Intracellular Localization of the 74- and 53-kDa Forms ofl-Histidine Decarboxylase in a Rat Basophilic/Mast Cell Line, RBL-2H3

Abstract

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. 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 1The abbreviations used are: HDC,l-histidine decarboxylase; PAGE, polyacrylamide gel electrophoresis; GST, glutathione S-transferase; ER, endoplasmic reticulum; PDI, protein-disulfide isomerase; SLO, streptolysin-O; PIPES, 1,4-piperazinediethanesulfonic acid; RIPA, radioimmune assay; PBS, phosphate-buffered saline; TBS, Tris-buffered saline; FP2, NADPH-cytochrome P-450 reductase. ; 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 (1Beaven M.A. Histamine: Its Role in Physiological and Pathological Processes. Karger, Basel, Switzerland1978Google Scholar, 2Code C.F. Fed. Proc. 1965; 24: 1311-1321PubMed Google Scholar, 3Schwartz J.C. Pollard H. 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Endocrinology. 1996; 137: 4435-4442Crossref PubMed Scopus (19) Google Scholar, 11Saxena S.P. Brandes L.J. Becker A.B. Simons K.J. LaBella F.S. Gerrard J.M. Science. 1989; 243: 1596-1599Crossref PubMed Scopus (102) Google Scholar, 12Liao W. Rudling M. Angelin B. Endocrinology. 1997; 138: 1863-1870Crossref PubMed Scopus (17) Google Scholar). HDCs have been purified from the soluble fraction of various tissues and found to be a dimer consisting of two identical 53-kDa subunits (13Ohmori E. Fukui T. Imanishi N. Yatsunami K. Ichikawa A. J. Biochem. (Tokyo). 1990; 107: 834-839Crossref PubMed Scopus (62) Google Scholar, 14Taguchi Y. Watanabe T. Kubota H. Hayashi H. Wada H. J. Biol. Chem. 1984; 259: 5214-5221Abstract Full Text PDF PubMed Google Scholar, 15Watabe A. Fukui T. Ohmori E. Ichikawa A. Biochem. Pharmacol. 1992; 43: 587-593Crossref PubMed Scopus (21) Google Scholar, 16Martin S.A.M. Bishop P.M. Biochem. J. 1986; 234: 349-354Crossref PubMed Scopus (38) Google Scholar), while the size of cDNA-deduced HDC is around 74 kDa (17Joseph D.R. Sullivan P.M. Wang Y.M. Kozak C. Fenstermacher D.A. Behrendsen M.E. Zahnow C.A. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 733-737Crossref PubMed Scopus (163) Google Scholar, 18Yamauchi K. Sato R. Tanno Y. Ohkawa Y. Maeyama K. Watanabe T. Satoh K. Yoshizawa M. Shibahara S. Takishima T. Nucleic Acids Res. 1990; 18: 5891Crossref PubMed Scopus (51) Google Scholar, 19Yamamoto J. Yatsunami K. Ohmori E. Sugimoto Y. Fukui T. Katayama T. Ichikawa A. FEBS Lett. 1990; 276: 214-218Crossref PubMed Scopus (93) Google Scholar). 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 (20Yamamoto J. Fukui T. Suzuki K. Tanaka S. Yatsunami K. Ichikawa A. Biochim. Biophys. Acta. 1993; 1216: 431-440Crossref PubMed Scopus (35) Google Scholar, 21Yatsunami K. Tsuchikawa M. Kamada M. Hori K. Higuchi T. J. Biol. Chem. 1995; 270: 30813-30817Crossref PubMed Scopus (35) Google Scholar). 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 (22Tanaka S. Fukui T. Yamamoto J. Shima Y. Kume T. Ohgo M. Ichikawa A. Biochim. Biophys. Acta. 1995; 1253: 9-12Crossref PubMed Scopus (22) Google Scholar). 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 (23Tanaka S. Funakoshi E. Kawahara A. Nemoto K. Fukui T. Suzuki T. Igarashi K. Ichikawa A. Methods Find. Exp. Clin. Pharmacol. 1995; 17: 25-29PubMed Google Scholar). 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 (24Snyder S.H. Brown B. Kuhar M.J. J. Neurochem. 1974; 23: 37-45Crossref PubMed Scopus (62) Google Scholar) and rat brain (25Martres M.P. Braudry M. Schwartz J.C. Brain Res. 1975; 83: 261-275Crossref PubMed Scopus (90) Google Scholar, 26Braudry M. Martres M.P. Schwartz J.C. Agents Actions. 1973; 3: 175-176Crossref PubMed Scopus (3) Google Scholar, 27Toledo A. Sabria J. Rodriguez R. Brandner R. Rodriguez J. Palacios J.M. Blanco I. J. Neurochem. 1988; 51: 1400-1406Crossref PubMed Scopus (7) Google Scholar, 28Toledo A. Rodriguez R. Sabria J. Rodriguez J. Blanco I. J. Neurochem. 1988; 56: 380-384Crossref Scopus (3) Google Scholar). 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. Anti-NADPH-cytochrome P-450 reductase (anti-FP2) antiserum was the generous gift of Dr. R. Masaki (Kansai Medical University, Osaka, Japan). Anti-GST fusion HDC antiserum was prepared as described previously (29Asahara M. Mushiake S. Shimada S. Fukui H. Kinoshita Y. Kawanami C. Watanabe T. Tanaka S. Ichikawa A. Uchiyama Y. Narushima Y. Takasawa S. Okamoto H. Tohyama M. Chiba T. Gastroenterology. 1996; 111: 45-55Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). The following materials were purchased from the sources indicated: Amberlite CG-50 (Type I, 200–400 mesh) from Rohm and Haas (Philadelphia, PA); leupeptin, aprotinin, and digitonin from Wako Pure Chemicals (Tokyo, Japan); polyvinylidene difluoride membranes from Millipore Corp. (Tokyo, Japan); fluorescein isothiocyanate-conjugated anti-rabbit goat IgG antibody and rhodamine-conjugated anti-mouse goat IgG antibody from Leinco Technologies, Inc. (St. Louis, MO); biotinylated anti-mouse IgG antibody and ABC kit from Vector Laboratories (Burlingame, CA); anti-protein-disulfide isomerase antibody from Stress Gen Biotechnologies Corp. (Victoria, Canada); anti-mannosidase II antibody from Babco (Richmond, CA); [35S]methionine (1000 Ci/mmol) from NEN Life Science Products; and streptolysin-O, Protein A-Sepharose CL-4B, and Percoll from Amersham Pharmacia Biotech. All other chemicals were commercial products of reagent grade. RBL-2H3 cells were grown in RPMI 1640 medium supplemented with 10% fetal calf serum (complete medium) in 5% CO2 at 37 °C in a fully humidified atmosphere. Exponentially growing cells were used in all experiments. Cells were starved for 30 min in methionine-free RPMI 1640 medium supplemented with 10% dialyzed fetal calf serum and then pulse-labeled with [35S] methionine (10 μCi/ml) for designated periods. In chase experiments after pulse labeling, cells were rinsed in the complete medium once and incubated for appropriate periods. [35S]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, leupeptin, aprotinin, and 1 was the mixture was at min at of Protein A-Sepharose was to the was then incubated for 1 at of the supernatant was at for min at Anti-GST fusion HDC antiserum was and then the mixture was incubated for 1 at of Protein A-Sepharose was and the mixture was incubated for 1 at of the mixture was at for min at and the was washed with 1 ml of RIPA The pellet was in an of buffer mm pH containing 10% and 0.1% and for The was to to PubMed Scopus Google Scholar). The gel was and with a Cells were with [35S]methionine for 30 min and were harvested and in mm HEPES-NaOH, pH 7.3, containing mm mm mm 1 mm 1 and the mixture of protease inhibitors described The homogenate was at for min at The supernatant was at for 1 at and the supernatant fraction was designated as the In the fraction at was in buffer mm HEPES-NaOH, pH 7.3, containing mm 1 mm 1 mm and the mixture of protease and was at for 1 at The supernatant fraction was designated as the and the was designated as the The fraction at for min was in buffer and at for 30 min at The supernatant was designated as the and the was the fraction and was with anti-GST fusion HDC antiserum and with as described Cells were with by for min on ice with mm in PBS at °C for this the cells were rinsed with PBS and then incubated at 37 °C for min to the In immunofluorescence experiments for of buffer was Percoll was as described U. A. Nilsson E. A. I. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar) with a All were at cells were in ml of buffer consisting of pH 7.3, mm and the mixture of protease inhibitors and by with a cells and were by at for and the supernatant was ml of Percoll containing mm pH 7.3, and the mixture of protease inhibitors on of 1 ml of was at for a were 1 ml from the using a were ml from the in For the enzyme fraction was with an of mm pH 7.3, containing Triton X-100, incubated for 1 at and dialyzed against buffer (10 mm pH 7.3, containing mm mm mm fluoride, and mm at °C for h. For with anti-GST fusion HDC 1 ml of and ml of RIPA buffer were to the mixture was incubated for 1 at °C and at for min at The supernatant was with anti-GST fusion HDC antiserum as described decarboxylase activity was as described previously (13Ohmori E. Fukui T. Imanishi N. Yatsunami K. Ichikawa A. J. Biochem. (Tokyo). 1990; 107: 834-839Crossref PubMed Scopus (62) Google Scholar). the mixture of of of of of and The was at 37 °C and was by ml of Histamine was on a of Amberlite CG-50 and then by the A. J. Pharmacol. Exp. Google Scholar). The was by the of J. Biol. Chem. Full Text PDF PubMed Google Scholar) with serum as a activity of lactate NADPH-cytochrome and were as described M. Methods 1994; PubMed Scopus (7) Google Scholar, T. S. J. Biochem. (Tokyo). PubMed Scopus Google Scholar, B. J. S. Biochem. J. PubMed Scopus Google Scholar). was on was to and the proteins were to a polyvinylidene difluoride in mm containing mm and at for min at The was rinsed in Tris-buffered mm pH containing 150 mm and then in containing 5% at The was then incubated with antibody anti-mannosidase II antibody in containing 5% for 1 at 37 The was washed with containing at The was incubated with biotinylated anti-mouse IgG antibody in for 1 at and then with an ABC Cells were grown to a low on in Cells with were rinsed with PBS and in mm sodium pH containing 0.1% and on ice for 30 were with mm sodium pH containing sodium chloride, 0.1% and 0.1% Triton at for min incubated in PBS containing with goat serum at for 30 and incubated in with anti-GST fusion HDC antiserum antibody and with antibody at °C were once rinsed with mm sodium pH containing sodium and 0.1% for min and then by for 1 at with fluorescein isothiocyanate-conjugated goat anti-rabbit IgG antibody and rhodamine-conjugated goat anti-mouse IgG antibody The cells were with a and were with and of the cells with [35S]methionine were to and J. K. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). the cells with [35S]methionine were washed and incubated in buffer 1 mm pH with digitonin for min at with 1% Triton for 30 min at cells washed once in buffer and the soluble by at were incubated in the and of prepared in buffer for min at The was by RIPA buffer containing the mixture of protease inhibitors and the mixture was to as described In with the expressed recombinant 74-kDa HDC in Sf9 cells (20Yamamoto J. Fukui T. Suzuki K. Tanaka S. Yatsunami K. Ichikawa A. Biochim. Biophys. Acta. 1993; 1216: 431-440Crossref PubMed Scopus (35) Google Scholar), we found that the 74-kDa form of RBL-2H3 cells was in the soluble fraction Only a 74-kDa HDC was found in the However, the 53-kDa form was in the particulate fraction and The of 74-kDa HDC in pulse-labeled cells was while the 53-kDa form min and in the of an of From these results, it was that the 74-kDa form of HDC is to the 53-kDa form in RBL-2H3 cells 1 The molecular form of HDC in the soluble fraction of RBL-2H3 cells was by is known to G. W. K.J. M. Methods Biol. 1989; PubMed Scopus (111) Google Scholar). The of the was by the of of lactate of approximately 90% of lactate dehydrogenase activity, approximately 40% of HDC activity was from the cells the cells were with the molecular of released HDC were by shown in only the 74-kDa form of HDC was detected in the of RBL-2H3 cells the and 74-kDa forms of HDC in cells after the the of 74-kDa HDC in the cells was that in the cells treatment of activity of HDC of in a To the localization of the and 53-kDa forms of HDC, the cells were pulse-labeled for 30 and then by Percoll The HDC in fraction was by to the molecular shown in the 74-kDa form was recovered in and 90% of the lactate dehydrogenase activity the 53-kDa form was in fraction where NADPH-cytochrome activity and both the of isomerase and the of II were detected the in the cells pulse-labeled over The of the 53-kDa form in fraction was that in cells pulse-labeled for 30 min In in cells pulse-labeled for the 53-kDa form in fraction 1 where histamine and HDC activity and activity To the localization of HDC in specialized organella of RBL-2H3 immunofluorescence with anti-GST fusion HDC antiserum were the staining of the cells the treatment with 0.1% Triton II and the of cells with and with Triton X-100, With 90% of the cells were found to be as by staining not with the cell staining the cells with with anti-GST fusion HDC antiserum antibody However, the of was with antiserum the other the cells with and then with Triton after of staining with of the antiserum against fusion HDC, PDI, and These results of HDC with and Furthermore, the of the of HDC that the of HDC is to that of but not to that of NADPH-cytochrome P-450 reductase. The of HDC and was with The in double of HDC with antibody and with antiserum the localization of HDC in the luminal of the ER. To the molecular form of the luminal HDC, digitonin-permeabilized cells were with to the of and J. K. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). this most of the lactate dehydrogenase activity but of the was to be released from the cells on the of the observation by not In the prepared from digitonin-permeabilized only the 74-kDa form of HDC was detected treatment of the digitonin-permeabilized cells with the remains of the 74-kDa form but the 53-kDa form of HDC was the other the treatment of Triton resulted in the of the and 53-kDa forms of HDC and both of were to the digestion and the 53-kDa known as a of from various tissues (13Ohmori E. Fukui T. Imanishi N. Yatsunami K. Ichikawa A. J. Biochem. (Tokyo). 1990; 107: 834-839Crossref PubMed Scopus (62) Google Scholar, 14Taguchi Y. Watanabe T. Kubota H. Hayashi H. Wada H. J. Biol. Chem. 1984; 259: 5214-5221Abstract Full Text PDF PubMed Google Scholar, 15Watabe A. Fukui T. Ohmori E. Ichikawa A. Biochem. Pharmacol. 1992; 43: 587-593Crossref PubMed Scopus (21) Google Scholar, 16Martin S.A.M. Bishop P.M. Biochem. J. 1986; 234: 349-354Crossref PubMed Scopus (38) Google Scholar), RBL-2H3 cells were found to the 74-kDa to the HDC of the 74-kDa form in RBL-2H3 cells leaked treatment of SLO, that the 74-kDa form is in the Furthermore, 74-kDa HDC is a as shown by in the the 74-kDa form lacks an that is after the translocation the (20Yamamoto J. Fukui T. Suzuki K. Tanaka S. Yatsunami K. Ichikawa A. Biochim. Biophys. Acta. 1993; 1216: 431-440Crossref PubMed Scopus (35) Google Scholar), it be synthesized by in the as a the of the 74-kDa form of HDC, we the of the by the that the of the 74-kDa form is by such as and S. Nemoto K. E. S. Ichikawa A. FEBS Lett. 1997; PubMed Scopus Google Scholar). the of the 74-kDa form of HDC be post-translational the It is that of the 74-kDa form was in the particulate has been to in the ER, as from the of the 74-kDa HDC and the The of the 74-kDa form in the particulate fraction of RBL-2H3 cells was by the of digestion of the cells. 74-kDa HDC was Triton treatment and treatment of the digitonin-permeabilized cells From these results, it is that the 74-kDa form on the of the ER. membrane-associated HDC activity, (24Snyder S.H. Brown B. Kuhar M.J. J. Neurochem. 1974; 23: 37-45Crossref PubMed Scopus (62) Google Scholar) a low but of rat HDC activity with the membranous the of the activity was found in the supernatant were Furthermore, (25Martres M.P. Braudry M. Schwartz J.C. Brain Res. 1975; 83: 261-275Crossref PubMed Scopus (90) Google Scholar), Braudry M. Martres M.P. Schwartz J.C. Agents Actions. 1973; 3: 175-176Crossref PubMed Scopus (3) Google Scholar), and A. Sabria J. Rodriguez R. Brandner R. Rodriguez J. Palacios J.M. Blanco I. J. Neurochem. 1988; 51: 1400-1406Crossref PubMed Scopus (7) Google Scholar) reported HDC activity in rat A. Rodriguez R. Sabria J. Rodriguez J. Blanco I. J. Neurochem. 1988; 56: 380-384Crossref Scopus (3) Google Scholar) reported that the HDC activity was by However, these reports did not refer to the molecular size of the HDC. From results, it is that membrane-associated HDC activity reported previously from the membrane-associated 74-kDa form. the 74-kDa form has not been it is it has full activity. In the recombinant mouse 74-kDa form a low catalytic activity with purified HDC, a dimer of the 53-kDa (20Yamamoto J. Fukui T. Suzuki K. Tanaka S. Yatsunami K. Ichikawa A. Biochim. Biophys. Acta. 1993; 1216: 431-440Crossref PubMed Scopus (35) Google Scholar). Yatsunami reported that the 54-kDa form of recombinant HDC, expressed in the has activity to that of the purified enzyme K. Tsuchikawa M. Kamada M. Hori K. Higuchi T. J. Biol. Chem. 1995; 270: 30813-30817Crossref PubMed Scopus (35) Google Scholar). In the we that 40% of HDC activity in RBL-2H3 cells was by treatment and this activity is to from the 74-kDa form of HDC These results that the 74-kDa form has activity. In to the we previously reported that the recombinant 74-kDa form of HDC expressed in Sf9 cells was recovered in the particulate while the 53-kDa HDC in the expressed cells was the soluble form (20Yamamoto J. Fukui T. Suzuki K. Tanaka S. Yatsunami K. Ichikawa A. Biochim. Biophys. Acta. 1993; 1216: 431-440Crossref PubMed Scopus (35) Google Scholar, 21Yatsunami K. Tsuchikawa M. Kamada M. Hori K. Higuchi T. J. Biol. Chem. 1995; 270: 30813-30817Crossref PubMed Scopus (35) Google Scholar). the of the 74-kDa HDC only on treatment with a mixture of and it is that the recombinant 74-kDa HDC in Sf9 cells is an form of the to have been reported in a R.E. L. A. and R. Scholar). In we showed that the 53-kDa form of HDC is in the particulate fraction in RBL-2H3 cells. The particulate 53-kDa form is to be in the luminal of the ER, the 53-kDa form was in the fraction containing NADPH-cytochrome reductase and PDI, and it was to digestion of digitonin-permeabilized cells but was to that of Triton cells. In the study with anti-GST fusion HDC antiserum that HDC is in the luminal of the ER, was of with but not with is a typical marker localization in the space of the E. R. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar). the other antiserum was against the of NADPH-cytochrome P-450 has a region with a S. G. M. L. M. Proc. Natl. Acad. Sci. U. S. A. 1980; PubMed Scopus Google Scholar). the of HDC in the be from the luminal 53-kDa form of HDC. it is assumed that the translated 74-kDa form of HDC, synthesized on the of is to and translocated the and in the luminal space of the it is post-translationally processed to the 53-kDa form. The 53-kDa form of HDC was not detected in the extract of and digitonin-permeabilized RBL-2H3 cells the other purified HDC, consisting of a dimer of the 53-kDa has been from the supernatant fraction of of various tissues and cells. It is that particulate HDC is the of tissues and cells. In the a of the 53-kDa form of HDC with a typical was recovered in the supernatant fraction of the homogenate as shown in Histamine is in the in cells and and is released after (1Beaven M.A. Histamine: Its Role in Physiological and Pathological Processes. Karger, Basel, Switzerland1978Google Scholar). that the 53-kDa form of HDC, in the and Golgi, is to the fraction containing HDC activity, and and it is that the 53-kDa form of HDC has a role in the of histamine in the granules. In the 74-kDa form of HDC is the the 53-kDa form is the particulate enzyme in RBL-2H3 and both in the luminal of the ER.