purified recombinant cAPX showed the homologous 28-kDa protein (Fig. III-4). The enzyme was found to be a monomeric form by gel filtration analysis (data not shown).
cAPX isoenzymes may be grouped into two classes in terms of the structure of the subunit. One class exists as a monomeric form in the cAPXs of spinach (Tanaka et al.
1991), potato tuber (Elia et al. 1992), Brassica rapa (Ishikawa et al. 1996a), and Euglena (Ishikawa et al. 1996b). The other class is a homodimer in the cAPXs of pea (Mittler and Zilinskas 1991a) and legume root nodules (Dalton et al. 1987), in which each subunit fails to associate via disulfide bonds. The recombinant pea and soybean cAPX isoenzymes also existed as homodimers (Patterson and Poulos 1994, Dalton et al. 1996).
Characterization of recombinant sAPX, tAPX and cAPX
Table III-2 shows comparison of enzymatic properties of both recombinant and native APX isoenzymes. The properties of recombinant sAPX, tAPX and cAPX produced in E. coli were indistinguishable from those of the respective native isoenzymes. The recombinant chlAPX isoenzymes utilized AsA at a higher rate as electron donor and showed no or low activity for the other electron donors such as GSH, NAD(P)H, Cyt c, guaiacol, and pyrogallol. The relative activities among the
94 67 43 30 20.1
sAPX (33 kDa) cAPX (28 kDa)
1 2 3
pyrogallol as electron donor, and the value was similar to that of the native cAPX.
The Km values of both recombinant sAPX and cAPX for AsA (0.33and 0.52mM) and H2O2 (0.04 and 0.01 mM) were similar to those of each native isoenzyme. The optimum pH of both the purified recombinant sAPX and cAPX was 7.0, which well corresponded to each value of the native enzymes. Miyake et al. have reported that the properties of the native tAPX are very similar to those of sAPX with respect to high specificity for AsA and Km values for H2O2 and AsA. The sole difference in terms of properties between tAPX and sAPX is the higher molecular weight of the membrane-bound enzyme compared to the sAPX; the tAPX is bound to thylakoid membranes in such a form that the active site of the enzyme is exposed to the stroma for the access of the substrate (Miyake et al. 1993, Ishikawa et al. 1996c, 1997). I could confirm that not only the native sAPX and tAPX isoenzymes but also the recombinant enzymes share the similar characterizations.
Table III-2 Comparison of some enzymatic properties of recombinant sAPX, tAPX, and cAPX isoenzymes with those of spinach native isoenzymes
Recombinant Native
sAPX tAPX cAPX sAPX tAPX cAPX
Molecular mass (kDa)
Gel filtration 33 n.d. 28 30±1# 40±2& 31$
SDS-PAGE 33 38 28 30# 40& 31$
(33,239 Da)¶ (37,710 Da) (27,560 Da) Donor specificity (%)*
AsA 100 100 100 100 100 100
Iso-AsA 58 65 32 59 60 38
GSH 0 0 0 0 0 0
Cyt c 0 0 0 0 0 0
NAD(P)H 0 0 0 0 0 0
Pyrogaroll 19 15 350 25 13 368
Guaiacol 0.6 0.3 3.9 1.4 0.1 4.3
Km (mM)
AsA 0.33 0.35 0.52 0.30# 0.57& 0.60
H2O2 0.04 0.04 0.01 0.03# 0.08& 0.01
Optimum pH 7.0 7.0 7.0 7.0 7.0 7.0
*The peroxidase activity for AsA was shown as 100%.
¶Data in parentheses are calculated from deduced amino acid sequences (Ishikawa et al. 1995, 1996c).
n.d.; not determined; #, Nakano and Asada 1987; &, Miyake et al. 1993; $, Tanaka et al. 1991.
APX has been known as a labile enzyme in an AsA-depleted medium.
Compared with the cAPX isoenzyme, the chlAPX isoenzymes have a half-time of within a minute in that medium (Asada 1992). When the purified recombinant sAPX
was diluted with the AsA-depleted medium, the enzyme showed a rapid inactivation, whose half time was approxmately 15 s. The half-inactivation time of the purified recombinant cAPX was approximately 60 min, which value was very similar to that reported for the native cAPX (Asada 1992). I have found that mAPX, which was highly homologous to cAPX, was also relatively stable in the AsA-depletion medium (Ishikawa et al. 1998, Chapter II). It is of interest that in spite of no detectable amount of AsA in E. coli cells, the recombinant APX isoenzymes are overexpressed as an active form. Miyake and Asada (Miyake et al. 1993, Miyake and Asada 1996) have reported that the inactivation of sAPX isoenzyme in an AsA-depleted medium is caused by the instability of Compound I to H2O2 when AsA is not available for Compound I and that no inactivation of sAPX occurs under anaerobic conditions.
One possible explanation for this expression, therefore, is that E. coli cells maintain highly reduced and anaerobic conditions in vivo.
As shown in Figs. III-5A and B, the absorption spectra of the purified recombinant sAPX and cAPX closely resembled those of native enzymes form several sources (Miyake et al. 1993, Chen and Asada 1989, Nakano et al. 1991, Dalton et al.
1987). Characteristic absorption maxima of a Soret band at 403 and 401 nm were found for the recombinant sAPX and cAPX, respectively. The Soret peaks of the recombinant sAPX and cAPX were shifted to 418 and 403 nm, respectively, by reduction with dithionite, and respective additional peaks appeared at 537 and532nm.
Wave length (nm) 0.4
0.2
0
0.05
0.025
400 500 600 7000
403420418 537
Wave length (nm) 0.4
0.2
0
0.05
0.025
400 500 600 7000
401 403 419
534 532
A B
Absorbance Absorbance
Fig. III-5 Absorption spectrum of purified recombinant sAPX (A) and cAPX (B). The sample cuvette contained 115 µg of the purified sAPX or 130 µg of the purified cAPX in 1 ml of 50 mM potassium phosphate, pH 7.0, 20% (w/v) sorbitol, 0.1% PMSF, and 1 mM EDTA.
———, native form; ---, reduced form; -•-•-•-, CN-form.
The absorbance coefficients of the oxidized forms of sAPX (403 nm) and cAPX (401 nm) were7.2 x 104 M-1/cmand6.7 x 104 M-1/cm, respectively. The cyanide complex
of the oxidized sAPX and cAPX gave peaks at 420 and 537 nm (Fig. III-5A) and 419 and 534 nm (Fig. III-5B), respectively.
Immunological cross-reactivity of the recombinant APX with the native APX isoenzymes from spinach
On immunoblot analysis, polyclonal antibodies against the recombinant sAPX and cAPX isoenzymes reacted with the respective recombinant APX isoenzyme proteins (Figs. III-6A, B). The polyclonal antibody against sAPX from tea leaves cross-reacted with both sAPX and cAPX isoenzymes (Miyake et al. 1993, Chen and Asada 1989). In contrast, the polyclonal antibody against the native pea cAPX and the mAb against the native spinach cAPX failed to cross-react with the respective chlAPX isoenzymes (Mittler and Zilinskas 1991a, Saji et al. 1990). Polyclonal antibodies against the recombinant spinach sAPX and cAPX isoenzymes could efficiently cross-reacted with both chlAPXs and cAPX. This result is not surprising
tAPX (38 kDa) sAPX (33 kDa) cAPX (28 kDa)
A B
4 5 6 1 2 3
94 67
43 30 20.1 kDa
Fig. III-6 Production and cross-reactivity of anti-recombinant sAPX (A) and cAPX (B) polyclonal antisera. Purified recombinant cAPX (lanes 1, 4), purified recombinant sAPX (lanes 2, 5) and solubilized recombinant tAPX (lanes 3, 6) were separated by SDS-PAGE and then subjected to immunoblot analysis using anti-recombinant sAPX and cAPX serum, respectively, as described in Materials and Methods section. Relative molecular mass standards are indicated.
because of the existence of highly conserved domains between chlAPXs and cAPX (Fig. III-1). Recently, we have defined that the Euglena monoclonal antibody
(EAP1), which cross-reacts with both chlAPXs and cAPXs from higher plants, recognizes the common epitope at the site around the proximal His residue in APX isoenzymes (Ishikawa et al. 1996b).
Conclusions
These results presented here supported the fact that chloroplatic APXs and cAPXs can be distinguished by their specificity for the electron donor, stability in AsA-depleted medium, and amino acid sequences. It is an interesting problem how the chlAPX isoenzymes provide a possible characteristic structure allowing the specific binding of AsA and their lability. These questions raise a structural problem, which can only be solved by obtaining the three-dimensional structure of the enzymes.
Jespersen et al. (1997) have proposed that the Trp-175 residue near the proximal His residue is a strong candidate related to the specificity of chlAPX isoenzymes toward AsA. The pET/sAPX plasmid is an excellent expression system for spinach chlAPX isoenzyme. The yields of the recombinant enzyme are relatively high, and its enzymatic properties are identical to those of the native sAPX isolated from spinach chloroplasts. In our laboratory I am progressing toward facilitating a crystallographic and site-directed mutagenesis study of spinach sAPX.
Summary
The spinach sAPX, tAPX, and cAPX isoenzymes were overexpressed in Escherichia coli and their enzymatic properties were compared with the respective native isoenzymes. The purification of the recombinant sAPX and cAPX using the conventional column chromatography yielded 0.73 mg and 2.2 mg of protein/liter of bacteria culture with enzyme activities of 800 and 486 µmol min-1 mg protein-1, respectively. In every respect, the recombinant sAPX, tAPX, and cAPX isoenzymes exhibited identical enzymatic properties with each native isoenzyme. Specifically, the recombinant sAPX and tAPX showed high utilization of AsA as an electron donor and had a very short life-time in AsA-depleted medium. Polyclonal antibodies raised against both purified recombinant sAPX and cAPX isoenzymes were prepared. Both antibodies showed a cross-reaction with the recombinant and native APX isoenzymes.
CHAPTER IV
Characterization of Monoclonal Antibodies against
Ascorbate Peroxidase Isoenzymes: Purification and Epitope-mapping Using Immunoaffinity Column Chromatography
I have isolated cDNA clones encoding APX isoenzymes from a spinach cDNA library and developed their recombinant expression systems in E. coli (Chapter III, Ishikawa, et al. 1995 1996c, 1998, Yoshimura et al. 1998). Recently, I have reported that biotic and abiotic stresses induce the expression of APX genes (Yoshimura et al.
2000). Considering the scavenging capacity of APX isoenzymes for H2O2 generated in plant cells under various stress conditions, analysis of the changes in the levels of their proteins and activities as well as gene expressions is important to understand their critical functions and actual responses to environmental stresses. However, the study of protein levels of APX has been limited by the lack of a high sensitive antibody probe to detect APX isoenzymes. Previously, we generated two mAbs (EAP1 and EAP2) against Euglena cAPX (Ishikawa et al. 1996b). Although EAP1 cross-reacted with both sAPX and cAPX from spinach leaves and EAP2 efficiently cross-reacted with the cAPX rather than the sAPX, they did not recognize plant APX well in crude extracts.
In this study, I have developed a panel of mAbs that selectively recognize the different APX isoenzymes. The utility of these mAbs for cross-reactivity, purification using an immunoaffinity column, and analysis of proteolytic fragments including their epitopes are demonstrated.
Materials and Methods
Materials
The cDNAs for spinach APX isoenzymes were as previously described (Chapter III, Ishikawa, et al. 1995 1996c, 1998). Recombinant APX (sAPX, tAPX, cAPX, and mAPX) proteins were expressed in E. coli and purified as described previously (Chapter III, Ishikawa, et al. 1998, Yoshimura et al. 2000). All of the other reagents were of analytical grade and were purchased from commercial sources.
Preparation of mAbs
mAbs to spinach APX isoenzymes were generated by the multiple immunization of BALB/c mice with each purified recombinant enzyme. Mice showing the highest titer of anti-sAPX and cAPX immunoreactivity were used to create fusions with myeloma cells using the standard protocols (Harlow and Lane 1988). Positive
hybridomas were cloned twice by the limiting dilution. These hybridomas were also screened against sAPX and cAPX. Positive hybridomas recognizing the respective APX isoenzyme were then injected into mice to produce ascites fluid. IgGs were purified by Protein G Sepharose (Amersham Pharmacia Biotech, Uppsala, Sweden).
ELISA
One hundred µl of TBS (20 mM Tris-HCl, pH 7.4, 2.7 mM KCl, and 100 mM NaCl) containing 1 µg of antigen protein was placed in each well of a 96-well microtiter plate and incubated for 18 h at 4˚C. After removing the antigen fluid, the wells were blocked with TBS containing 2% BSA for 2 h at room temperature. After washing the wells three times with TBS containing 0.05% Tween-20, the monoclonal antibody was reacted for 2 h at room temperature, followed by peroxidase-conjugate goat anti-mouse Igs antibody for 2 h at room temperature. Finally, the remaining peroxidase activity was determined using 5-aminosalicylic acid as substrate. The results were monitored spectorophotometrically as optical density at 410 nm.
SDS-polyacrylamide gel electrophoresis and immunoblot analysis
Partially purified recombinant APX proteins were mixed with a SDS-loading buffer (150 mM Tris-HCl, pH 6.8, 4% SDS, and 10% 2-mercaptoethanol) and boiled for 5 min. The leaves of spinach, tobacco, and Mesembryanthemum crystallium were homogenized with the SDS-loading buffer. Each homogenate was boiled for 5 min and centrifuged at 10,000 g for 10 min. The supernatants were quantified with respect to protein contents. Samples were separated by SDS-polyacrylamide gel electrophoresis (12.5% polyacrylamide) and transferred onto a PVDF membrane (Bio-Rad Laboratories) as described in chapter II. The mAbs (chl-mAb3, chl-mAb6, and cyt-mAb1) to spinach APX isoenzymes were diluted 1:1,000 in TBS containing 0.1%
BSA, and the membrane was incubated with each mAb at room temperature for 1 hr.
The membrane was washed in TPBS (PBS containing 0.05% Tween-20), followed by incubation for 30 min in alkaline phosphatase-conjugated goat anti-mouse-Igs (Bio-Rad) diluted 1:3,000 in TBS containing 0.1% BSA. The membrane again was washed in TPBS, and the immunoreactive proteins were visualized using nitrobluetetrazorium (Bio-Rad) and 5-bromo-4-chloro-3-indlyl phosphate (Sigma, MO, U.S.A.). Proteins in the gel were stained with a silver-staining regent (Wako, Osaka, Japan).
Immunoaffinity purification of recombinant APX isoenzymes
The mAbs were covalently coupled tothe NHS-activated Sepharose (Amersham).
Each mAb (4 mg) in 0.1 ml of 0.2 M NaHCO3, pH 8.3, containing 0.5 M NaCl, was injected on the column and allowed to stand for 4 h at 4˚C. The following washing and deactivation of the column were carried out with a buffer (0.5 M ethanolamine, pH 8.3, containing 0.5 M NaCl) and then a buffer (0.1 M sodium acetate, pH 4.0, containing 0.5 M NaCl) according to the manufacturer's instructions (Amersham). The NHS-activated Sepharose columns immobilized with each mAb were neutralized with 0.1 M NaH2PO4, pH 7.0, containing 0.15 M NaCl and 1 mM AsA. The amount of mAb covalently linked to the matrix was 1.0 mg/ml NHS-activated Sepharose.
The spinach recombinant APX isoenzymes (sAPX and cAPX) were produced in E.
coli(Chapter III, Ishikawa et al. 1998, Yoshimura et al. 1998) and then purified by immunoaffinity column chromatography. The recombinant E. coli cells (0.3 g FW) expressing spinach cAPX or sAPX were resuspended in 5 ml of 0.1 M NaH2PO4, pH 7.0, containing 0.15 M NaCl and 1 mM AsA, and sonicated (10 kHz) for a total of 1 min with four intervals of 15 sec each. This lysate was centrifuged at 15,000 g for 20 min.
The 0.5 ml (1.38 mg protein) of the supernatant obtained as a crude extract was loaded onto immunoaffinity columns equilibrated with 0.1 M NaH2PO4, pH 7.0, containing 0.15 M NaCl and 1 mM AsA. The column was washed with 3 ml of 0.2 M NaHCO3, pH 8.3, containing 0.5 M NaCl and 1 mM AsA, and then eluted with 0.1 M glycine-HCl, pH 3.0, containing 0.5 M NaCl and 1 mM AsA. The APX activity was spectrophotometrically assayed as described in chapter II.
Proteolysis of APX and analysis of fragments
The purified recombinant APX isoenzymes by the immunoaffinity columns were collected and digested with some endoproteinases. Endoproteinase Asp-N treatment was performed (Wako) at an enzyme (500 pmol): substrate ratio of 1:100 for 18 h at 37°C in a 50 mM potassium phosphate buffer, pH 8.0. Achromobacter lysyl endopeptidase (Wako) treatment was performed at an enzyme (500 pmol): substrate ratio of 1:200 for 6 h at 37°C in a 100 mM Tris-HCl buffer, pH 9.0. The proteolytic products were then subjected to immunoaffinity columns equilibrated with 0.1 M NaH2PO4, pH 7.0, containing 0.15 M NaCl. The columns were then washed with 3 ml of the coupling buffer and then eluted with 0.1 M glycine-HCl, pH 3.0, containing 0.5 M NaCl. The peptide mixtures were collected and then transferred onto a PVDF membrane according to the instruction manual of ProSorbTM kit (Applied Biosystems).
Amino acid sequencing of the peptides was performed as described in chapter II.
Results and Discussion
Development of mAbs
The recombinant sAPX and cAPX isoenzymes of spinach were purified and then used for the immunization of mice. Hybridoma cultures were screened for the production of antibodies specific to each isoenzyme by ELISA. Finally, I obtained two chlAPX mAbs (chl-mAb3 and chl-mAb6) and one cAPX mAb (cyt-mAb1). The isotype analysis showed that all of them are the IgG1 subclass. The ascites fluids from these mAbs were titrated by 1/(2n) dilutions under optimal conditions. The chl-mAb3, chl-mAb6, and cyt-mAb1 gave 10-5, 10-3, and 10-3 dilutions, respectively, to 50%
binding in the ELISA.
Cross-reactivity of chl-mAb3, chl-mAb6, and cyt-mAb1
To examine the specificity of the mAbs, I performed the immunoblot analysis of APX isoenzymes. Fig. IV-1 shows immunoreactivities of chl-mAb3, chl-mAb6, and cyt-mAb1. Each left panel shows the immunoreactivity with recombinant spinach APX proteins prepared from E. coli. The chl-mAb3 and chl-mAb6 for chlAPX recognized both recombinant sAPX and tAPX proteins with high specificity, but not cAPX and mAPX proteins. The coding region up to the amino acid residue 365 of spinach tAPX was identical to that of sAPX because chlAPXs were encoded on the same gene and were regulated by alternative splicing of 3'-terminal exons (Chapter V, Ishikawa et al.
1997, Yoshimura et al. 1999). These facts indicated that the chl-mAb3 and chl-mAb6 recognize their epitopes within the common coding region of spinach chlAPX isoenzymes. As shown in the right panel of Fig. IV-1, the chl-mAb3 and chl-mAb6 could detect the tAPX and sAPX proteins (38 and 33 kDa bands, respectively) even in the crude extract prepared from spinach leaves. Unlike chl-mAb3, chl-mAb6 also showed a cross-reaction with chlAPX isoenzymes from tobacco and Mesembryanthemum crystallium (Fig. IV-1). These results indicate that chl-mAb3 and chl-mAb6 recognize distinct epitopes.
The cyt-mAb1 for cAPX isoenzyme reacted preferentially with recombinant and native cAPX proteins from spinach (Fig. IV-1). Although the amino acid sequence of cAPX contained highly identical regions with that of mAPX (64.2% identity) (Chapter II, Ishikawa et al. 1998), the cyt-mAb1 did not react with the recombinant mAPX protein, suggesting that the epitope of cyt-mAb1 should be located within an inherent region of cAPX itself. The cyt-mAb1 cross-reacted with the native cAPX proteins in crude extracts prepared from not only spinach but also tobacco and Mesembryanthemum crystallium (Fig. IV-1). Incubation of the purified recombinant APX isoenzymes with their respective mAbs had little effect on the activities,
suggesting that all the antibodies are not directed toward the active site of APX proteins (data not shown).
chl-mAb6 tAPX (38 kDa)
sAPX (33 kDa)
cAPX (28 kDa)
Fig. IV-1 Immunoblot analysis of recombinant and native APX proteins with chl-mAb3, chl-mAb6, and cyt-mAb1. Cell extracts of E. co li that are expressed recombinant APX isoenzymes and extracts from plant leaves were subjected to SDS-polyacrylamide gel electrophoresis (12.5% gel) and analyzed by immunoblot analysis with the respective mAbs as detailed in the section titled Materials and Methods. Arrows indicate protein bands of tAPX, sAPX, and cAPX. Lane 1, recombinant sAPX; lane 2, recombinant tAPX; lane 3, recombinant cAPX; lane 4, recombinant mAPX; lane 5, spinach leaf extract; lane 6, tobacco leaf extract; lane 7, Mesembryanthemum crystallium leaf extract.
Immunoaffinity purification of recombinant APX isoenzyme proteins
Immunoaffinity columns were used to obtain large quantities of purified recombinant proteins of sAPX and cAPX isoenzymes in a single step. The columns were constructed by coupling the respective mAb (chl-mAb3, chl-mAb6, or cyt-mAb1) to the NHS-activated Sepharose as described in the Materials and Methods section. Fig.
IV-2 showed the purification of the recombinant APX proteins from the E. coli crude
extracts by each immunoaffinity column. All of the columns immobilizing chl-mAb3, chl-mAb6, and cyt-mAb1 were extremely effective for the purification of recombinant sAPX or cAPX. The densitometric analysis of silver-stained gels estimated that the yields of the purified recombinant sAPX protein using chl-mAb3 and chl-mAb6 columns were 34 and 12%, respectively (Fig. IV-2). The recombinant cAPX corresponding to the 28-kDa band was purified with the yields of 28% from the cyt-mAb1 column (Fig. IV-2). These facts indicated that three mAbs efficiently interacted with the respective undenatured form of the proteins. N-terminal amino acid sequences of the purified recombinant isoenzymes were in completeagreement with the deduced amino acid sequences from the respective cDNA sequences (data not shown).
One of the characteristic properties of APX isoenzymes is that they are very labile in an AsA-depleted medium (Asada 1997). Therefore, AsA was added to all of the solutions during the immunoaffinity purification. However, no enzyme activity of any APX isoenzyme could be detected in the purified enzyme solution eluted from the respective immunoaffinity columns because proteins adhering to the columns were eluted with the acidic glycine solution (pH 3.0). Although the purified APX isoenzymes were not in active forms, I obtained satisfactory quantities of each isoenzyme protein for the next analysis.
30
14.4 94 67 43 kDa
sAPX (33 kDa)
1 2 3 4 5 6 7
30
14.4 94 67 43 kDa
cAPX (28 kD a)
Fig. IV-2 Immunoaffinity purification of recombinant APX isoenzyme proteins. Cell extracts of E. coli having sAPX and cAPX genes and immunoaffinity-purified enzymes were subjected to SDS-polyacrylamide gel electrophoresis (12.5% gel). Protein bands were stained using the silver-stain method. Arrows indicate protein bands of sAPX and cAPX. Lanes 1 and 5, molecular mass standards; lane 2, cell extract from E. coli expressed recombinant sAPX; lane 3, purified sAPX by the chl-mAb6 column; lane 4, purified sAPX by the chl-mAb3 column; lane 6, cell extract from E. coli expressed recombinant cAPX; lane 7, purified cAPX by the cyt-mAb1 column.
Detection of proteolytic APX fragments using immunoaffinity columns
As mentioned above, the immunoblot analysis of APX isoenzymes showed significant differences in epitope specificity of mAbs chl-mAb3, chl-mAb6, and cyt-mAb1. We have reported that two mAbs (EAP1 and EAP2) raised against Euglena cAPX recognized cAPX, chlAPXs, and mAPX proteins from higher plants as well as Euglena cAPX protein (Ishikawa et al. 1996b, 1998). The results indicated that the mAbs recognize a common epitope among APX isoenzymes of plant and Euglena. To identify more precisely differences in recognition among these antibodies including Euglena mAbs, I determined proteolytic APX fragments responsible for each mAb.
The purified recombinant sAPX and cAPX isoenzymes were digested with endopeptidases and the digests were applied to the respective immunoaffinity columns.
Each peptide adhered to the immunoaffinity columns was eluted by the glycine-HCl buffer, pH 3.0, and the amino acid sequences were then analyzed. Fig. IV-3 shows the alignment of deduced amino acid sequences of spinach APX isoenzymes and the locations of peptide fragments adhered to the respective immunoaffinity columns. As anticipated, the results constitute strong evidence that all mAbs recognize different epitopes (Fig. IV-3). In the experiment using an Endoproteinase Asp-N-cleaved sAPX, the chl-mAb6 column could bind to a single peptide fragment consisting of six residues, DIKEKR, indicating that the epitope recognized by the chl-mAb6 is located in an inherent region of chlAPX isoenzymes. In contrast to chl-mAb6, no fragment was found to elute from the chl-mAb3 column. Although I tried to use different types of protease (lysyl endopeptidase, Staphylococcus aureus V8 protease, Arginine endopeptidase, trypsin, or chymotrypsin) for cleavage of sAPX protein, no fragment could adhere to the chl-mAb3 column. These results suggest that the chl-mAb3 recognizes some specific conformation of sAPX, which is destroyed by proteolytic cleavage, whereas the chl-mAb6 recognizes a shorter unique sequence.
In the lysyl endopeptidase-cleavage of cAPX, the epitope for cyt-mAb1 was determined to be located in the sequence of 22 residues, QCAPLMLRLAWHSAGTF DCTSK, which contains the distal His residue of a common heam-binding site among APX isoenzymes (Asada 1997). It should be noted that the peptide sequence upstream of the distal His residue (CAPLMLRLAWH) is identical to that of the mAPX isoenzyme. Since the cyt-mAb1 did not recognize any APX isoenzymes except for the cAPX (Fig. IV-1), the cyt-mAb1-defined epitope would be located within the peptide of 10 residues, SAGTFDCTSK, consistent with the downstream of the distal His residue.
The pea cAPX has been modeled as a tertiary structure based on X-ray crystal structures (Patterson and Poulos 1994, Patterson et al. 1995). The predicted tertiary structure of spinach cAPX indicated that the downstream peptide region of the distal His residue constructs -helix and locates on the surface of the cAPX protein (data not shown). This
should support the fact that, in the immunoaffinity purification, cyt-mAb1 efficiently interacts with undenatured recombinant cAPX (Fig. IV-2).
Consistent with the immunoblot analysis of our previous report (Ishikawa et al.
1996b), immunoaffinity column immobilizing mAb EAP1 was found to adhere to the proteolytic fragments of both cAPX and sAPX. The amino acid sequences of the peptide fragments from cAPX and sAPX were QMGLTDQDIVALSGGH and DIVALSGAHTLG,respectively, indicating that the EAP1 bound to the site on the
sAPX 1:MASFTTTTAAAASRLLPSSSSSISRLSLSSSSSSSSSLKCLRSSPLVSHLFLRQRGGSAYVTKTRF tAPX 1:MASFTTTTAAAASRLLPSSSSSISRLSLSSSSSSSSSLKCLRSSPLVSHLFLRQRGGSAYVTKTRF cAPX : mAPX :
sAPX 67:STKC---YASDPAQLKNAREDI--KELLQSKFCHPIMVRLGWHDAGTYNKDIKEWPQRGGA tAPX 67:STKC---YASDPAQLKNAREDI--KELLQSKFCHPIMVRLGWHDAGTYNKDIKEWPQRGGA cAPX 1: MGKSYPTVSENYQKSIEKARRKL--RGLIAEKQCAPLMLRLAWHSAGTFDCTSKT----GGP cyt-mAb1
mAPX 1: MAM--PVVNTEYLKEIDKARRDL--RALISNRNCAPLMLRLAWHDAGTYCAKTKT----GGP
sAPX 123:NGSLSFDVELKHGANAGLVNALKLLQPIKDKYSGVTYADLFQLASATAIEEAGGPTIPMKYGRVDA tAPX 123:NGSLSFDVELKHGANAGLVNALKLLQPIKDKYSGVTYADLFQLASATAIEEAGGPTIPMKYGRVDA cAPX 57:FGTMKHQAELAHGANNGLVIAVRLLEPIKEQFPEITYADFYQLAEFVAVEVTGGPEVPFHPGREDK EAP2
mAPX 55:NASIRNEEECAHGANNGLKKAIDWCEEVKSKHPKITYADLYQLAGVVAVEVTGGPTVDFVPGRKDS
sAPX 189:TGPEQCPEEGRLPDAGPPSPAQHLRDVF-YRMGLDDKDIVALSGAHTLGRSRPERSGWGKPETKYT EAP1
tAPX 189:TGPEQCPEEGRLPDAGPPSPAQHLRDVF-YRMGLDDKDIVALSGAHTLGRSRPERSGWGKPETKYT cAPX 133:PEP---PQEGRLPDATKG--CDHLRDVFIKQMGLTDQDIVALSGGHTLGRCHKDRSGFEGA--- EAP1
mAPX 121:NAC---PKEGRLPDAKQG--APHLRDIF-YRMGLTDKDIVALSGGHTLGRAHPERSGFDGP---
sAPX 254:KDGPGAPGGQSWTAEWLKFDNSYFKDIKEKRDADLLVLPTDAALFEDPSFKVYAEKYAADQEAFFK chl-mAb6
tAPX 254:KDGPGAPGGQSWTAEWLKFDNSYFKDIKEKRDADLLVLPTDAALFEDPSFKVYAEKYAADQEAFFK cAPX 189:---WTTNPLVFDNTYFKELLSGEKEGLLQLPSDKALLSDPVFRPLVEKYAADEDAFFA mAPX 176:---WTQEPLKFDNSYFVELLKGESEGLLQLPTDKTLVEDPAFRPFVELYAKDEDVFFR
sAPX 320:DYAEAHAKLSNQGAKFDPAEGITLNGTPAGAAPEKFVAAKYSSNKD
tAPX 320:DYAEAHAKLSNQGAKFDPAEGITLNGTPAGAAPEKFVAAKYSSNKRSELSDSMKEKIRAEYEGFGG cAPX 244:DYAEAHLKLSELGFADA
mAPX 230:DYAVSHKKLSELGFTPSGGKSSCQSAVGVLVTAAVVICSYIYEVRKRSK
tAPX 385:SPRNKPLPTNYFLNIMIVIGVLAVLSYLAGN
Fig. IV-3 Location of the proteolytic APX fragments responsible for the respective mAbs. The sequence alignment is shown for the stromal APX (sAPX), the thylakoid-bound APX (tAPX), the cytosolic APX (cAPX), and the microbody-bound APX (mAPX). The gaps are introduced to optimize the alignment. Amino acid numbers are shown on the left side of the figure. The consensus amino acids are shaded. The proteolytic APX fragments bound to the immunoaffinity columns immobilizing chl-mAb6 or cyt-mAb1 are shown by double lines and indicated by the name of each mAb. The proteolytic APX fragments bound to EAP1 and EAP2 immunoaffinity columns (Ishikawa et al. 1996b) are shown by lines.
proximal His, another common heam-binding site among APX isoenzymes (Fig. IV-3).
Since EAP1 also cross-reacts with the mAPX isoenzyme (Chapter II, Ishikawa et al.
1998), the EAP1-defined epitope would be located within the common peptide of nine residues, DIVALSGG(A)H. Unlike EAP1, EAP2 showed higher cross-reactivity with cAPX than with sAPX (Ishikawa et al. 1996b). The location of the epitope to the EAP2 on the cAPX isoenzyme was determined in the same manner as those of other antibodies.
The amino acid sequence of the fragment obtained from the cAPX was HQAELAHGANNGLVIAVRLLEPIK (Fig. IV-3). The results showed that the EAP2 recognized a relatively homologous region downstream of the distal His residue in the APX isoenzymes.
In conclusion, I have developed a panel of mAbs that recognize all plant APX isoenzymes. There are a lot of studies about mRNA expression levels of APX isoenzymes and the enzyme activities in response to oxidative stresses (Mittler and Zilinskas 1994, Conklin and Last 1995, Vansuyt et al. 1997, Creissen et al. 1999, Ye and Gressel 2000). In addition to these experiments, I expect that these mAbs will become a powerful tool to analyze the regulation of each APX protein. Actually, I have recently used these antibodies to analyze the changes in protein levels separately of spinach APX isoenzymes during high light stress (Yoshimura et al. 2000). Moreover, the mAbs should greatly facilitate future studies on the distribution and structure of APX isoenzyme proteins.
Summary
I have developed three monoclonal antibodies against spinach chloroplastic (chl-mAb3 and chl-mAb6) and cytosolic (cyt-mAb1) ascorbate peroxidase (APX) isoenzymes to analyze the cross-reactivity and the structure of the epitopes for each monoclonal antibody. All three antibodies reacted specifically with their respective isoenzymes, but none cross-reacted with the others. Immunoreactive fragments in proteolytic recombinant APX isoenzymes were detected by means of the adsorption on the corresponding immunoaffinity column. The cyt-mAb1 reacted with a peptide fragment containing the distal His region obtained by the lysylendopeptidase digestion.
The chl-mAb6 was capable of binding to the fragment, D-I-K-E-K-R, which is consistent with an inherent region of chloroplastic isoenzymes. No fragments reacting to the chl-mAb3 could be found in this study, suggesting that the chl-mAb3 recognizes a conformationally constituted epitope of the chloroplastic APX molecule, which may be destroyed by the enzymatic cleavage. I concluded that the peptides identified as epitopes are characteristic evidence of monoclonal antibodies.
CHAPTER V
Expression mechanism of Chloroplastic Ascorbate Peroxidase Isoenzymes in Spinach
Chloroplasts of higher plants develop two APX isoenzymes which exist as stromal soluble (sAPX) and thylakoid-bound (tAPX) forms (Asada 1997, Chen and Asada 1989, Miyake and Asada 1992). Miyake and Asada (1995) have reported that the enzymological properties of tAPX are very similar to those of sAPX with respect to high specificity for AsA, Km values for H2O2 and AsA, inhibition by cyanide, thiol-modifying reagents, thiols and suicide inhibitors, such as hydroxyurea, and inactivation in AsA-depleted medium. The sole difference in their properties between tAPX and sAPX is the higher molecular weight of the membrane-bound enzyme compared to the soluble enzyme. Thus, tAPX seems to be bound to thylakoid membranes in such a form that the active site of the enzyme is exposed to the stroma for the access of the substrate. In the previous study, the first complete cloning and molecular characterizations of sAPX and tAPX from spinach have shown that the nucleotide sequence encoding the tAPX isoenzyme is identical to that of sAPX through the coding region up to amino acid position 364, where the remainder of the C-terminal coding region is substituted by a different sequence that encodes 50 amino acids which constructs a hydrophobic thylakoid membrane binding domain (Fig.
V-1) (Ishikawa et al. 1996c). These data and the presence of different polyadenylation tracts in cDNAs encoding the sAPX and tAPX variants suggest that both enzymes are generated by a common pre-mRNA from an identical gene by alternative splicing of the 3'-terminal exons. The cDNAs encoding cAPX isoenzymes have been isolated and characterized from several plant sources including spinach (Ishikawa et al. 1995, Mittler and Zilinskas 1992, Kubo et al. 1992). The gene from pea (Mittler and Zilinskas 1992) and Arabidopsis (Kubo et al. 1992) has been isolated and characterized. Accordingly, it seems likely that the APX of spinach is a multigene family and there are at least four APX genes. In order to elucidate the occurrence of an identical nuclear gene by alternative splicing of the 3'-terminal exons and to understand the gene regulation of chlAPX isoenzymes in higher plants, I sought to clone and characterize the nuclear gene (ApxII) from spinach.