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(Journal of Leukocyte Biology. 2001;69:963-968.)
© 2001 by Society for Leukocyte Biology

Involvement of cytosolic prolyl endopeptidase in degradation of p40-phox splice variant protein in myeloid cells

Takeshi Hasebe*, Jian Hua*, Akimasa Someya*, Philippe Morain{dagger}, Frédéric Checler{ddagger} and Isao Nagaoka*

* Department of Biochemistry, Juntendo University, School of Medicine, Bunkyo-ku, Tokyo 113-8421, Japan; and
{dagger} Division D of Medical Chemistry, Institute de Recherche Servier, 92150 Suresnes, and
{ddagger} IPMC du CNRS, UPR411, 06560 Valbonne, France

Correspondence: Isao Nagaoka, Department of Biochemistry, Juntendo University, School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan. E-mail: nagaokai{at}med.juntendo.ac.jp


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ABSTRACT
 
Our previous studies indicated that an alternatively spliced variant mRNA of p40-phox, a cytosolic component of NADPH oxidase, is expressed but its protein is hardly detected in myeloid cells such as promyelocytic HL-60 cells and neutrophils. Here, we have examined the stability of p40-phox variant protein in undifferentiated HL-60 cells. When in vitro-translated proteins were incubated with subcellular fractions of HL-60 cells, p40-phox variant protein but not native p40-phox was degraded by the cytosol and granule fractions. The degradation of variant protein by the granule fraction was observed using sonicated but not intact granules, suggesting that the variant protein is unlikely to be degraded by the granules in intact cells. To identify the enzyme(s) involved, we examined the effects of various enzyme inhibitors on the degradation of variant protein by the cytosol fraction. Degradation was completely inhibited by proline-specific serine protease (prolyl endopeptidase) inhibitors but not by proteasome, calpain, and metalloprotease inhibitors. Furthermore, the variant protein was degraded by a purified prolyl endopeptidase, and the degradation was protected by treating HL-60 cells with a cell-permeable inhibitor (S17092-1) for prolyl endopeptidase. These observations suggest that a cytosolic prolyl endopeptidase is involved in the degradation of p40-phox variant protein in myeloid cells.

Key Words: NADPH oxidase • alternative splicing • proteolysis • HL-60


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INTRODUCTION
 
The NADPH oxidase is a multicomponent enzymatic system responsible for the production of microbicidal superoxide anion by phagocytes [1 ]. It consists of the membrane-bound cytochrome b558 and cytosolic factors (p47-phox, p67-phox, p40-phox, and small guanosine triphosphate-binding proteins Rac-1/2). In resting cells, these components are distributed in the membrane and cytosol, and the enzyme is dormant. On activation, the cytosolic components associate with cytochrome b558 and form the catalytically active reduced nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase complex.

p40-phox has been identified by our group [2 ] and other investigators [3 , 4 ], and its role in phagocytic NADPH oxidase activation has been examined. The involvement of p40-phox in the activation of NADPH oxidase has been suggested, because the synthetic peptide corresponding to the C-terminal amino acid sequence of p40-phox and an antibody to the C terminus of p40-phox inhibit the translocation of cytosolic factors and NADPH oxidase activation [5 , 6 ]. Furthermore, p40-phox is phosphorylated and translocated to the plasma membrane during neutrophil activation [7 8 9 ]. In addition, it has been revealed that p40-phox increases the affinity of p47-phox for cytochrome b558 [10 ]. In contrast, overexpressed p40-phox has been reported to down-regulate NADPH oxidase activity [11 , 12 ]. These observations suggest that p40-phox has a role in modulating the activity of NADPH oxidase.

The human p40-phox gene is a single copy in chromosomal location 22q13.1, and spans approximately 18 kb with 10 exons [13 ]. Recently, we have found that two mRNA species are produced in myeloid cells after transcription [14 ]. One is identical to a known p40-phox mRNA that encodes a protein of 339 residues. Another mRNA contains an additional 245-bp intron 8 sequence in the open reading frame and encodes a protein of 348 residues. N-terminal 253-amino-acid residues are identical between p40-phox and the splice variant protein, whereas C-terminal 254- to 348-amino-acid residues of the variant protein share low homology with p40-phox. It is interesting that Western blot analysis has revealed that the variant protein is hardly expressed in myeloid cells [14 ], although the splice variant mRNA is abundantly expressed in the cells. In this study, we have examined the stability of p40-phox variant protein in myeloid cells and the enzyme(s) involved in the degradation.


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MATERIALS AND METHODS
 
Reagents
Diisopropyl fluorophosphate (DFP) and phenylmethylsulfonyl fluoride (PMSF) were purchased from Wako Pure Chemicals, Osaka, Japan. Carbobenzoxyl-L-leucyl-L-leucyl-L-leucinal (MG-132) and N-acetyl-leucyl-L-leucyl-L-norleucinal (ALLN) were purchased from Calbiochem-Nova Biochem Co., San Diego, CA. Succinyl-glycyl-L-proline 4-methylcoumaryl-7-amide (suc-Gly-Pro-MCA) and carbobenzoxyl-glycyl-L-proline (Z-Gly-Pro) were purchased from Peptide Institute Inc., Osaka, Japan. Soybean trypsin inhibitor was purchased from Life Technologies, Rockville, MD. S17092-1 was synthesized as described previously [15 , 16 ]. Actinomycin D, dimethyl sulfoxide (DMSO), and 4ß-phorbol 12-myristate 13-acetate (PMA) were obtained from Sigma Chemical Co., St. Louis, MO.

Cell cultures
Human promyelocytic leukemia HL-60 cells were obtained from the American Type Culture Collection (CCL-240; Manassas, VA). The cells were maintained in RPMI-1640 medium (Nisui Pharmaceutical Co. Ltd., Tokyo, Japan) supplemented with 10% fetal bovine serum, 100 U/mL of penicillin, and 0.1 mg/mL of streptomycin [17 ]. Cell viability was determined using the trypan blue exclusion technique and was >95%.

Preparation of subcellular fractions
Subcellular fractionation of undifferentiated HL-60 cells was carried out as described previously [18 , 19 ]. Briefly, cells were suspended at a concentration of 5 x 107 cells/mL in ice-cold relaxation buffer [100 mM KCl, 3 mM NaCl, 1 mM ATP, 3.5 mM MgCl2, 10 mM piperazine-N,N'-bis(2-ethanesulfonic acid), pH 7.2]. Then, cells were disrupted by nitrogen cavitation [350 lb/in2 (2,411.5 kPa), 20 min; Parr Instrument Co., Moline, IL), and homogenized in a Teflon-glass homogenizer at 0°C. The homogenate was centrifuged at 420 g for 10 min, and the sedimented fraction was termed the nuclear fraction. The supernatant was further centrifuged at 8,200 g for 15 min, and the resultant pellet was termed the granule fraction. The supernatant was centrifuged at 105,000 g for 2 h, and the resulting sediment was termed the membrane fraction. The remainder constituting the supernatant was termed the cytosol fraction. After washing once with relaxation buffer, all pellets were suspended in the same buffer containing 0.25 M sucrose at a concentration of 5 x 107 cell equivalents/mL.

Evaluation of fraction markers
The activities of ß-glucuronidase, lactate dehydrogenase (LDH), myeloperoxidase (MPO), and prolyl endopeptidase were determined as described earlier [20 21 22 23 ]. In brief, ß-glucuronidase activity was measured by quantitating the release of phenolphthalein from phenolphthalein glucuronidate in the presence of 0.1% Triton X-100. LDH was assayed by measuring the oxidation of nicotinamide adenine dinucleotide (reduced form) at 340 nm on conversion of pyruvate to lactate. MPO activity was determined by measuring the color reaction of o-tolidine at 440 nm. Prolyl endopeptidase activity was determined by measuring the release of 7-amino-4-methyl-coumarin from the fluorogenic substrate suc-Gly-Pro-MCA. One unit of activity was defined as the amount of enzyme activity necessary to split 1 µmol of substrate in 1 min under the conditions used. DNA content was measured using the diphenylamine reaction [24 ]. In, brief, subcellular fractions were hydrolyzed in 0.25 N perchloric acid at 70°C for 15 min. After centrifugation at 10,000 g for 5 min, the supernatant was mixed with 2 v of diphenylamine reagent (1.5% diphenylamine, 1.5% sulfuric acid, 0.08 mg/mL of acetaldehyde in glacial acetic acid) and heated at 37°C for 16 h, and the resulting color reaction was measured spectrophotometrically at 600 nm.

In vitro translation of p40-phox and variant protein
p40-phox and variant RNAs were synthesized in vitro by transcription of p40-phox and variant complementary DNAs (cDNAs) subcloned into pBluescript SK(-) plasmid using T7 RNA polymerase (Stratagene, La Jolla, CA) [14 ]. RNAs (500 ng) were translated in vitro with rabbit reticulocyte lysate (Novagen Inc., Madison, WI) at 30°C for 1 h, and then RNA templates were digested with 10 µg/mL of RNase A at room temperature for 5 min.

Degradation of p40-phox variant protein by subcellular fractions of HL-60 cells
To examine the stability of p40-phox and variant protein, the in vitro-translated proteins were incubated with each of subcellular fractions from undifferentiated HL-60 cells at 30°C for 0.5–10 h. The reaction mixture consisted of 5 µL of in vitro-translated protein and each subcellular fraction (2 x 104 cell equivalents) in a total volume of 50 µL of relaxation buffer containing 0.25 M sucrose. After incubation, the mixture was added to an equal volume of sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) sample buffer (6.25 mM Tris-HCl, pH 6.8, 2% SDS, 5% 2-mercaptoethanol, 0.02% bromophenol blue, 10% glycerol). Aliquots (4 x 103 cell equivalents) were subjected to 12% SDS-PAGE [25 ] and transferred to the polyvinylidene fluoride membrane (Millipore Corp., Bedford, MA) [26 ]. The membrane was blocked in Block Ace (Dainippon Pharmaceutical Co. Ltd., Tokyo, Japan) and probed with a 1:4,000 dilution of rabbit antisera against p40-phox N-terminal peptide [6 ], p40-phox C-terminal peptide [6 ], or p40-phox variant C-terminal peptide [14 ]. Anti-p40-phox N-terminal peptide antiserum can recognize both p40-phox and the variant protein, whereas anti-p40-phox C-terminal peptide antiserum and anti-p40-phox variant C-terminal peptide antiserum can specifically recognize p40-phox and the variant protein, respectively. The membrane was further probed with a 1:5,000 dilution of horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G (ICN Pharmaceuticals Inc., Irvine, CA), and the proteins were finally detected with Supersignal® West Pico Chemiluminescent Substrate (Pierce Chemical Co., Rockford, IL). The detected bands were quantified using a scanning densitometer (MasterScan System; Scanalytics, Inc., Fairfax, VA).

Antisera against p40-phox N-terminal peptide [6 ], p40-phox C-terminal peptide antiserum [6 ], and p40-phox splice variant C-terminal peptide [14 ] were raised in rabbits by injection of keyhole limpet hemocyanin-conjugated synthetic peptides corresponding to M1AVAQQLRAESDFEQ15, H325ITQKDNYRVYNTMP339, and T336SRWRKISAA345, respectively.

Effects of protease inhibitors on the degradation of p40-phox variant protein
To identify the enzyme(s) involved in the degradation of the variant protein, the in vitro-translated protein was incubated with cytosol or granule fractions (2 x 104 cell equivalents) of undifferentiated HL-60 cells for 10 h in the presence of protease inhibitors: 1 mM DFP; 1 mM PMSF [27 ]; 5 µM ALLN [28 ]; 50 µM MG-132 [29 ]; 2 mM ethyelenediaminetetraacetic acid (EDTA); 2 mM ethyleneglycoltetraacetic acid (EGTA); 1 mg/mL soybean trypsin inhibitor [27 ]; 3 mM Z-Gly-Pro [18 ]; and 1 µM S17092-1 [16 ]. The concentrations of inhibitors used in this study were determined based on the previous reports [16 , 18 , 27 28 29 ]. Moreover, to confirm whether the prolyl endopeptidase is involved in the degradation of the variant protein, this protein was incubated with 0.4 mU/mL of prolyl endopeptidase purified from Flavobacterium meningosepticum (Seikagaku Co., Tokyo, Japan) at 30°C for 10 h [30 ].

In addition, to examine the involvement of prolyl endopeptidase in the degradation of the variant protein by intact cells, undifferentiated HL-60 cells were treated with 6.25 µM S17092-1, a cell-permeable prolyl endopeptidase inhibitor, for 3 days [16 ]. Then cells were treated with 5 mM DFP, and disrupted in ice by sonication (Tomy Ultrasonic Disruptor UD-201, Tominaga Works, Ltd., Tokyo, Japan). The sonicates were subjected to SDS-PAGE and Western blotting.

Furthermore, we examined that the effects of S17092-1 on the superoxide-producing activity of differentiated HL-60 cells. In brief, HL-60 cells were incubated with 1.3% DMSO in the presence or absence of 6.25 µM S17092-1 for 5 days, and superoxide-producing activity was evaluated by superoxide dismutase-inhibitable cytochrome c reduction assay after PMA stimulation [31 ]; the assay mixtures consisted of 1 x 106 cells/mL, 60 µM cytochrome c, 1 mM CaCl2, 1 mM MgCl2, and 1 µg/mL of PMA with or without 20 µg/mL of superoxide dismutase in a total volume of 200 µL of phosphate-buffered saline (137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4, 1.5 mM KH2PO4, pH 7.4). After stimulation at 37°C for 5 min, the mixtures were centrifuged at 800 g for 5 min. Cytochrome c reduction was calculated by the absorbance difference at 540–550 nm using an absorption coefficient of 21,000 M-1cm-1. In addition, DMSO-differentiated HL-60 cells with or without S17092-1-treatment were subjected to SDS-PAGE and Western blotting, and the expression of p40-phox variant protein and other NADPH oxidase components was evaluated using rabbit anti-gp91-phox, anti-p22-phox, anti-p40-phox C-terminal peptide; p40-phox variant C-terminal peptide or Rac-2 antibody, and mouse anti-p67-phox or anti-p47-phox antibody, as described previously [31 ].

In some experiments, to examine the stability of p40-phox and its splice variant mRNAs, undifferentiated HL-60 cells were treated with 5 µg/mL of actinomycin D, an RNA synthesis inhibitor, at 37°C for 2 and 4 h [32 ]. Total cellular RNA was isolated by an acid guanidinium thiocyanate-phenol-chloroform extraction method [33 ], and the amounts of p40-phox mRNA (1.3 kb) and the splice variant mRNA (1.5 kb) were determined by Northern blot analysis using a digoxigenin-labeled 539-bp p40-phox cDNA probe [14 ]. The detected bands were quantified using a scanning densitometer (MasterScan System).


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RESULTS
 
Comparison of the stability of p40-phox and its variant protein in HL-60 cell lysate
Our previous studies indicated that an alternative splice variant mRNA of p40-phox was expressed but its protein was hardly detected in HL-60 cells [14 ]. So, to compare the stability of p40-phox and its variant protein, the in vitro-translated proteins were incubated with HL-60 cell lysate. As shown in Figure 1 , p40-phox was hardly degraded by incubation with the lysate, whereas the variant protein was degraded in a time-dependent manner. These results suggested that the variant protein is unstable in myeloid cells.



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  		Figure 1. Degradation of p40-phox and its variant protein by HL-60 cell lysates. The in vitro-translated p40-phox and its variant protein were incubated with sonicates of HL-60 cells (2 x 104 cell equivalents) for 2.5 or 5 h, and aliquots (4 x 103 cell equivalents) were subjected to SDS-12% PAGE. Both proteins were detected using anti-p40-phox N-terminal peptide antiserum (A). Protein bands detected were quantified using the MasterScan system and expressed as relative amounts of p40-phox (•) and variant protein ({circ}) (B). When aliquots (4 x 103 cell equivalents) of HL-60 cell lysates without in vitro-translated proteins were analyzed by Western blotting, neither p40-phox nor its variant protein were detected under our conditions (data not shown). Data are the means ± SD of three separate experiments.

Further to compare the stabilities of p40-phox and variant mRNAs, HL-60 cells were incubated with 5 µg/mL of actinomycin D for 2 and 4 h, and the expression of p40-phox and variant mRNAs was analyzed by Northern blotting. By actinomycin D treatment, the expression of p40-phox and variant mRNAs was decreased to 60.2 ± 10.7% and 64.5 ± 20.8%, respectively, at 2 h and 24.5 ± 16.7% and 24.8 ± 17.7%, respectively, at 4 h compared with control cells without actinomycin D treatment. These observations indicate that the stability of variant mRNA is the same as that of p40-phox mRNA, and they suggest that the low expression of the variant protein can not be explained by the difference in the stabilities between p40-phox and variant mRNAs.

Degradation of p40-phox variant protein by subcellular fractions of HL-60 cells
To examine the subcellular fraction(s) involved in the degradation, we performed the subcellular fractionation of HL-60 cells. Figure 2A shows the distribution patterns of markers in the subcellular fractions of HL-60 cells. In the cytosol fraction, most of LDH, prolyl endopeptidase, and p40-phox were recovered. MPO activity (>80%) was detected in the granule fraction, and >95% of DNA was detected in the nuclear fraction.



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  		Figure 2. Degradation of p40-phox variant protein by subcellular fractions of HL-60 cells. Distribution patterns of fraction markers in subcellular fractions of HL-60 cells are shown (A). Contents of DNA and p40-phox and activities of myeloperoxidase (MPO), lactate dehydrogenase (LDH), and prolyl endopeptidase (PEP) were determined among the nuclear (N), granule (G), membrane (M), and cytosol (C) fractions of HL-60 cells, and expressed as the percentages of the total contents or activities recovered. Data represent the means ± SD of three separate experiments. The in vitro translated variant protein was incubated without (Control) or with each of the four subcellular fractions or the lysate for 5 h, and then subjected to 12% SDS-12% PAGE (B). Western blotting was performed using an anti-p40-phox N-terminal peptide antiserum.

Next, to examine the degradation of the variant protein by subcellular fractions, the in vitro-translated variant protein was incubated with each of sonicated subcellular fractions (Fig. 2B ). After 5 h of incubation, the variant protein was markedly degraded by the granule fraction and slightly degraded by the cytosol fraction. In contrast, the variant protein was not degraded by the nuclear and membrane fractions. When the variant protein was incubated with intact granules without sonication, the degradation activity was reduced by >90%, compared with sonicated granules (data not shown). These observations likely indicate that the variant protein is mainly degraded in the cytosol but not in the granules of intact cells.

Effects of protease inhibitors on the degradation of p40-phox variant protein
To identify enzyme(s) involved in the degradation, we examined the effects of protease inhibitors on the degradation of the variant protein. The degradation by the cytosol fraction was completely inhibited by a serine protease inhibitor DFP and slightly inhibited by another serine protease inhibitor PMSF (Fig. 3A ). However, the degradation was not inhibited by other inhibitors for proteasome (MG-132), calpain (ALLN), metalloprotease (EDTA, EGTA), and trypsin (soybean trypsin inhibitor). These results suggested that the variant protein is degraded by serine protease(s) in the cytosol. In contrast, the degradation by the granule fraction was completely inhibited by DFP, PMSF, and soybean trypsin inhibitor (Fig. 3B ), suggesting that the granule enzyme(s), which can degrade the variant protein, is serine protease(s) but is different from that in the cytosol fraction.



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  		Figure 3. Effects of protease inhibitors on the degradation of p40-phox variant protein by the cytosol fraction of HL-60 cells. The in vitro-translated variant protein was incubated without (Control) or with the cytosol (A) or granule (B) fraction in the absence (without inhibitor) or presence of inhibitors (DFP, PMSF, EDTA, EGTA, MG-132, and Soybean trypsin inhibitor) at 30°C for 10 h. The variant protein was detected by SDS-PAGE and Western blotting using anti-p40-phox N-terminal peptide antiserum.

Role of prolyl endopeptidase in the degradation of p40-phox variant protein
Since the variant protein contains a proline-rich sequence in the C-terminal region [14 ], it is likely that the variant protein can be degraded by a cytosolic proline-specific serine protease, namely, prolyl endopeptidase. It is interesting that prolyl endopeptidase activity has been reported to be completely inhibited by DFP but slightly inhibited by PMSF [23 ]. So, we examined whether the degradation of the variant protein is inhibited by prolyl endopeptidase-specific inhibitors such as Z-Gly-Pro and S17092-1. The degrading activity of the cytosol fraction was completely inhibited by these inhibitors as well as DFP (Fig. 4A ). Moreover, the degradation was inhibited by 3 mM suc-Gly-Pro-MCA, a substrate for prolyl endopeptidase (data not shown). Under these conditions, prolyl endopeptidase activity was reduced by DFP, Z-Gly-Pro, and S17092-1 to 3.68 ± 0.69%, 8.10 ± 0.52%, and 0.62± 0.45% of the control activity without inhibitors, respectively. In addition, EDTA, EGTA, MG-132, ALLN, and soybean trypsin inhibitor, which could not protect the variant protein from the degradation, had no effect on the cytosolic prolyl endopeptidase activity (data not shown). These observations indicate that prolyl endopeptidase is likely involved in the degradation of the variant protein by the cytosol fraction.



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  		Figure 4. Degradation of p40-phox variant protein by prolyl endopeptidase. The in vitro-translated variant protein was incubated without (Control) or with the cytosol fraction in the absence (Without inhibitor) or presence of DFP and prolyl endopeptidase inhibitors (Z-Gly-Pro and S17092-1) at 30°C for 10 h (A). The variant protein was detected by SDS-PAGE and Western blotting using anti-p40-phox N-terminal peptide antiserum. The in vitro-translated p40-phox and variant protein were incubated without (Control) or with 0.4 mU/mL of prolyl endopeptidase purified from Flavobacterium meningosepticum for 10 h (B). p40-phox and variant protein were detected by SDS-PAGE and Western blotting using anti-p40-phox N-terminal peptide antiserum. (C) HL-60 cells were cultured without (Control) or with 6.25 µM S17092-1 for 3 days. After incubation, the cells (105 cell equivalents) were subjected to SDS-PAGE and Western blotting. p40-phox and variant protein were detected using anti p40-phox C-terminal peptide-specific antiserum and anti-p40-phox variant C-terminal peptide-specific antiserum, respectively.

Next, we examined whether the variant protein could be degraded by a purified Flavobacterium prolyl endopeptidase. As shown in Figure 4B , the variant protein was degraded by the purified prolyl endopeptidase. In contrast, p40-phox was not degraded by the prolyl endopeptidase. These results suggest that the variant protein can be a substrate for prolyl endopeptidase.

When the variant protein was incubated with the cytosol fraction of HL-60 cells or Flavobacterium prolyl endopeptidase, three degradation products 10.5, 34.0, and 37.0 kDa in size were detected by Western blot analysis after short-time incubation (2.5–5 h) with both cytosolic prolyl endopeptidase and exogenous prolyl endopeptidase (data not shown). Prediction of prolyl endopeptidase cleavage sites suggested that the variant protein is likely digested at Pro-87 or Pro-90, Pro-304 or Pro-307, and Pro-330 or Pro-333 in the primary sequence of p40-phox variant protein, which could produce the 10.5-, 34.0-, and 37.0-kDa degradation products, respectively [14 ]. In contrast, when the variant protein was incubated with granule fraction, only one degradation product of 20.0 kDa was detected after short-time incubation (0.5–2.5 h) (data not shown). These observations further suggest that the enzymes, which can degrade the variant protein, are different between the cytosol and granule fractions.

Further to confirm whether prolyl endopeptidase is involved in the degradation of the variant protein in intact cells, undifferentiated HL-60 cells were incubated with the cell- permeable prolyl endopeptidase inhibitor S17092-1, and the expression of the variant protein was examined by Western blot analysis. The degradation of the variant protein was apparently protected by S17092-1, although the expression of p40-phox was not affected by S17092-1 (Fig. 4C ). We also confirmed that prolyl endopeptidase activity in S17092-1-treated cells was decreased to 3.32 ± 0.47% of control cells (n = 3) Taken together, these observations indicate that cytosolic prolyl endopeptidase is a candidate for protease(s) involved in the degradation of p40-phox variant protein in myeloid cells

We previously revealed that an alternatively spliced variant mRNA of p40-phox was increased, but the variant protein could not be detected in DMSO-differentiated HL-60 cells [14 ]. In preliminary experiments, we incubated HL-60 cells with 1.3% DMSO for 5 days and examined prolyl endopeptidase activity. However, we could not detect the significant difference in the prolyl endopeptidase activities between undifferentiated and differentiated HL-60 cells [40.98 ± 3.89 mU/107 undifferentiated HL-60 cells vs. 38.0 ± 1.24 mU/107 DMSO-treated HL-60 cells (mean ± SD of three separate experiments)]. These results suggest that the activity of endogenous prolyl endopeptidase is enough to degrade the variant protein in both undifferentiated and differentiated HL-60 cells.

Furthermore, we incubated HL-60 cells with 1.3% DMSO in the presence or absence of 6.25 µM S17092-1 for 5 days, and we examined superoxide-producing activity by cytochrome c reduction assay after PMA stimulation. By S17092-1-treatment, expression of the variant protein was increased, but expression of other NADPH oxidase components (gp91-phox, p22-phox, p67-phox, p47-phox, and Rac-2) was not affected (data not shown). It is interesting, however, that the superoxide-producing activities were not significantly changed by S17092-1 treatment in DMSO-differentiated HL-60 cells [32.7 ± 7.2 nmol/107 cells/min by S17092-1-treated HL-60 cells vs. 34.9 ± 3.5 nmol/107 cells/min by S17092-1-untreated HL-60 cells (mean ± SD of three separate experiments)]. These results suggest that the variant protein is unlikely to be involved in the activation of NADPH oxidase. However, under our conditions, expression of the variant protein was 10- to 20-fold lower than that of p40-phox, suggesting that the amount of the variant protein is too small to affect NADPH oxidase activation. The function of the variant protein could be evaluated in future using the variant protein-overexpressing cells.


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DISCUSSION
 
Here we have reported our study of the stability of p40-phox variant protein in myeloid cells and the enzyme(s) involved in the degradation. p40-phox variant protein was degraded by HL-60 cell lysate, and subcellular fractionation study indicated that the variant protein was degraded by the granule and the cytosol fractions. However, the enzyme(s) involved in the degradation appeared to be distinctly different between the granule and cytosol fractions, because the inhibition patterns of degrading activities of these fractions were different among the enzyme inhibitors used.

Cytosolic proteins can be degraded through a chaperone-mediated transport into the lysosomal granules [34 ]. In this system, a target signal sequence is required for the transport of cytosolic proteins, and the substrates containing Lys-Phe-Glu-Arg-Gln sequences can associate with a molecular chaperone, a 73-kDa heat shock cognate protein [35 ]. Then the complex is internalized to lysosomes by the action of a 96-kDa lysosomal integral membrane glycoprotein [36 ]. However, the prediction of protein-sorting signals and subcellular localization sites in the amino acid sequences using PSORT software (Human Genome Center, Institute of Medical Science, University of Tokyo, Japan) [37 ] has indicated that p40-phox variant protein does not contain the sorting sequence Lys-Phe-Glu-Arg-Gln for internalization and transport to the granules and that p40-phox variant protein is distributed in the cytosol and/or nucleus but not in the granules. Furthermore, p40-phox variant protein was hardly degraded by the intact granules without sonication. These observations indicate that the variant protein is degraded in the cytosol but not in the granules of intact cells.

Cytosolic and nuclear proteins could be degraded by proteasome [38 ], calpain [39 ], and other proteases in the cytosol. The proteasome-dependent degradation is regulated by the ubiquitination of the substrates, which contain the polypeptide sequences enriched in proline (P), glutamic acid (E), serine (S), and threonine (T) [40 ]. The prediction of a Pro-Glu-Ser-Thr (PEST) region [41 ] has revealed that both p40-phox and splice variant protein contain possible PEST sequences with a PESTfind score of +6.73 in the 228–241 amino acid residues, suggesting that both proteins could be degraded by proteasome. However, in vitro degradation analysis indicated that the variant protein was degraded but p40-phox was not degraded by the cytosol fraction. Thus, the degradation of variant protein is unlikely to be dependent on a proteasome-mediated pathway. Actually, the degradation of the variant protein could not be protected by a proteasome inhibitor (MG-132). Calpain is a Ca2+-dependent cysteine protease in the cytosol, which contributes to the degradation of the cytosolic proteins [39 ]. However, the degradation of the variant protein was not protected by a calpain inhibitor (ALLN) and a calcium-chelating agent (EGTA), suggesting that calpain is not involved in the degradation.

In this study, we revealed that prolyl endopeptidase inhibitor protected the degradation of a variant protein by the cytosol fraction in intact cells and that a purified prolyl endopeptidase degraded the variant protein. These observations indicate that cytosolic prolyl endopeptidase might be the protease(s) involved in the degradation of p40-phox variant protein in myeloid cells. Presumably, p40-phox variant protein, an abnormal protein, may be eliminated by prolyl endopeptidase in the cells, because the variant protein is assumed to form an aberrant random coil structure in the proline-rich C-terminal region, which has been predicted by a self-optimized method from a protein secondary structure prediction program [42 ].

Received November 17, 2000; revised January 25, 2001; accepted January 26, 2001.


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REFERENCES
 
    1
  1. Babior, B. M. (1999) NADPH oxidase: an update Blood 93,1464-1476[Free Full Text]
    2. 2
  2. Someya, A., Nagaoka, I., Yamashita, T. (1993) Purification of the 260 kDa cytosolic complex involved in the superoxide production of guinea pig neutrophils FEBS Lett 330,215-218[Medline]
    3. 3
  3. Wientjes, F. B., Hsuan, J. J., Totty, N. F., Segal, A. W. (1993) p40-phox, a third cytosolic component of the activation complex of the NADPH oxidase to contain src homology 3 domains Biochem. J. 296,557-561
    4. 4
  4. Tsunawaki, S., Mizunari, H., Nagata, M., Tatsuzawa, O., Kuratsuji, T. (1994) A novel cytosolic component, p40-phox, of respiratory burst oxidase associates with p67-phox is absent in patients with chronic granulomatous disease who lack p67-phox Biochem. Biophys. Res. Commun. 199,1378-1387[Medline]
    5. 5
  5. Someya, A., Nagaoka, I., Nunoi, H., Yamashita, T. (1996) Translocation of guinea pig p40-phox during activation of NADPH oxidase Biochim. Biophys. Acta 1277,217-225[Medline]
    6. 6
  6. Tsunawaki, S., Kagara, S., Yoshikawa, K., Yoshida, L. S., Kuratsuji, T., Namiki, H. (1996) Involvement of p40-phox in activation of phagocyte NADPH oxidase through association of its carboxy-terminal, but not its amino-terminal, with p67-phox J. Exp. Med. 184,893-902[Abstract/Free Full Text]
    7. 7
  7. Fuchs, A., Bouin, A. P., Rabilloud, T., Vignais, P. V. (1997) The 40-kDa component of the phagocyte NADPH oxidase (p40phox) is phosphorylated during activation in differentiated HL60 cells Eur. J. Biochem. 249,531-539[Medline]
    8. 8
  8. Bouin, A. P., Grandvaux, N., Vignais, P. V., Fuchs, A. (1998) p40-phox is phosphorylated on threonine 154 and serine 315 during activation of the phagocyte NADPH oxidase J. Biol. Chem. 273,30097-30103[Abstract/Free Full Text]
    9. 9
  9. Someya, A., Nunoi, H., Hasebe, T., Nagaoka, I. (1999) Phosphorylation of p40-phox during activation of neutrophil NADPH oxidase J. Leukoc. Biol. 66,851-857[Abstract]
    10. 10
  10. Cross, A. W. (2000) p40-phox participates in the activation of NADPH oxidase by increasing the affinity of p47-phox for flavocytochrome b558 Biochem. J. 349,113-117[Medline]
    11. 11
  11. Sathyamoorthy, M., de Mendez, I., Adams, A. G., Leto, T. L. (1997) p40-phox down-regulates NADPH oxidase activity through interactions with its SH3 domain J. Biol. Chem. 272,9141-9146[Abstract/Free Full Text]
    12. 12
  12. Vergnaud, S., Paclet, M. H., Benna, L. E., Pocidalo, M. A., Morel, F. (2000) Complementation of NADPH oxidase in p67-phox-defient CGD patients: p67-phox/p40-phox interaction Eur. J. Biochem. 267,1059-1067[Medline]
    13. 13
  13. Zhan, S., Vazquez, N., Zhan, S., Wientjes, F. B., Budarf, M. L., Schrock, E., Ried, T., Green, E. D., Chanock, S. J. (1996) Genomic structure, chromosomal location, start of transcription, and tissue expression of the human p40-phox, a new component of the nicotinamide adenine dinucleotide phosphate-oxidase complex Blood 88,2714-2721[Abstract/Free Full Text]
    14. 14
  14. Hasebe, T., Someya, A., Nagaoka, I. (1999) Identification of a splice variant mRNA of p40-phox, an NADPH oxidase component of phagocytes FEBS Lett 455,257-261[Medline]
    15. 15
  15. Portevin, B., Benoist, A., Remond, G., Hervé, Y., Vincent, M., Lepagnol, J., de Nanteuil, G. (1996) New prolyl endopeptidase inhibitors: in vitro and in vivo activities of azabicyclo[2.2.2]octane, azabicyclo[2.2.1]heptane, and perhydroindole derivatives J. Med. Chem. 39,2379-2391[Medline]
    16. 16
  16. Barelli, H., Petit, A., Hirsch, E., Wilk, S., De Nanteuil, G., Morain, P., Checler, F. (1999) S17092-1, a highly potent, specific and cell permeant inhibitor of human proline endopeptidase Biochem. Biophys. Res. Commun. 257,657-661[Medline]
    17. 17
  17. Collins, S. J., Gallo, R. C., Gallagher, R. E. (1977) Continuous growth and differentiation of human myeloid leukaemic cells in suspension culture Nature 270,347-349[Medline]
    18. 18
  18. Nagaoka, I., Yamashita, T. (1985) Possible involvement of aminopeptidase, an ecto-enzyme, in the inactivation of bradykinin by intact neutrophils Biochim. Biophys. Acta 847,67-76[Medline]
    19. 19
  19. Nagaoka, I., Tsutsumi-Ishii, Y., Yomogida, S., Yamashita, T. (1997) Isolation of cDNA encoding guinea pig neutrophil cationic antibacterial polypeptide of 11 kDa (CAP11) and evaluation of CAP11 mRNA expression during neutrophil maturation J. Biol. Chem. 272,22742-22750[Abstract/Free Full Text]
    20. 20
  20. Avila, J. L., Convit, J. (1973) Studies on human polymorphonuclear leukocyte enzymes. I. Assay of acid hydrolases and other enzymes Biochim. Biophys. Acta 293,397-408[Medline]
    21. 21
  21. Bergmeyer, H. U., Bernt, E. (1974) Lactate dehydrogenase Bergmeyer, H. U. eds. Methods of Enzymatic Analysis 2,574-579 Academic Press New York.
    22. 22
  22. Baggiolini, M., Hirsch, J. G., de Duve, C. (1969) Resolution of granules from rabbit heterophil leukocytes into distinct populations by zonal sedimentation J. Cell. Biol. 40,529-541[Abstract/Free Full Text]
    23. 23
  23. Kato, T., Nakano, T., Kojima, K., Nagatsu, T., Sakakibara, S. (1980) Changes in prolyl endopeptidase during maturation of rat brain and hydrolysis of substance P by the purified enzyme J. Neurochem. 35,527-535[Medline]
    24. 24
  24. Thompson, C. T., Dvorak, J. A. (1989) Quantitation of total DNA per cell in an exponentially growing population using the diphenylamine reaction and flow cytometry Anal. Biochem. 177,353-357[Medline]
    25. 25
  25. Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4 Nature 227,680-685[Medline]
    26. 26
  26. Towbin, H., Staehelin, T., Gordon, J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications Proc. Natl. Acad. Sci. USA 76,4350-4354[Abstract/Free Full Text]
    27. 27
  27. Sheterline, P., Hopkins, C. R. (1981) Transmembrane linkage between surface glycoproteins and components of the cytoplasm in neutrophil leukocytes J. Cell. Biol. 90,743-754[Abstract/Free Full Text]
    28. 28
  28. Figrueirodo-Pereira, M. E., Banik, N., Wilk, S. (1996) Comparison of the effect of calpain inhibitors on two extralysosomal proteinases: the multicatalytic proteinase complex and m-calpain J. Neurochem. 62,1989-1994[Medline]
    29. 29
  29. Tsubuki, T., Saito, Y., Tomioka, M., Ito, H., Kawashima, S. (1996) Differential inhibition of calpain and proteasome activities by peptidyl aldehydes of di-leucine and tri-leucine J. Biochem. 119,572-576[Abstract/Free Full Text]
    30. 30
  30. Yoshimoto, T., Walter, R., Tsuru, D. (1980) Proline-specific endopeptidase from Flavobacterium J. Biol. Chem. 255,4786-4792[Free Full Text]
    31. 31
  31. Hua, J., Hasebe, T., Someya, A., Nakamura, S., Sugimoto, K., Nagaoka, I. (2000) Evaluation of the expression of NADPH oxidase components during maturation of HL-60 cells to neutrophil lineage J. Leukoc. Biol. 68,216-224[Abstract/Free Full Text]
    32. 32
  32. Baer, M. R., Augustinos, P., Kinniburgh, A. J. (1992) Defective c-myc and c-myb RNA turnover in acute myeloid leukemia cells Blood 79,1319-1326[Abstract/Free Full Text]
    33. 33
  33. Chomczynski, P., Sacchi, N. (1987) Single-step method of RNA isolation by acid guanidinium thiocyate-phenol-chloroform extraction Anal. Biochem. 162,156-159[Medline]
    34. 34
  34. Dice, J. F. (1990) Peptide sequences that target cytosolic proteins for lysosomal proteolysis Trends. Biochem. Sci. 15,305-309[Medline]
    35. 35
  35. Chiang, H. L., Terlecky, S. R., Plant, C. P., Dice, J. F. (1989) A role for a 70-kilodalton heat shock protein in lysosomal degradation of intracellular proteins Science 246,382-385[Abstract/Free Full Text]
    36. 36
  36. Cuervo, A. M., Dice, J. F. (1996) A receptor for the selective uptake and degradation of proteins by lysosomes Science 273,501-503[Abstract]
    37. 37
  37. Nakai, K., Horton, P. (1999) PSORT: a program for detecting sorting signals in proteins and predicting their subcellular localization Trends Biochem. Sci. 24,34-35[Medline]
    38. 38
  38. Kornitzer, D., Ciechanover, A. (2000) Modes of regulation of ubiquitin-mediated protein degradation J. Cell. Physiol. 182,1-11[Medline]
    39. 39
  39. Carafoli, E., Molinari, M. (1998) Calpain: a protease in search of a function? Biochem. Biophys. Res. Commun. 247,193-203[Medline]
    40. 40
  40. Rogers, S., Wells, R., Rechsteiner, M. (1986) Amino acid sequences common to rapidly degraded proteins: the PEST hypothesis Science 234,364-368[Abstract/Free Full Text]
    41. 41
  41. Rechsteiner, M., Rogers, S. W. (1996) PEST sequences and regulation by proteolysis Trends. Biochem. Sci. 21,267-271[Medline]
    42. 42
  42. Geourjon, C., Deleage, G. (1994) SOPM: a self-optimized method for protein secondary structure prediction Protein Eng 7,157-164[Abstract/Free Full Text]



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