruit, but high levels of mRNA were also detected in roots and aerial parts of field-grown plants (Fig. 3A). The mRNA levels within each type of tissue remained relatively unchanged during plant development and fruit ripening. We analyzed AcCYS1 gene expression in different subtissues of a field-grown plant at the time of fruit harvesting (approximately 20 months after planting) and in a flowering bud from a 14-month-old plant (Fig. 3B). The fruit flesh contained high cystatin mRNA levels, which were most abundant in the shell. Expression was lower in field-grown roots. Tissue print hybridization was used to assess AcCYS1 mRNA levels in adventitious roots grown directly from a crown in water (Fig. 3C). Similar levels of AcCYS1 expression were observed along the length of the root, with especially high expression in the root tip.
A Smaller AcCYS1 Species Is Detected in Ripe Fruit and Not in Roots, Leaves, and Green Fruit
The expression of AcCYS1 was further studied by determining protein size and levels in vivo using immunoblot analysis with an AcCYS1 antiserum. The polyclonal antiserum was generated against a peptide epitope, FDKEDLARFAVREYN (Figs. 1 and 2A). A distinct 27-kD protein was detected in all tissues examined (fruit, roots, and leaves; Fig. 4A
). Other protein bands coinciding with 25, 30, and 32 kD were also detected in leaves as well as a 25-kD species in fruit. The larger species in leaves could contain unique posttranslational modifications that decrease its mobility and do not occur in the other tissues. In addition, an abundant approximately 15-kD protein Small Fruit Cystatin (SFC) was detected in mature ripe fruit extracts but not in immature green fruit or roots and leaves (Fig. 4A). The 27-kD size estimated from the blot is higher than the theoretical size of AEPC that was calculated to be 17.2 kD (Fig. 2A). The larger 27-kD band could be due to posttranslational modifications; however no N-glycosylation sites were present in the sequence, although an O-glycosylation site is present. Interestingly, recombinant unmodified AEPC (rAEPC) expressed in Escherichia coli, lacking posttranslational modifications, migrated similar to, but slightly higher than, the endogenous 27-kD protein in pineapple tissue (Fig. 4A). It contains a 6X-His tag that contributed approximately 0.8 kD to the mobility. Recombinant MPC (rMPC) and CPC (rCPC), which are calculated to be 15.5 and 10.8 kD, migrated at approximately 21 and 16.5 kD, respectively. The slower mobility is most likely due to the many negatively charged amino acids (Asp and Glu) throughout rAEPC, rMPC, and rCPC, which can retard migration in SDS-PAGE, leading to an overestimation of the molecular mass (Armstrong and Roman, 1993
The Smaller Cystatin in Mature Fruit Is Created by Proteolytic Processing of the NTT We sought to determine the origin of the smaller cystatin (approximately 15 kD) present in mature fruit. We conducted protein processing assays using a mature fruit extract and rMPC. The rMPC (Fig. 2A) was produced and affinity purified from E. coli (Fig. 6B) and incubated with the mature fruit extract (Fig. 4B). The mixture was then subjected to immunoblot analysis using the cystatin antiserum (Fig. 4B, left) or the monoclonal anti-HIS antiserum (Fig. 4B, right). The rMPC appeared as an approximately 21-kD protein on the blot (Fig. 4, A and B). The mature fruit extract contained the endogenous approximately 15-kD cystatin (SFC; Fig. 4, A and B). When rMPC was incubated with the fruit extract, a prominent, smaller, approximately 16-kD protein (labeled as 1 versus the endogenous fruit cystatin labeled as 2) was observed (Fig. 4B). The approximately 16-kD band corresponded in size to the endogenous ripe fruit protein (approximately 15 kD) plus the C-terminal His-tag (approximately 0.8 kD; Fig. 4B). The anti-HIS antiserum, which only binds to the polypeptide retaining the His-tag at the C terminus, detected rMPC and a single processed product of 16 kD, but it did not detect the endogenous SFC that lacks the His-tag, thereby confirming that the processed 16-kD product has the NTT removed. The next experiment mapped the processing site and unequivocally verified that the NTT was removed at a key region of AcCYS1. The processed product was subjected to liquid chromatography/tandem mass spectrometry analyses, and three N-terminal peptides were identified (Fig. 4C). The original mass spectrometry data spectra are presented in Supplemental Figure S1 online. The primary cleavage site (>80%) occurred after Gly-95, which is one residue after the conserved essential Gly-94. The secondary cleavage site occurred immediately after Gly-94 (15%), and a weak minor cleavage occurred four residues down between Asp-98 and Ala-99. Therefore, the endogenous ripe fruit cystatin is 1.6 kD smaller than rCPC. The smaller size is accounted for by the lack of 6X-His tag (0.8 kD) and the residues 93 to 98 (MGGIYD) that are in the first β-strand.
Immunolocalization of Pineapple Cystatin in the Apoplast of Root Cells
We next studied the subcellular localization of AcCYS1 using immunolabeling with affinity-purified (described in Methods) anticystatin antiserum combined with fluorescence and electron microscopy. Fruit tissue was problematic to fix for microscopy, but transverse sections (1 µm and 100 nm for epifluorescence and micron electroscopy, respectively) of adventitious roots grown in water from crowns were highly suitable for fixation, embedding, and immunolocalization. Specific fluorescence (bright green) was detected in many cell types around the apoplasm of the root when only the primary cystatin antiserum was used with the Alexa Fluor anti-rabbit secondary antibody (Fig. 5, A and B
). The AcCYS1 protein was primarily found in the apoplast of xylem, phloem, and pith cells (Fig. 5B). Light-yellow autofluorescence was observed with no antisera (Supplemental Fig. S2). The use of the secondary antibody alone did not enhance the autofluorescence or change the fluorescence pattern (Supplemental Fig. S2B). High-resolution immunogold labeling of fixed root (100-nm sections) also indicated the apoplastic and cell wall localization of pineapple cystatin (Fig. 5C). No gold labeling was observed when the secondary immunogold antiserum was used alone (Supplemental Fig. S2, C and D). Interestingly, root border-like cells (Hamamoto et al., 2006
rAEPC and rMPC Effectively Inhibit Bromelain Compared to rCPC We tested the effect of AcCYS1 on papain and stem and fruit bromelains and defined the influence of the unique extended AE-rich NTT on AcCYS1 inhibitory potency. Papain and stem bromelain were from commercial sources, whereas fruit bromelain was purified as described in "Materials and Methods." We expressed three versions of pineapple cystatin in E. coli: rAEPC, rMPC, and rCPC, which differ in the length of their N termini and share the same C termini (Figs. 2A and 6 ). The rAEPC, rMPC, and rCPC were affinity purified through a C-terminal His-tag, their purity was verified by Coomassie Brilliant Blue staining after SDS-PAGE (Fig. 6B), and their identity confirmed by immunoblot analysis with cystatin antiserum (Fig. 6C). The effects of equimolar rAEPC, rMPC, and rCPC were initially tested on papain because it is a standard reference Cys protease. Chicken cystatin was included as a standard reference control. rCPC and chicken cystatin (250 and 500 pmol) proved to be more effective inhibitors of papain compared to the same amounts of rMPC and rAEPC (Fig. 7A ). However, the inhibition of papain increased using 500 pmol of rMPC and rAEPC. For stem bromelain, 250 to 500 pmol rMPC and rAEPC inhibited it >95% (Fig. 7B), whereas rCPC was less (80% inhibition) and chicken cystatin nominally effective (40% inhibition), respectively. The rAEPC competitively inhibited bromelain as determined via a Lineweaver Burk plot (Supplemental Fig. S3).
Because the expression of the AcCYS1 gene was elevated in fruit (Fig. 2), the AcCYS1 protein was also processed in mature fruit (Fig. 4), and fruit and stem bromelains have differentiated in amino acid sequence, we tested the effect of AcCYS1 on purified fruit bromelain (Fig. 7C). In contrast to stem bromelain, 250 to 500 pmol of rCPC was nominally effective (20%–25% inhibition) against fruit bromelain, whereas the same amounts of rMPC and rAEPC were highly effective (98% inhibition). To determine the concentration of rAEPC and rMPC that would produce a similar degree of inhibition as rCPC, we tested a dilution series of the cystatins. rAEPC or rMPC (50 pmol) were as effective as 500 pmol of rCPC, indicating a 10 times greater effectiveness of rAEPC and rMPC as inhibitors.
To investigate the characteristics of the inhibitor-enzyme interaction more closely, surface plasmon resonance analysis was employed to measure the affinity of each pineapple cystatin for stem bromelain (Table I ). The rCPC, rMPC, and rAEPC had moderately increasing association rate constants and decreasing dissociation constants; however, all exhibited tight binding to stem bromelain. The calculated dissociation equilibrium constant (Ki) values showed that rAEPC and rMPC were 1.3- to 1.9-fold more potent inhibitors of stem bromelain than rCPC (Table I).
This research addresses a central issue in the regulation of enzymes needed during a terminal phase of plant development, specifically, fruit ripening. The hydrolytic enzymes associated with ripening and senescence must be tightly regulated and inhibited to prevent damage to the cellular biosynthetic apparatus prior to ripening. Subsequently, the inhibition must be released when hydrolysis is required. One mechanism is the use of the propeptide to inhibit the protease whereby propeptide removal activates the protease (Taylor et al., 1995 ). However, not all propeptides function as intramolecular protease inhibitors. For some proteases, the propeptides act primarily as intramolecular protein folding chaperones that are needed for protease activity (Subbian et al., 2005; Schilling et al., 2009). Moreover, the bromelains that accumulate in the stem and fruit lack the propeptide (Yamada et al., 1976; Ritonja et al., 1989; Harrach et al., 1998). This raises the question: How are proteases lacking the propeptide inhibited at their cellular destinations? We propose that the highly expressed fruit cystatin, AcCYS1, undertakes this inhibitory role in immature fruit (Fig. 8
). In the model, as fruit ripen, the proteolytic removal of the unique inhibitory NTT region of AcCYS1 significantly increases fruit bromelain activity (Fig. 8). Bromelain is the most abundant protease in fruit (Yamada et al., 1976), and the resulting increase in its activity after cleavage of its inhibitor, AcCYS1, is expected to stimulate proteolysis. Likewise, a noncystatin bromelain inhibitor (Hatano et al., 1996; Sawano et al., 2002) was recently shown to be abundant in green fruit but not in ripe fruit (Moyle et al., 2005).
Although cell wall polysaccharide hydrolysis is key for fruit softening, the degradation of proteins, such as extensin crosslinkers, cell-wall-associated kinases, and arabinogalactan proteins in the extracellular matrix (Baluska et al., 2003 ), is also involved in fruit softening and decay (Smith and Harris, 1995; Brummell et al., 1999; Yong et al., 2005) and in the separation of cells from surrounding living tissue as observed for root epidermal cells (Fig. 5, D–F). As described for other diverse tissues and fruits (Soda et al., 1986; Alonso and Cranell, 1995; Itai et al., 2000; Matarasso et al., 2005), Cys proteases of the secretory pathway are required during ripening and senescence for the degradation and recycling of proteins (Solomon et al., 1999; Matarasso et al., 2005; Ondzighi et al., 2008). Fruit bromelain contains a strong signal peptide (http://www.uniprot.org/uniprot/O23791), indicating that it enters the secretory pathway, as does AcCYS1 (Fig. 8, and discussed below). Cys proteases are secreted in pineapple suspension cultures and have been detected in the pineapple (Pérez et al., 2004) and tomato (Solanum lycopersicum) apoplasms (Tian et al., 2007).
We presented three lines of evidence that the secretory signal peptide in the N terminus of AcCYS1 is functional: (1) Microsomal membranes processed the signal peptide from the SPPC polypeptide co- and posttranslationally in vitro; (2) immunoblot analysis using an AcCYS1 antiserum on in vivo proteins from three tissues detected a cystatin that coincided in electrophoretic mobility with rAEPC that is equivalent to the processed form; and (3) the AcCYS1 protein was immunolocalized to the apoplast of root cells indicative of the protein entering the secretory pathway, which is an attribute of signal-peptide-containing proteins. Carrot (Daucus carota) cystatin and human cystatin F are also secreted proteins (Ojima et al., 1997
This work also concerns the coevolution of inhibitors with their target enzymes. Plant proteases are presumed to coevolve with their cognate inhibitors in the same cellular environment (Otlewski et al., 2005
Structural differences in both the NTT and core region affect their potency and specificity of animal (Hall et al., 1995
Plant Growth Conditions Ananas comosus (cv Smooth Cayenne) field plants were grown at Del Monte (Del Monte Produce Hawaii) in central Oahu with a temperature range of 18°C to 32°C. The same variety was also grown in sand in the greenhouse at 23°C to 30°C. Water-grown plants were derived from crowns removed from mature fruit. The crowns were placed in water in one-liter beakers at 25°C and grown for 25 d.
Plants were collected, briefly washed, dry-blotted, cut, and immediately frozen in the field with liquid N2 to prevent induction of stress-related genes in the tissues. Root RNA from field- and sand-grown plants was isolated from fresh tissue at the root apices only (distal 4–5 cm). The woody basal ends gave RNA of poor quality and low yields. Aerial tissues were used either entirely (3.5-month-old plants) or partly (older plants) after cutting into halves. Fruits and other plant organs were also cut into halves to obtain RNA that represented the whole organ. Water-grown roots were dry-blotted before freezing. RNA was isolated as described (Neuteboom et al., 2002
For each cystatin (rCPC, rMPC, and rSPPC, cloned in pBluescript), circular plasmid was added to coupled transcription-translation extract (reticulocyte lysate) containing [35S]Met with and without canine pancreatic microsomal membranes according to the manufacturer (Promega). The samples were incubated at 30°C for 90 min posttranslational or 60 min cotranslational and quenched on ice before loading with equal volume of 2x loading buffer on a 12% resolving, 5% stacking SDS-PAGE gel. After running for 1 h at 60 V, then 2.5 h at 80 V in 1x Tris-Gly buffer, pH 8.3, the gel was vacuum dried at 80°C for 1 h and exposed to x-ray film overnight.
RNA concentrations were determined spectrophotometrically. Ten micrograms of total cellular RNA was denatured with glyoxal and loaded on a 1.5% agarose gel (Sambrook et al., 1989
Tissue printing was carried out on roots from water-grown plants, dry-blotted, and then sectioned with a razor. The exposed planes were blotted onto GeneScreen Plus (NEN Life Science Products) and hybridized with [
Proteins were extracted from pineapple fruit (immature and mature), leaf, and root. Each tissue was frozen in liquid N2 and ground with a mortar and pestle, and 200 mg was suspended in 500 µL protein extraction buffer (50 mM Tris, pH 8, 250 mM Suc, 2 mM dithiothreitol, 2 mM EDTA, and 1 mM PMSF) rotated 50 rpm for 2 h at 4°C. The protein samples were centrifuged for 20 min at 18 K rpm at 4°C and the supernatant transferred to a new tube. Ten microliters (5 µg protein) of mature fruit extract was mixed with rCPC (2 µg) and rMPC (2 µg), and the reaction was incubated for 15 min at room temperature before addition of an equal volume of protein sample loading buffer. Proteins were analyzed via SDS-PAGE (12% resolving; 5% stacking) and electrotransferred onto PROTRAN nitrocellulose membranes (Perkin-Elmer Life Sciences). Immunoblot analysis was conducted as described (Lu and Christopher, 2006 For mapping the proteolytic processing site, 24 µg of rMPC was added to 120 µg of pineapple fruit extract and incubated for 5 min followed by separation via SDS-PAGE (12% resolving; 5% stacking). The protein bands were stained with Coomassie Brilliant Blue. Standard trypsinization of the processed protein gel band followed by nano-liquid chromatography/mass spectrometry analysis was conducted at Midwest Bio Services.
Roots were fixed with 4% paraformaldehyde and 0.2% glutaraldehyde in 0.1 M sodium cacodylate buffer containing 2 mM CaCl2, pH 7.4, for 1 h at room temperature and washed in 0.1 M cacodylate with 2 mM CaCl2 for 2 x 10 min at room temperature. The roots were then dehydrated in a graded series of ethanol (10%, 30%, 50%, 70%, 85%, 95%, and 100%) and infiltrated with 1:1 ethanol/LR White resin, 2:1 ethanol/LR White resin, both for 1 to 2 h at room temperature on a rotator followed by 100% LR White embedding that was polymerized at 50°C. Semithin (1 µm) and ultrathin (80 nm) sections were obtained on a Reichert Ultracut E ultramicrotome with glass and diamond knives, respectively. For epifluorescence, the thin sections were mounted on glass slides with Vectashield (Vector Laboratories), blocked for 1 h with PBST plus 5% nonfat milk (blocking solution), incubated for 1 h with 1:100 primary antisera (anti-pineapple cystatin) in blocking solution, washed for 3 x 3 min in blocking solution, further incubated for 1 h with 1:100 secondary antisera (Alexa Fluor 488 goat anti-mouse IgG; Molecular Probes), and then washed for 5 min in blocking solution followed by PBS for 2 x 5 min, and deionized water for 2 x 10 min. The immunolabeled sections were covered with fluoromount and a coverslip and examined on an Olympus BX51 upright compound microscope with a 488-nm excitation filter. Images were recorded with an Optronics Macrofire SP CCD camera. For transmission electron microscopy, the ultrathin sections were mounted on nickel grids, treated with and without 5% sodium metaperiodate for 2 x 5 min, and washed 3 x 3 min with deionized water. Subsequent immunolabeling were the same as described above for the epifluorescence with the exception that the secondary antibody was 10-nm gold-conjugated anti-mouse H+L (Ted Pella). The tissues were poststained with 5% uranyl acetate and 3% lead citrate and viewed on an LEO 912 EFTEM at 100 kV and photographed with a Proscan frame-transfer CCD camera.
To express AEPC in Escherichia coli, it was necessary to add a methione immediately preceding the AE-rich NTT. In addition, the Met-51 was substituted with Ile because earlier trials in vitro showed undesired initiation of translation from the internal Met-51 from the SPPC construct leading to production of MPC (Fig. 2B). The cystatins, CPC and MPC, were cloned into expression vector pET25b+ with the C-terminal 6X-HIS tag (Novagen). The pET25b+ vector was modified by removal of the pelB leader by digesting with NdeI/NcoI, blunt-end filling with Klenow, and self-ligation. AEPC was also cloned in the same modified pET25b+ vector with the exception that the Met at Met-53 site was altered to become an Ile (Epoch Biolabs), and the digestion sites were NdeI/HindIII. Transformation into Novablue cells for construct confirmation followed by transformation into BL21DE3 cells for expression was conducted. The cystatins were overexpressed by growing cultures at 37°C with 225 rpm shaking to OD260 = 0.6, then induced with 1 mM isopropylthio-β-galactoside. Total cell protein was extracted with rLysozyme, benzonase nuclease, and protease inhibitor cocktail (without EDTA) in addition to BugBuster extraction buffer. The cystatins were purified on nickel-nitrilotriacetic acid agarose His-Bind Resin columns according to the manufacturer (Novagen). Protein concentrations were determined with a Bio-Rad kit. Both total cell and purified proteins were analyzed on duplicate SDS-PAGE (12% resolving; 5% stacking) followed by either Coomassie Brilliant Blue staining or immunoblotting with pineapple cystatin or His-tag antibodies (as described above). The resulting recombinant proteins were designated rAEPC, rMPC, and rCPC.
Fruit bromelain was purified to >90% purity from ripe fruit as described for stem bromelain (Ritonja et al., 1989
Affinity-purified rCPC, rMPC, rAEPC, and chicken cystatin (Sigma-Aldrich) were assayed against papain and stem bromelain (76220 and B5144; Sigma-Aldrich) and fruit bromelain (EC 3.4.22.33) using the EnzChek Protease Assay Kit (Molecular Probes). Each recombinant cystatin concentration was verified spectrophotometrically and equivalency via Coomassie Brilliant Blue staining of SDS-PAGE separated proteins. A total of 0 to 500 pmol of each cystatin was added to 0 and 0.2 units of each protease in a 200-µL reaction. Addition of 200 µL of 10 µg/mL BODIPY casein substrate was added to the above mixture, vortexed, and incubated in the dark for 1 h at room temperature. The fluorescence was read in a fluorometer (Turner BioSystems) with a fluorescein filter (excitation at 485 ± 12.5 nm and emission at 530 ± 15 nm) over time. The background fluorescence was measured in the absence of protease and was subtracted from the values with protease. Enzyme activity was represented as nanograms of fluorescent BODIPY product produced per microliter per minute. The assays were conducted in three experiments and the data analyzed via ANOVA. Surface plasmon resonance analysis was conducted at using a Biacore T100 instrument at the company's facility (Biacore, a subsidiary of GE Healthcare). Each cystatin (rCPC, rMPC, and rAEPC) was separately conjugated to a sensor chip until the point of saturation using amine-coupled anti-HIS and anti-HSV antisera. Bromelain was injected across the captured cystatin surface at a range of concentrations from 50 to 0.78 nM with 0.125 mg mL–1 total E. coli proteins (containing empty pET25b+ vector) added simultaneously as a negative control. A reference surface containing the captured antiserum alone was used to monitor nonspecific binding and subtract bulk refractive index. The analysis was conducted using a 1:1 ratio of inhibitor to enzyme concentration, with real-time monitoring of pre-steady-state and steady-state kinetics of association and dissociation, which are appropriate parameters for measuring inhibitor affinity (Koiwa et al., 2001 Sequence data from this article can be found in the GenBank/EMBL data libraries under accession number EU937516.
The following materials are available in the online version of this article.
We thank Tina Weatherby at the University of Hawaii Biological Electron Microscope Facility for expert microscopy assistance. Received June 2, 2009; accepted July 20, 2009; published July 31, 2009.
1 This work was supported by grants from the U.S. Department of Agriculture-Tropical and Subtropical Agricultural Research Program (HAW00516–1016S) and the National Sciences Foundation (MCB–03–48028) to D.A.C. 
2 These authors contributed equally to the article. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: David A. Christopher (dchr{at}hawaii.edu).
[W] The online version of this article contains Web-only data.
[OA] Open access articles can be viewed online without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.109.142232 * Corresponding author; e-mail dchr{at}hawaii.edu.
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M, Repellin A, Gareil M, d'Arcy-Lameta A, Pham Thi AT, Zuily-Fodil Y (2004) A multicystatin is induced by drought-stress in cowpea (Vigna unguiculata (L.) Walp.) leaves. FEBS Lett 577: 545–550