(Received for publication, March 6, 1995; and in revised form, October 30, 1995)
From the
We have expressed and characterized human recombinant 74-kDa (rHDC74) and 54-kDa (rHDC54) L-histidine decarboxylases (HDCs) in Sf9 cells. By immunoblot analysis, rHDC74 and rHDC54 were shown to be localized predominantly in the particulate and soluble fractions, respectively. rHDC74 exhibited histamine-synthesizing activity equivalent to that of rHDC54. The existence of 74- and 54-kDa HDCs was also confirmed in the particulate and supernatant fractions of the cell lysate, respectively, from the human basophilic leukemia cell line KU-812-F. The ratio of HDC activity to immunoreactivity was similar for the two forms of the enzyme. The specific activity of purified rHDC54 (1.12 µmol/mg/min) was comparable to those of HDCs from other mammalian tissues or cells. The purified rHDC54 was eluted as a monomer form from a Superdex-200 column; the molecular mass of the enzyme was approximately 54 kDa on SDS-polyacrylamide gel electrophoresis without 2-mercaptoethanol. The HDC activity of rHDC54 significantly decreased on dialysis against buffer without pyridoxal 5`-phosphate; addition of pyridoxal 5`-phosphate to the dialysate readily increased in the enzyme activity to the original activity. Taken together, these results suggest that human HDC functions as both 74- and 54-kDa forms having equivalent HDC activity, which are localized in the particulate and soluble fractions, respectively, and that the latter form exhibits its activity as a monomer form.
Histamine plays a pivotal role in a wide variety of reactions,
such as inflammation, gastric acid secretion, neurotransmission, and
cell growth (Kahlson and Rosengren, 1968; Beaven, 1978; Woolley, 1993). L-Histidine decarboxylase (HDC, ()EC 4.1.1.22) is
the only enzyme that catalyzes the formation of a biogenic amine from
its precursor, L-histidine (Schayer, 1966). HDCs fall into two
classes: those that use pyridoxal 5`-phosphate (PLP) as a cofactor and
those that have a pyruvoyl coenzyme (Recsei and Snell, 1984). The
sequences of the HDCs in the first class from Enterobacter
aerogenes, Klebsiella planticola, Morganella
morganii, Drosophila melanogaster, mouse, rat, and human
show strong homology (Sandmeier et al., 1994). There is
spectral or chemical evidence for PLP in a number of these enzymes, but
not in the human enzyme. The sequence of the HDC from Lactobacillus 30a, which contains the pyruvoyl coenzyme, is quite different.
Many investigators demonstrated that HDC regulates the level of histamine in tissues or cells, and that modulation of HDC activity under various conditions markedly affects the level of the amine (Schneider et al., 1987; Mamune-Sato et al., 1990; Yatsunami et al., 1993). It is thus of great importance to clarify the mechanism underlying regulation of the HDC activity.
Recently, we reported the structure of the human HDC gene, and proposed the possibility that HDC activity is transcriptionally regulated (Yatsunami and Ohtsu et al., 1994). Accumulated evidence suggests that HDC activity is also regulated at the translational level. HDC cDNAs have been isolated, and characterized, from human basophilic leukemia cells (Yamauchi et al., 1990), human erythroleukemia cells (Zahnow et al., 1991), fetal rat liver (Joseph et al., 1990), and mouse mastocytoma P-815 cells (Yamamoto et al., 1990). The molecular masses of HDC enzymes (74 kDa), calculated from amino acid sequences derived from the respective cDNAs, are larger than those of the purified HDC subunits (53-54 kDa) by about 20-21 kDa. It is, therefore, conceivable that primary translated products are processed after translation to yield mature enzymes and that such post-translational processing of HDCs changes the activity or nature of the enzymes.
Until recently, however, it had not been clarified whether or not a primary translated 74-kDa HDC has activity, because it has never been isolated from any tissue or cells. Most recently, mouse recombinant 74-kDa HDC exhibiting enzyme activity was expressed in Sf9 cells (Yamamoto et al., 1993). However, it was not clearly shown whether or not the enzyme activity was due to the recombinant 74-kDa HDC itself, because the mouse recombinant 74-kDa HDC was unstable and its purification was unsuccessful. In addition, it is totally unknown whether 74-kDa HDCs from other mammalian sources have a molecular nature similar to that of mouse 74-kDa HDC. Although the 54-kDa HDCs purified so far characteristically resemble each other, the nature of 74-kDa HDCs might differ among man, mouse, and rat in some respects, because their C-terminal regions are not so homologous (Mamune-Sato et al., 1992).
To clarify whether or not a 74-kDa HDC has HDC activity, and whether and how 74- and 54-kDa HDCs take part in histamine synthesis, we have expressed cDNAs for human 74- and 54-kDa HDCs using a baculovirus system. To our surprise, the human 74- and 54-kDa HDCs exhibited equivalent enzyme activity, and the latter existed as a monomer form.
Figure 1: Structure and construction of recombinant transfer vectors pVLHDC74 and pVLHDC54. Human HDC cDNA in the Okayama-Berg vector was digested with PstI and BamHI. A long 5`-untranslated region or the 3`-end corresponding to the C-terminal 20 kDa of HDC was deleted using the polymerase chain reaction protocol, as described under ``Materials and Methods.'' The resulting HDC fragments (pcHDC74 and pcHDC54) were subcloned downstream of the polyhedrin promoter in pVL1392. The solid bars indicate the untranslated 5`- and 3`- ends of human HDC cDNA. A bold arrow indicates the polyhedrin promoter.
For production of antiserum, three female Japanese white rabbits were immunized by intradermal injection of MAP (0.1 mg in phosphate-buffered saline) in complete Freund's adjuvant (1:1), and given boosters in incomplete Freund's adjuvant (1:1) at intervals of 2 or 3 weeks. After the fourth booster, each rabbit was bled and antiserum was obtained.
Sf9 cells
(approximately 1.5 10
cells) transfected with
vHDC54 were sonically disrupted as described above. The sonicate was
then centrifuged at 100,000
g for 60 min. The
resulting supernatant was used as the crude extract. rHDC54 was
purified from the crude extract (559 mg) by ammonium sulfate
fractionation and three successive column chromatographies with
phenyl-Sepharose HP, Resource Q, and Superdex 200. The purification
conditions employed for each step were essentially identical to those
used for the purification of mouse HDC (Ohmori et al., 1990),
except that 80 mM NaCl was used to elute HDC from the
phenyl-Sepharose HP column (mouse HDC was eluted from the column by 5
mM NaCl). The purified enzyme (34 µg) was stored at
-80 °C.
Gel filtration on a Superdex-200 FPLC column was performed using a gel filtration calibration kit (Pharmacia) as standards: blue dextran (2,000 kDa), ferritin from horse spleen (440 kDa), aldolase from rabbit muscle (158 kDa), bovine serum albumin (66 kDa), ovalbumin (43 kDa), chymotrypsinogen A from bovine pancreas (25 kDa), and ribonuclease A from bovine pancreas (13.7 kDa).
A crude extract from
Sf9 cells, which had been infected with vHDC74, was fractionated by
centrifugation at 100,000 g for 60 min. An aliquot of
each fraction was subjected to HDC assaying and immunoblot analysis. As
shown in Fig. 2A, a strong band for rHDC74 was observed
for the precipitate fraction. The intensities of the bands of rHDC74 in
respective lanes were nicely correlated with the amounts of the
respective HDC activities loaded. No other protein bands that were
correlated with the amounts of HDC activity were detected.
Figure 2:
Subcellular distribution of recombinant
74- and 54-kDa HDCs. Sf9 cells were infected with vHDC74 (A)
or 54 (B), harvested, and then lysed. The homogenate (800
g supernatant) was fractionated by centrifugation at
100,000
g for 60 min and then subjected to immunoblot
analysis. The total amounts of HDC activities loaded were 3.78, 0.48
and 2.78 pmol/min/lane, respectively (A), and 7.84, 5.92 and
0.44 pmol/min/lane, respectively (B). Hom.,
homogenate; 100 k, 100,000
g; S,
supernatant; P, precipitate; M, molecular mass
markers.
Fig. 2B shows the subcellular distribution of
rHDC54. In contrast to rHDC74, an intense band for rHDC54 was also
exclusively observed for the 100,000 g supernatant
fraction. Only 5% of the total HDC activity in the crude extract was
recovered in the 100,000
g precipitate fraction.
When the intensities of the bands were compared between rHDC74 and rHDC54 on a same gel, equivalent amounts of HDC activity of the two species were found to give protein bands with apparently similar intensities (data not shown).
Figure 3:
Detection of the insoluble and active
74-kDa HDC in KU-812-F cells. A homogenate (800 g supernatant) from KU-812-F cells was fractionated by
centrifugation as described under ``Materials and Methods,''
and then assayed for the activities of HDC and lactate dehydrogenase.
Each value is the mean ± S.E. for four determinations (A). The supernatant and precipitate fractions were subjected
to immunoblot analysis (B). The intensities of bands
corresponding to the 74- and 54-kDa HDCs, indicated by arrows in B, were calculated with an Image analyzer (PDI,
Huntington Station, NY) using the software Quantity One®. The
result is presented as a percentage of the value obtained for 54-kDa
HDC (C). Sup., supernatant; Ppt.,
precipitate.
Figure 4:
Superdex 200 FPLC column chromatography of
the recombinant 54-kDa HDC. The active fractions (114 µg) from a
Resource Q column were applied to a Superdex 200 FPLC column, and then
gel filtration was carried out as described under ``Materials and
Methods.'' Aliquots of fractions from the Superdex 200 FPLC column
were subjected to SDS-polyacrylamide gel electrophoresis in the absence
of 2-mercaptoethanol, and then the gel was silver-stained (inset). , enzyme activity; -, A
. The molecular mass markers were: blue dextran
(2,000 kDa), ferritin (440 kDa), aldolase (158 kDa), bovine serum
albumin (66 kDa), ovalbumin (43 kDa), chymotrypsinogen A (25 kDa), and
ribonuclease A (13.7 kDa).
Fractions from the Superdex 200 column were subjected to SDS-polyacrylamide gel electrophoresis in the absence of 2-mercaptoethanol and the gel was silver-stained. As shown in Fig. 4(inset), a single intense band corresponding to a molecular mass of about 54 kDa was exclusively obtained for the active fractions. Immunoblot analysis showed that the anti-HDC antibody only reacted with the single 54-kDa subunit protein (data not shown).
Mammalian HDCs have been characterized and purified from various types of cells and tissues (Ohmori et al., 1990; Taguchi et al., 1984; Martin and Bishop, 1986; Grzanna, 1984). These studies, however, focused only on a soluble 53-54-kDa HDC, although the primary translated HDCs are 74 kDa in size. The purpose of this work was to clarify whether and how 74- and 54-kDa HDCs take part in histamine synthesis. Resolution of this question will provide a clue as to the mystery that mammalian HDCs have to exist in two forms.
An anti-HDC peptide antibody, which recognizes only denatured HDC, was raised and used for the detection of human recombinant 74- and 54-kDa HDCs. The peptide sequence, Ser-218 to Lys-232, in the human HDC sequence is highly hydrophilic as well as antigenic and was selected to completely match those of the mouse (Ser-225 to Lys-239) (Yamamoto et al., 1990) and rat (Ser-221 to Lys-235) (Joseph et al., 1990) HDCs. Immunoblot analysis using the antibody demonstrated that 87% of the total human rHDC74 expressed in Sf9 cells existed as an insoluble form with histamine-synthesizing activity. Although quantitative determination by immunoblot analysis has limitations, equal amounts of HDC activity of the two species apparently gave bands of equal intensity of rHDC74 and rHDC54, suggesting that the specific activities of the two HDCs are equivalent. It is unlikely that rHDC74 was cleaved to yield rHDC54 during the enzyme assay, and the latter exhibited HDC activity because no processing from rHDC74 to rHDC54 was observed during incubation of the former with histidine for 3.5 h at 37 °C (data not shown). When 74- and 54-kDa human HDC cDNAs were subjected to in vitro transcription and translation, synthesis of the respective HDC proteins was observed. In addition, both the in vitro translated proteins exhibited HDC activity (data not shown). Overall, we concluded that human rHDC74 possesses as much histamine-synthesizing ability as rHDC54.
Fig. 3suggests that the insoluble HDC is not an artifact in the present expression study, but that a fairly large amount of an insoluble 74-kDa HDC exists and functions. Approximately 29% of the total HDC activity was found to exist in the insoluble fraction from human leukemia cells, KU-812-F, which is the source of human HDC cDNA. Possible contamination by soluble HDC of these fractions was ruled out because less than 3% of the lactate dehydrogenase activity was detected in either fraction. Immunoblot analysis using an extract of KU-812-F cells supports the existence of 74-kDa HDC in the cells, and the same ratio of activity to immunoreactivity for the 74-kDa and 54-kDa forms. Although the figure contains multiple bands corresponding to higher and lower molecular masses, there is no contradiction between the intensities of the two immunoreactive bands and the respective HDC activities. We have no evidence ruling out the possibility that these bands were derived from aggregation or processing of the respective enzymes, but we tentatively conclude that these bands appeared nonspecifically for the following reasons. First, the activity to immunoreactivity ratio of the two forms is not contradictory. Second, an anti-peptide antibody often gives nonspecific bands when samples are very crude and the amounts of samples, applied to a gel, are very large, which was the case in this experiment.
If, as we have suggested, an insoluble 74-kDa HDC does contribute to histamine synthesis in physiological tissues and cells, how might soluble 54 and particulate 74-kDa HDCs participate in it? This is a difficult question because the molecular nature of the particulate HDC is totally unknown, and we could not determine its cellular location by means of immunohistochemistry using the anti-HDC peptide antibody we raised. We assume that a particulate 74-kDa HDC binds to cellular membranes and promptly responds to signals from outside the cell. In contrast to stored histamine, newly formed histamine is believed to act soon after its synthesis at the site or in the cell where it is formed (Kahlson and Rosengren, 1968). The newly formed histamine, sometimes called induced histamine or nascent histamine, plays an important role in cell growth (Kahlson and Rosengren, 1968; Brandes and LaBella, 1993), platelet aggregation (Saxena et al., 1989), and gastric acid secretion (Kahlson and Rosengren, 1968; Glavin and Brandes, 1988). It is possible that a particulate 74-kDa HDC synthesizes newly formed histamine, whereas a soluble 54-kDa HDC produces stored histamine. In any case, the hydrophobic C-terminal 20-kDa regions of mammalian 74-kDa HDCs probably participate in anchoring of the proteins to cellular membranes. These assumption requires further investigation.
The HDC activity of rHDC54 significantly decreased on dialysis for 16 h against buffer without PLP (Table 1). The HDC activity was not completely abolished on the dialysis, probably because 16 h was not long enough to completely remove PLP from the sample. The addition of PLP to the dialysate readily increased the enzyme activity to the original level. These results strongly suggest that the human HDC is PLP-dependent.
Human rHDC54 was purified by the same procedure as that used for the purification of HDC from mouse mastocytoma cells (Ohmori et al., 1990). In addition, the specific activity of the purified human rHDC54 (1.12 µmol/mg/min) was proved to be equivalent to that of the mouse and rat HDCs (800 and 260 nmol/mg/min, respectively), suggesting that human rHDC54 is functionally similar to native HDCs from mammalian tissues or cells. This is reasonable because the homology of the 54-kDa HDC molecules is considerably high among three mammals (Mamune-Sato et al., 1992). However, the activity of 74-kDa HDCs might differ among them because the homology in their C-terminal 20-kDa is not so striking. Assuming that the C-terminal regions of HDC molecules affect their folding and thereby influence the respective HDC activities, the activity of 74-kDa HDC in mouse or rat might not be similar to that of the respective 54-kDa HDC.
Purified human rHDC54 was shown to be eluted as a monomer form with an apparent molecular mass of 54 kDa on gel filtration on a Superdex-200 column. Only a single band of 54-kDa HDC was detected on SDS-polyacrylamide gel electrophoresis of the eluted protein, even in the absence of 2-mercaptoethanol. Until recently, HDC has been purified to homogeneity only from rat liver (Taguchi et al., 1984) and mouse mastocytoma cells (Ohmori et al., 1990). In both cases, active HDCs were reported to exist as a dimer form. A monomer and active HDC was neither detected nor isolated. Human HDC is the first example of an HDC existing as a monomer form. It is unlikely that the active monomer HDC, found in the present study, was an artifact produced during the process of purification of the recombinant HDC, because HDC activity derived from cultured KU-812-F cells was also eluted at the same position as the purified rHDC54 was on a Superdex-200 gel filtration column. The specific activity of the purified human monomer, rHDC54, was equivalent to that of the dimer HDCs from mouse and rat. However, we cannot rule out the possibility that the C terminus of 54-kDa human HDC is not identical to those of other HDCs, which could contribute to their dimerization. Further studies are needed to clarify this point.