Intracellular localization of the 74- and 53-kDa forms of L-histidine decarboxylase in a rat basophilic/mast cell line, RBL-2H3.

To clarify the process of post-translational modification of L-histidine decarboxylase (HDC), we investigated the conversion of the 74-kDa form of HDC into the 53-kDa form in specialized organella of a rat basophilic/mast cell line (RBL-2H3). With treatment of streptolysin-O, RBL-2H3 cells released approximately 40% of HDC activity accompanied by over 90% of lactate dehydrogenase activity. Only the 74-kDa form of HDC was detected in the leaked fraction by SDS-polyacrylamide gel electrophoresis. The 74-kDa form in the homogenate of pulse-labeled cells was recovered in both the supernatant and particulate fractions, while the 53-kDa form was detected only in the particulate fraction containing marker proteins of microsomes, Golgi, and lysosomal granules. Confocal microscopic observation using double staining immunofluorescence with anti-GST fusion HDC antiserum showed that most of the HDC coexists with protein-disulfide isomerase, a typical marker of the luminal space of the ER. With treatment of digitonin, RBL-2H3 cells released only 74-kDa HDC. Trypsin digestion of digitonin-permeabilized cells resulted in the disappearance of the 74-kDa form but not the 53-kDa form. From these results, it is assumed that the 74-kDa form of HDC, synthesized in the cytosol, is translocated into the lumen of the ER, where it is converted to the 53-kDa form.

L-Histidine decarboxylase (HDC 1 ; EC 4.1.1.22) catalyzes decarboxylation of L-histidine to histamine. It is the only enzyme that forms histamine in mammals. Histamine is well known to act as an important physiological modulator (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12). HDCs have been purified from the soluble fraction of various tissues and found to be a dimer consisting of two identical 53-kDa subunits (13)(14)(15)(16), while the size of cDNA-deduced HDC is around 74 kDa (17)(18)(19). The recombinant 74-kDa form expressed in the particulate fraction of Sf9 cells had a low activity, while the recombinant 54-kDa form that lacks the C-terminal region of the 74-kDa form had a full activity (20,21). Furthermore, the particulate recombinant 74-kDa form was converted into soluble 53-kDa form with a high catalytic activity by porcine pancreatic elastase in vitro (22). These results suggest that the translated HDC is post-translationally processed to active mature form. Very recently, we have shown the existence of the converting enzyme-like activity in the extract of mouse tissues (23). Like the role of post-translational processing of proteins, the intracellular translocation of proteins is known. However, the intracellular localization of HDC remains to be clarified. In addition to the soluble form of HDC, membrane-associated HDC activity has been reported in various tissues, such as rat hypothalamus (24) and rat brain (25)(26)(27)(28). However, these reports did not refer to the molecular size of the membranous HDC. The aim of this study was to investigate the conversion of the 74-kDa form into the 53-kDa form in specialized organella of rat basophilic/mast (RBL-2H3) cells.
Cell Culture-RBL-2H3 cells were grown in RPMI 1640 medium supplemented with 10% fetal calf serum (complete medium) in 5% CO 2 at 37°C in a fully humidified atmosphere. Exponentially growing cells were used in all experiments.
Biosynthetic Labeling-Cells were starved for 30 min in methioninefree RPMI 1640 medium supplemented with 10% dialyzed fetal calf serum and then pulse-labeled with [ 35 S] methionine (10 Ci/ml) for designated periods. In chase experiments after pulse labeling, cells were rinsed in the complete medium once and incubated for appropriate periods.
Immunoprecipitation-[ 35 S]Methionine-labeled cells were harvested and washed in PBS twice. The cell pellet was suspended in 1 ml of RIPA buffer (30 mM HEPES-NaOH, pH 7.3, containing 150 mM sodium chloride, 1% Triton X-100, 1% deoxycholate and 0.1% SDS) and incubated on ice for 1 h. For protection against proteolytic degradation, a mixture of protease inhibitors (0.2 mM phenylmethylsulfonyl fluoride, 100 M benzamidine, 10 g/ml leupeptin, 10 g/ml aprotinin, 10 g/ml E-64, and 1 g/ml pepstatin A) was added. Thereafter, the mixture was centrifuged at 10,000 ϫ g for 10 min at 4°C. Fifty microliters of Protein A-Sepharose CL-4B (1:1 slurry) was added to the resulting supernatant, which was then incubated for 1 h at 4°C. An aliquot of the supernatant was centrifuged at 8,000 ϫ g for 5 min at 4°C. Anti-GST fusion HDC antiserum was added (1:200), and then the mixture was incubated for 1 h at 4°C. Fifty microliters of Protein A-Sepharose CL-4B was added, and the mixture was incubated for 1 h at 4°C. An aliquot of the incubation mixture was recentrifuged at 8000 ϫ g for 5 min at 4°C, and the resulting precipitate was washed five times with 1 ml of RIPA buffer. The pellet was resuspended in an equal volume of 2ϫ SDS-sample buffer (125 mM Tris-HCl, pH 6.8, containing 4% SDS, 10% 2-mercaptoethanol, 20% glycerol, and 0.1% bromphenol blue) and boiled for 15 min. The sample was subjected to SDS-PAGE according to Laemmli (30). The gel was dried and analyzed with a Fujix BAS 2000 Bio-Imaging Analyzer.
Subcellular Fractionation-Cells (1.5 ϫ 10 7 cells) were prelabeled with [ 35 S]methionine for 30 min and were harvested and homogenized in 10 mM HEPES-NaOH, pH 7.3, containing 1.5 mM MgCl 2 , 10 mM KCl, 0.5 mM dithiothreitol, 1 mM EDTA, 1 mM EGTA, and the mixture of protease inhibitors described above. The homogenate was centrifuged at 1000 ϫ g for 10 min at 4°C. The resultant supernatant was recentrifuged at 100,000 ϫ g for 1 h at 4°C, and the supernatant fraction was designated as the S1 fraction. In contrast, the precipitate fraction at 10,000 ϫ g was dissolved in buffer H (20 mM HEPES-NaOH, pH 7.3, containing 25% glycerol, 0.5 M NaCl, 1.5 mM MgCl 2 , 1 mM EDTA, 1 mM EGTA, and the mixture of protease inhibitors) and was recentrifuged at 100,000 ϫ g for 1 h at 4°C. The resultant supernatant fraction was designated as the S2 fraction, and the precipitate was designated as the P1 fraction. The fraction precipitated at 1000 ϫ g for 10 min was dissolved in buffer H and centrifuged at 10,000 ϫ g for 30 min at 4°C. The supernatant was designated as the S3 fraction, and the precipitate was named the P2 fraction. Each fraction (S1, S2, S3, P1, and P2) was separately immunoprecipitated with anti-GST fusion HDC antiserum and analyzed with SDS-PAGE as described above.
Streptolysin-O Treatment-Cells were treated with 1000 units/ml streptolysin-O (SLO) (preactivated by incubation for 15 min on ice with 10 mM dithiothreitol) in PBS at 4°C for 10 min. After this SLO-binding step, the cells were rinsed twice with PBS and then incubated at 37°C for 3 min to effect the permeabilization. In immunofluorescence experiments for selective permeabilization of plasma membrane, detergentfree buffer was used.
Percoll Density Gradient Fractionation-Percoll density gradient fractionation was performed as described (31) with a minor modification. All steps were performed at 4°C. Briefly, 5 ϫ 10 7 cells were resuspended in 1.5 ml of homogenization buffer consisting of 0.34 M sucrose, 10 mM HEPES, pH 7.3, 0.3 mM EDTA, and the mixture of protease inhibitors and homogenized by 40 strokes with a Dounce glass homogenizer (Kontes, Vineland, NJ). Remaining unbroken cells and nuclei were pelleted by centrifugation at 500 ϫ g for 10 min, and the supernatant was layered onto 6 ml of 20% Percoll containing 15 mM HEPES, pH 7.3, 0.25 M sucrose, and the mixture of protease inhibitors on top of 1 ml of saturated sucrose. Centrifugation was performed at 32,000 ϫ g for 60 min, thus creating a density gradient. Fractions were collected 1 ml from the bottom using a peristaltic pump. (Fractions were collected 0.5 ml from the bottom in Fig. 3C.) For the enzyme assay, each fraction was diluted with an equal volume of 15 mM HEPES, pH 7.3, containing 0.2% Triton X-100, incubated for 1 h at 4°C, and dialyzed against buffer K (10 mM potassium phosphate, pH 7.3, containing 0.01 mM pyridoxal 5Ј-phosphate, 0.2 mM dithiothreitol, 2% polyethylene glycol 300, 0.2 mM phenylmethylsulufonyl fluoride, and 0.1 mM benzamidine chloride) at 4°C for 15 h. For immunoprecipitation with anti-GST fusion HDC antiserum, 1 ml of deionized water and 0.5 ml of 5ϫ RIPA buffer were added to each fraction. Then the mixture was incubated for 1 h at 4°C and centrifuged at 10,000 ϫ g for 15 min at 4°C. The resulting supernatant was immunoprecipitated with anti-GST fusion HDC antiserum as described above.
Histidine Decarboxylase Assay-Histidine decarboxylase activity was assayed as described previously (13). Briefly, the assay mixture (1 ml) comprised 0.8 mol of L-histidine, 0.2 mol of dithiothreitol, 0.01 mol of pyridoxal 5Ј-phosphate, 10 mg of polyethylene glycol 300, 100 mol of potassium phosphate (pH 6.8), and enzyme. The reaction was carried out at 37°C and was terminated by adding 0.04 ml of 60% perchloric acid. Histamine formed was isolated on a column of Amberlite CG-50 and then measured by the o-phtalaldehyde method (32).
Protein Assay-The protein concentration was determined by the method of Lowry et al. (33) with bovine serum albumin as a standard.
Immunoblot Analysis-SDS-PAGE was performed on slab gels (10%). A protein sample was subjected to SDS-PAGE, and the separated proteins were transferred electrophoretically to a polyvinylidene difluoride membrane in 25 mM Tris base containing 40 mM 6-aminohexanoic acid, 0.02% SDS, and 20% methanol at room temperature for 90 min at 15 V. The membrane was rinsed in Tris-buffered saline (TBS; 20 mM Tris-HCl, pH 7.5, containing 150 mM NaCl) and then preincubated overnight in TBS containing 5% nonfat milk at 4°C. The membrane was then incubated with anti-PDI antibody (1:1000) or anti-mannosidase II antibody (1:1000) in TBS containing 5% nonfat milk for 1 h at 37°C. The membrane was washed three times with TBS containing 0.05% Tween 20 (TTBS) at room temperature. The membrane was incubated with biotinylated anti-mouse IgG antibody in TTBS for 1 h at room temperature and then stained with an ABC kit.
Immunofluorescence-Cells were grown to a low density on round cover glasses in 12-well culture dishes under standard conditions. Cells treated with or without SLO were rinsed with PBS and fixed in 100 mM sodium phosphate, pH 7.5, containing 2% paraformaldehyde, 0.1% glutaraldehyde, and 3% sucrose on ice for 30 min. Then they were permeabilized with 10 mM sodium phosphate, pH 7.5, containing 0.5 M sodium chloride, 0.1% Tween 20, and 0.1% Triton X-100 at room temperature for 10 min three times, incubated in PBS containing 0.05% Tween 20 (TPBS) with 2% normal goat serum at room temperature for 30 min, and incubated in TPBS with anti-GST fusion HDC antiserum (1:500) or anti-FP2 antibody (1:500) and with anti-PDI antibody (1:500) at 4°C overnight. Thereafter, they were once rinsed with 10 mM sodium phosphate, pH 7.5, containing 0.5 M sodium chloride and 0.1% Tween 20 for 5 min and then stained by incubation for 1 h at room temperature with fluorescein isothiocyanate-conjugated goat anti-rabbit IgG antibody (1:200) and rhodamine-conjugated goat anti-mouse IgG antibody (1:150). The stained cells were viewed with a fluorescence microscope, and original photographs were obtained with Ektachrome 400HC film.
Digitonin Permeabilization and Trypsinization-Digitonin-permeabilization and trypsinization of the cells labeled with [ 35 S]methionine were performed according to Macri and Adeli (37). Briefly, the cells labeled with [ 35 S]methionine were washed and incubated in CSK buffer (0.3 M sucrose, 0.1 M KCl, 2.5 mM MgCl 2 , 1 mM sodium-free EDTA, 10 mM PIPES, pH 6.8) with 100 g/ml digitonin for 10 min at room temperature or with 1% Triton X-100 for 30 min at 4°C. Permeabilized cells washed once in CSK buffer and the soluble fractions obtained by centrifugation at 800 ϫ g were incubated in the presence and absence of trypsin (200 g/ml) prepared in CSK buffer for 10 min at room temperature. The reaction was stopped by adding 5ϫ RIPA buffer containing the mixture of protease inhibitors (described above), and the reaction mixture was subjected to immunoprecipitation as described above.  (20), we found that the 74-kDa form of RBL-2H3 cells was largely present in the soluble fraction (Fig.  1A, lane 1). Only a little 74-kDa HDC was found in the 100,000 ϫ g particulate fraction. However, the 53-kDa form was essentially localized in the particulate fraction (lanes 3 and 5).

74-kDa HDC in Sf9 cells
The content of [ 35 S]methionine-labeled 74-kDa HDC in pulse-labeled cells was abolished within 60 min, while the 53-kDa form appeared within 5 min and gradually increased in the presence of an excess amount of cold methionine. From these results, it was inferred that the 74-kDa form of HDC is immediately interconverted to the 53-kDa form in RBL-2H3 cells (Fig. 1B).
Localization of the 74-kDa HDC in the Cytosol-The molecular form of HDC in the soluble fraction of RBL-2H3 cells was extracted by SLO treatment, which is known to permeabilize plasma membrane selectively (38). The efficiency of the permeabilization was monitored by the amount of leakage of lactate dehydrogenase. Under conditions of approximately 90% leakage of lactate dehydrogenase activity, approximately 40% of HDC activity was discharged from the permeabilized cells when the cells were treated with SLO (Table I). After immunoprecipitation, the molecular sizes of released HDC were determined by SDS-PAGE. As shown in Fig. 2 Fractionation Study of the 74-and 53-kDa Forms of HDC-To determine the localization of the 74-and 53-kDa forms of HDC, the cells were pulse-labeled for 30 min, homogenized, and then fractionated by Percoll density gradient centrifugation. The immunoprecipitated radioactive HDC in each fraction was analyzed by SDS-PAGE to determine the molecular size. As shown in Fig. 3A, the 74-kDa form was mostly recovered in fractions 7 and 8, which contained more than 90% of the lactate dehydrogenase activity (Fig. 3D), whereas the 53-kDa form was in fraction 6, where microsomal NADPHcytochrome c reductase activity and both the 55-kDa band of protein disulfide isomerase (PDI) and the 135-kDa band of mannosidase II were detected (Fig. 3D). Fig. 3B shows the result in the cells pulse-labeled over 90 min. The level of the radioactive 53-kDa form in fraction 6 was much higher than that obtained in cells pulse-labeled for 30 min (Fig. 3, A versus  B). In addition, in cells pulse-labeled for 90 min, the 53-kDa form appeared in fraction 1 (Fig. 3B), where histamine and HDC activity and ␤-hexosaminidase activity co-existed (Fig. 3C).
Identification of HDC in the Particulate Fraction of RBL-2H3 Cells-To confirm the localization of HDC in specialized organella of RBL-2H3 cells, immunofluorescence studies with anti-GST fusion HDC antiserum were performed (Fig. 4). Lane I indicates the staining of the cells fixed before the treatment with 0.1% Triton X-100. Lanes II and III indicate the stainings of cells pretreated with SLO fixed and treated with or without Triton X-100, respectively. With SLO treatment, more than 90% of the cells were found to be permeabilized, as monitored by propidium iodide (5 g/ml) staining (data not shown). Compared with the whole cell staining pattern (lane I), the cells permeabilized with SLO exhibited no fluorescent signals when treated with anti-GST fusion HDC antiserum or anti-PDI antibody (Fig. 4A, lane II). However, the reticular pattern of signal was observed with anti-FP2 antiserum (Fig. 4B, lane II). On the other hand, the cells permeabilized with SLO and then treated with Triton X-100 after fixation, exhibited reticular patterns of staining with either of the antiserum against GST fusion HDC, PDI, or FP2 (Fig. 4, A and B, lane III). These results indicate co-localization of HDC together with PDI and FP2. Furthermore, the fluorescent pattern of the signal of HDC indicates that the membrane topology of HDC is similar to that of PDI but not to that of NADPH-cytochrome P-450 reductase.
The co-localization of HDC and PDI was confirmed with confocal microscopy (Fig. 5).
A Possible Localization of 53-kDa HDC in the Luminal Compartment-The distinct signal pattern in double stainings of HDC with anti-PDI antibody and with anti-FP2 antiserum indicates the possible localization of HDC in the luminal area of the ER. To identify the molecular form of the luminal HDC, digitonin-permeabilized cells were treated with trypsin according to the method of Macri and Adeli (37). Under this condition, most of the lactate dehydrogenase activity but none of the PDI protein was observed to be released from the cells on the basis of the observation by immunoblot analysis (data not shown). In the extracellular fluid prepared from digitonin-permeabilized cells, only the 74-kDa form of HDC was detected (Fig. 6, lane 1). Upon treatment of the digitonin-permeabilized cells with trypsin, the remains of the 74-kDa form disappeared, but the 53-kDa form of HDC was still detectable (lane 4). On the other hand, the treatment of Triton X-100 resulted in the solubilization of the 74-and 53-kDa forms of HDC (lanes 5 and 7), both of which were sensitive to the trypsin digestion (lanes 6 and 8).

DISCUSSION
Besides the 53-kDa form, known as a subunit of HDC-purified from various mammalian tissues (13)(14)(15)(16), RBL-2H3 cells were found to contain the 74-kDa form, corresponding to the nascent HDC molecule. Most of the 74-kDa form in RBL-2H3 cells leaked out upon treatment of SLO, indicating that the 74-kDa form is localized primarily in the cytosol. Furthermore, [ 35 S]methionine-labeled 74-kDa HDC is a short half-life protein as shown by its rapid degradation in the pulse-chase experiment. Since the 74-kDa form lacks an amino-terminal signal sequence that is cleaved after the translocation across the ER membrane (20), it may be synthesized by free ribosomes in the cytosol as a nonsecreted protein. Regarding the degradation of the 74-kDa form of HDC, very recently we demonstrated the involvement of the proteasome system by the finding that the degradation of the 74-kDa form is inhibited by proteasome inhibitors, such as carboxybenzyl-leucyl-leucylleucinal, n-acetyl-leucyl-leucyl-norleucinal, and lactacystin (39). Therefore, the amount of the cytosolic 74-kDa form of HDC may be regulated through post-translational steps including the proteasome system. It is notable that some of the 74-kDa form was also present in the particulate fraction, which has been revealed to exist in the ER, as judged from the distribution of the 74-kDa HDC and the PDI. The membrane topology of the 74-kDa form in the particulate fraction of RBL-2H3 cells was also analyzed by the technique of trypsin digestion of the permeabilized cells. Membrane-associated 74-kDa HDC was discharged upon Triton X-100 treatment and vanished upon trypsin treatment of the digitonin-permeabilized cells (Fig. 6). From these results, it is inferred that the 74-kDa form localizes on the surface of the ER. as a granule marker was measured in each fraction. D, the distribution of marker proteins was investigated. On each fraction, lactate dehydrogenase and NADPH-cytochrome c reductase activities were measured. The amounts of PDI and mannosidase II (Man II) were evaluated, respectively, by immunoblot analysis using their specific antibodies and determined densitometrically. but significant amount of rat hypothalamic HDC activity associated with the membranous fraction, although the majority of the activity was found in the supernatant when subcellular particles were osmotically lysed. Furthermore, Martres et al. (25), Braudry et al. (26), and Toledo et al. (27) also reported membrane-bound HDC activity in rat brain. Toledo et al. (28) also reported that the membrane-bound HDC activity was solubilized by Ca 2ϩ . However, these reports did not refer to the molecular size of the membrane-bound HDC. From our results, it is deduced that membrane-associated HDC activity reported previously originated from the membrane-associated 74-kDa form.
Since the 74-kDa form has not been purified, it is unknown whether it has full activity. In our previous report, the recombinant mouse 74-kDa form exhibited a low catalytic activity compared with purified HDC, comprising a dimer of the 53-kDa subunit (20). Yatsunami et al. reported that the 54-kDa monomer form of recombinant human HDC, being expressed in the cytosol fraction, has specific activity similar to that of the purified enzyme (21). In the present experiment, we observed that 40% of HDC activity in RBL-2H3 cells was discharged by SLO treatment (Table I), and this activity is inferred to chiefly originate from the 74-kDa form of HDC (Fig. 1). These results indicate that the 74-kDa form has significant enzymatic activity.
In contrast to the present study, we previously reported that the recombinant 74-kDa form of HDC expressed in Sf9 cells was mostly recovered in the particulate fraction, while the mutant 53-kDa HDC in the expressed cells was the soluble form (20,21). Considering the solubilization of the precipitated 74-kDa HDC only on treatment with a mixture of 6 M guanidine  1, 2, 5, and 6) were incubated in the presence (lanes 2, 4, 6, and 8) and absence (lanes 1, 3, 5, and 7) of trypsin (200 g/ml) prepared in CSK buffer for 10 min at room temperature. The reaction was stopped by adding 5ϫ RIPA buffer containing the mixture of protease inhibitors, and then aliquots were subjected to immunoprecipitation with anti-GST fusion HDC antiserum as described under "Experimental Procedures." and 10 mM dithiothreitol, it is quite possible that the precipitated recombinant 74-kDa HDC in Sf9 cells is an abnormal form of the protein similar to bacterial inclusion bodies, which have been reported in a baculovirus system (40).
In addition, we showed that the 53-kDa form of HDC is essentially localized in the particulate fraction in RBL-2H3 cells. The particulate 53-kDa form is regarded to be localized in the luminal area of the ER, because the 53-kDa form was present in the fraction containing NADPH-cytochrome c reductase and PDI, and it was extremely resistant to trypsin digestion of digitonin-permeabilized cells but was sensitive to that of Triton X-100-permeabilized cells. In addition, the immunofluorescent study with anti-GST fusion HDC antiserum indicates that HDC is present in the luminal area of the ER, because there was evidence of co-localization with PDI but not with FP2 (Fig. 4). PDI is a typical marker protein exhibiting specific localization in the intraluminal space of the ER (41). On the other hand, anti-FP2 antiserum was raised against the whole protein of NADPH-cytochrome P-450 reductase, which has a short putative N-terminal membrane-anchored region with a long cytosolic tail (42). Subsequently, the fluorescent signals of HDC in the ER may be mostly derived from the luminal 53-kDa form of HDC. Therefore, it is assumed that the initial translated 74-kDa form of HDC, synthesized on the surface of free ribosomes, is associated to and translocated across the ER membrane, and thereafter in the luminal space of the ER it is post-translationally processed to yield the 53-kDa form.
The 53-kDa form of HDC was not detected in the extract of SLO- (Fig. 2) and digitonin-permeabilized RBL-2H3 cells (Fig.  6). On the other hand, purified HDC, consisting of a dimer of the 53-kDa form, has been isolated from the supernatant fraction of homogenates of various tissues and cells. It is possible that particulate HDC is discharged during the homogenization of tissues and cells. In the present experiment, a significant amount of the 53-kDa form of HDC together with ␤-hexosaminidase, a typical granule marker, was recovered in the supernatant fraction of the homogenate as shown in Fig. 3.
Histamine is stored in the granules in mast cells and basophils and is released extracellularly accompanying degranulation after stimulation (1). We observed that the 53-kDa form of HDC, originally located in the ER and Golgi, is moved to the granule fraction containing histamine, HDC activity, and ␤-hexosaminidase (Fig. 3, B and C). Subsequently, it is inferred that the 53-kDa form of HDC has a role in the synthesis of histamine in the granules.
In summary, the 74-kDa form of HDC is the cytosolic enzyme, the 53-kDa form is the particulate enzyme in RBL-2H3 cells, and both are interconverted in the luminal area of the ER.