 |
INTRODUCTION |
Histamine is generated by the catalytic decarboxylation of
L-histidine. In mammals the enzyme that performs this
reaction, L-histidine decarboxylase
(HDC1; 4.1.1.22), is
initially translated as a 73-74-kDa protein. Studies to purify the
enzyme from native sources, however, led to the isolation of
100-110-kDa HDC complexes (fetal rat liver (1), mouse stomach (2),
mouse mastocytoma cells (3-5), mouse kidney (6)). These complexes
resolved to give 53-55-kDa protein bands on denaturing
SDS-polyacrylamide gels, suggesting that cellular expression of the
enzyme involved the dimerization of two processed isoforms.
Until recently therefore it was widely believed that post-translational
processing involved a single carboxyl-terminal processing step (7-9).
This was due in part to studies performed with one particular antiserum
that recognizes the full-length protein and a carboxyl-truncated
isoform that is 53-55 kDa (9, 10). Yet despite multiple reports of
purified 110-kDa complexes and 53-55-kDa processing, there were also
isolated reports of a ~120-140-kDa purified complex (11, 12).
Initially, these experiments were described as flawed (1); however more
recent studies employing newer antisera have independently demonstrated
the more complex nature of HDC post-translational processing. It is now
apparent therefore that a number of isoforms are generated from the
74-kDa primary translation product. This includes major additional
isoforms of sizes ~63, ~58, and ~36 kDa, as well as a number of
other minor bands (13, 14). However, these more recent data have raised further questions regarding which isoforms are responsible for enzymatic activity, as well as the precise sequence and regulation of
the proteolytic processing steps involved. Accordingly, the fundamental
physiological question as to why so many isoforms might be generated
remains largely unanswered.
To address some of these questions in the context of living cells that
process the enzyme, it is necessary to determine the contribution of
each isoform by specifically preventing the accumulation of that
isoform. However very little is known about HDC protein processing, and
no cleavage sites have yet been identified. Furthermore, while there
have been reports that porcine elastase is capable of digesting HDC
in vitro (15), the enzymes that specifically mediate HDC
processing in vivo have not yet been identified. The regulation of specific cleavage steps has not previously been demonstrated or addressed.
Although the reasons for generating multiple truncated isoforms remains
unclear, computer analysis has identified a degradation promoting
PEST domain within the carboxyl-terminal tail, between amino
acids ~500 and ~570 (10, 16). Reports that supported a single
proteolysis step and the generation of a single 53-55-kDa carboxyl-terminal processed isoform have logically argued that formation of the active dimer would involve the removal of this domain
and result in protein stabilization (10). The expression of recombinant
proteins supports this interpretation, and in transfected or infected
cell models the steady state accumulation of carboxyl truncated
53-55-kDa HDC isoforms was greater than for the full-length isoform
(7, 13, 14, 17). Nevertheless, the identification of processed isoforms
that potentially include part of the PEST domain has opened the
possibility for isoform-specific patterns of degradation (14). However,
studies have yet to specifically demonstrate that carboxyl-truncated
isoforms that lack the PEST domain can be regulated differently from
those that have, and the intracellular signaling pathways that might
influence this kind of regulation have yet to be determined.
Here we demonstrate that the inactive full-length HDC protein is
post-translationally processing into multiple isoforms. This was shown
in a number of in vivo cell models, including transfected COS-7 cells, as well as the stomach and fetal livers of rats. We
describe the properties of carboxyl-truncated HDC isoforms ranging in
size from 51 to 58 kDa and identify sequences that constitute part of
the 55-kDa processing site. Mutation of this cleavage site prevented
production of the 55-kDa isoform but did not affect enzymatic activity.
A carboxyl-truncated 53-kDa isoform of the rat HDC protein was unstable
and had no enzymatic activity.
 |
EXPERIMENTAL PROCEDURES |
Plasmid DNA Constructs--
Unless otherwise stated, all
expression constructs were generated by PCR amplification using
Pfu DNA polymerase (Stratagene) on a thermocycler
(PerkinElmer Life Sciences 9700) and using the CMV-HDC18 vector
as template (18). The pEP7-HA vector backbone used in this study was
generated by cloning double stranded oligonucleotides, for which
the sense strand was
5'-gctagcgtaatacgactcactatagggcctaccggactcagatctcgagctcaagctttcgaattctgcaggtaccggatccgcgtcgacgg actacaaagacgatgacgacaagtagacgcgtgcggccgc into the NheI and
NotI sites of the pEP-empty vector, which has already been
described (14). The pEP7-HDC1/460HA, pEP7-HDC1/472HA, pEP7-HDC1/477HA, pEP7-HDC1/486HA, pEP7-HDC1/498HA, pEP7-HDC1/505HA, pEP7-HDC1/516HA, and
pEP7-HDC1/656HA constructs were generated using the common sense
primer, 5'-gggaagcttgccaccatgatggagcccagtgaataccgtg, and the following
antisense primers, 5'-ccccgtcgacttggtggtgaactg, 5'-atgtcgactctcggatgagg, 5'-atgtcgacacaaggttagcagcctctcgg,
5'-atgtcgacgacggctgagaagt, 5'-ccccgtcgacaccggcggtggaataaggt,
5'-ccccgtcgacaggtctttggagtctctggtcac, 5'-atgtcgactcattgacagactccagg,
and 5'-tacagctgtggtaccggac, respectively, for PCR. PCR products
were cloned into the HindIII-SalI site of pEP7-HA
empty vector. This cloning strategy meant that the HA tag was in-frame
at the carboxyl terminus of expressed proteins.
Mutations were generated using the QuikChange site-directed mutagenesis
protocol (Stratagene). The sense primers used for SKD502/503/504PNS
(
502/3/4) mutants was 5'-ccggtgaccagagactccaaagacctgaccaatgggcta. The sense primer used for the K308G (
308) mutant was
5'-ccttcacctttaacccttccggatggatgatggtgcactttg. Mutants were generated
from the pEP7-HDC1/516HA or pEP7-HDC1/656HA vectors as described in the text.
Cell Culture--
COS-7 cells were maintained in complete
medium, which consisted of Dulbecco's modified Eagle's medium
(BioWhittaker) containing 10% fetal bovine serum and 1%
penicillin/streptomycin solution (Invitrogen). Cells were cultured in a
5% CO2 humidified incubator at 37 °C.
For transient transfection experiments, cells were seeded at a density
of 1 × 106 per 100-mm dishes. After 24 h, cells
were transfected for 3 h with 15 µg of test or control plasmid
DNA using Superfect as described by the manufacturer (Qiagen). When
appropriate 10 µM lactacystin (BioMol), 100 µM forskolin (Sigma), or 10 µM tunicamycin
(BioMol) were added after transfection. Unless otherwise stated cells
were harvested 36-48 h after transfection in 200 µl of 0.1 M sodium phosphate buffer, pH 7.4, supplemented with
complete protease inhibitors (Roche Molecular Biochemicals) and
sonicated. Protein concentration was determined using the method of Bradford.
Coupled Transcription/Translation Reactions--
In
vitro transcription/translation reactions were performed using
rabbit reticulocyte lysates with 1 µg of test or empty vector (
ve)
plasmids and were supplemented with radiolabeled (for gel fractionation) or cold (for enzyme assays) methionine as advised (TNT-Quick kit; Promega) and 0.1 mM pyridoxal
phosphate. For enzyme assays 35 µl of 0.1 M sodium
phosphate buffer, pH 7.4, supplemented with translation inhibitor
cycloheximide was added (final concentration 20 µg/ml) to 5 µl of
TNT reactions. Activities shown are mean ± S.D.,
n = three reactions. For denaturing gel
electrophoresis, 2 µl of reaction mix were fractionated on 8% gels.
RNA Analysis--
Cells to be analyzed for RNA were pelleted and
stored at
70 °C until required. Total RNA was extracted using
RNeasy kits (Qiagen). Northern blots were probed for HDC and
G3PDH mRNA expression as described elsewhere (14).
Recovery of Tissue from Rats--
Two groups of three male
Sprague-Dawley rats (~450 g) were fasted. 24 h later standard
dietary nuts were given to one group of animals, and the rats were
allowed to feed ad libitum for 2 h. Whole stomachs were
isolated and cleaned in PBS. Whole livers were harvested from rat
fetuses at day 20 of gestation. Rat tissue was successively homogenized
and then sonicated in 0.1 M sodium phosphate buffer, pH
7.4, supplemented with complete protease inhibitors. Protein
concentration was determined using the method of Bradford. Rat
experiments were preformed in accordance with local animal welfare regulations.
Assay of Histidine Decarboxylase Activity--
HDC activity in
40 µl of total cell or tissue lysates (200 µg) was determined from
the linear range of the activity curve as described elsewhere (13).
Unless otherwise stated, enzyme activities are shown above fractionated
protein samples from the same experiment (mean ± S.D.) and are
representative of three independent experiments.
Immunoblotting Analysis--
Whole cell or tissue lysates (100 µg) were diluted in 2× sample buffer and electrophoresed on
denaturing SDS-polyacrylamide (8%) or native (6%) polyacrylamide
gels. Fractionated proteins were transferred to a polyvinylidene
difluoride membrane, and membranes were immunoblotted by standard
procedures using an anti-HDC antibody (diluted 1:1000 in 2% non-fat
dried milk; Accurate Chemical and Scientific) or an anti-HA antibody
(diluted 1:200 in 2% non-fat dried milk; Santa Cruz Biotechnology).
Immunoreactive proteins were detected using the Renaissance kit
(PerkinElmer Life Sciences) and Biomax MS autoradiographic film
with intensifying screens (Eastman Kodak Co.). All immunoblots shown
were representative of at least three independent experiments.
Immunoprecipitation of HA-tagged Protein--
COS-7 cells were
seeded at a density of 1 × 106 cells per 100-mm dish
and transfected with 15 µg of pEP7-HDC1/472HA, pEP7-HDC1/486HA, or
pEP7-HDC1/516HA as described. 12 h after transfection the cells were washed twice with PBS and incubated in
Cys
/Met
medium, which consisted of
cysteine- and methionine-free Dulbecco's modified Eagle's medium
(BioWhittaker) supplemented with 10% dialyzed fetal bovine serum
(Invitrogen), 2 mM L-glutamine, and 1%
penicillin/streptomycin solution (Invitrogen). After 1 h the
medium was replaced with Cys
/Met
medium
supplemented with 200 µCi of Easytag express
[35S]methionine/cysteine mix (1175 mCi/mmol
[35S]methionine; PerkinElmer Life Sciences). After a 2-h
pulse, cells were washed twice with PBS, and Ultraculture medium was
added. When required the medium was supplemented with 0.1 µM phorbol 12-myristate 13-acetate or 10 µM
lactacystin. Cells were harvested at appropriate time points in 750 µl of radioimmune precipitation assay buffer (150 mM
NaCl, 20 mM Tris-HCl, pH 7.5, 2 mM EDTA, 0.1%
SDS, 0.25% deoxycholate, 1% Triton X-100) supplemented with protease
inhibitors (Roche Molecular Biochemicals). HA-tagged proteins were
immunoprecipitated using an anti-HA antibody (Santa Cruz Biotechnology)
and 30 µl of protein A-Sepharose CL-4B (30 mg/ml in PBS, 0.02%
sodium azide; Amersham Biosciences) as described elsewhere (14).
 |
RESULTS |
Carboxyl-terminal Processing of the ~74-kDa Primary Translation
Product Is Required for Rat HDC Activity--
Studies with
reticulocyte cell lysates unambiguously demonstrated that the
full-length rat HDC1/656HA protein is inactive in the absence of
physiological processing (Fig.
1A, top panel, lane 3). The expression of a carboxyl-truncated HDC1/516HA
isoform (~58 kDa), on the other hand, gave significant enzymatic
activity (Fig. 1A, top panel, lane
2).

View larger version (9K):
[in this window]
[in a new window]
|
Fig. 1.
A, the pEP7-HDC1/656HA and
pEP7-HDC1/516HA expression vectors were used as templates in coupled
transcription translation reactions. Cold expression reaction products
were analyzed for enzymatic activity (top panel, mean ± S.D., n = 3). 35S-Radiolabelled reaction
products were fractionated on denaturing SDS-polyacrylamide gels
(lower panel). B, lysates of fetal rat liver or
transfected COS-7 cells were compared for enzymatic activity
(upper panel, mean ± S.D.) or by denaturing SDS-PAGE
(lower panel) as indicated. Large and small
arrows are described in the text.
|
|
These studies demonstrated that physiological processing of the primary
translation product is necessary for activity, and in living cells
expression of the full-length rat HDC protein was associated with
physiological processing that resulted in the production of a number of
different sized isoforms and histidine decarboxylation activity. This
is shown in Fig. 1B, where HDC expression was compared
between extracts of fetal rat liver (lane 1) and transiently
transfected COS-7 cells expressing full-length rat HDC1/656HA protein
(lane 2). COS-7 cells, which do not express endogenous HDC,
were capable of processing the primary translation product, and in both
cases significant enzymatic activity (upper panel) and
multiple processed isoforms (lower panel) could be detected.
Although the ratio of the post-translationally processed isoforms
clearly differed between the two cell models, the sizes of the major
processed isoforms were similar, including the main 63-, 58-, 55-, and
36-kDa isoforms (large arrows). Other minor isoforms were
also detected (small arrows).
~74-kDa HDC Undergoes Carboxyl-terminal Processing in Transfected
COS-7 Cells to Generate the ~55-kDa Isoform--
On account of the
fact that so many different isoforms were being detected in our
immunoblots, we did not think it was possible to attribute activity to
one specific isoform. Furthermore, while our results with reticulocyte
cell lysates indicated that carboxyl-terminal processing is essential
for activity, we had no evidence to prove that the processed isoforms
specifically detected in transfected COS-7 cells were
carboxyl-terminally truncated. Nevertheless, numerous studies have
suggested that carboxyl-truncated ~53-55-kDa isoforms are
responsible for HDC catalysis in vivo (7-9). Accordingly, we were particularly interested in the major ~55-kDa isoform
generated by COS-7 cells and wanted to determine whether its expression was a result of carboxyl-terminal processing.
COS-7 cells were transfected to express the full-length HDC1/656HA and
the carboxyl-truncated HDC1/516HA protein (Fig.
2A, lanes 4 and
3, respectively). Forty-eight hours after transfection it
was apparent that the ~58-kDa primary translation product of HDC1/516HA was still being processed, and the major ~55- and 36-kDa isoforms were both being generated (Fig. 2A, see
arrows). This could only happen if the endogenous ~55- and
~36-kDa processing steps were carboxyl-terminal in nature.

View larger version (20K):
[in this window]
[in a new window]
|
Fig. 2.
COS-7 cells were transiently transfected with
pEP7-HDC1/656HA, pEP7-HDC1/516HA, or pEP7-HDC1/486HA as indicated.
ve refers to cell transfected with the pEP7-HA empty
vector. A, lysates were fractionated by denaturing SDS-PAGE
and analyzed for expression of HDC isoforms by immunoblotting with an
anti-HDC antibody. B, total RNA was extracted and probed for
expression of HDC (upper panel) and G3PDH (lower
panel). The immunoblot (A) and Northern blot
(B) results shown were derived from the same experiment and
are representative of three independent experiments.
|
|
A second truncated protein, HDC1/486HA, of ~54.4 kDa was also
expressed (Fig. 2A, lane 2). Gel fractionation
indicated that the expressed protein was shorter than the 55-kDa
cleavage site and did not therefore generate a processed 55-kDa
isoform. This allowed for a preliminary estimation of the processing
site to between amino acids 486 and 516.
Importantly, the experiments shown in Fig. 2A additionally
confirmed that carboxyl-terminal truncation was leading to an increase in steady state expression of the primary translation products. HDC
protein expression was greater for the truncated HDC1/486HA protein in
lane 2 than for the full-length protein in lane
4, for example. In representative experiments this was not because of increased mRNA levels (Fig. 2B, upper
panel) but instead coincided with truncations to remove the
degradation-promoting PEST domain located between amino acids 500 and
570 (10, 16).
Mutating Amino Acids 502, 503, and 504 Prevents Formation of the
55-kDa Isoform--
These data indicated that the ~55-kDa processed
isoform was generated by carboxyl-terminal truncations. Accordingly, we
sought to determine the catalytic contribution of such an isoform
specifically when processed from the 74-kDa primary translation product
and determine, as has been suggested in previous reports, whether HDC
activity is dependent on the production of such a carboxyl-truncated isoform. As a first step, we generated additional constructs that express carboxyl-truncated HDC1/498HA and HDC1/505HA proteins, which
more accurately encompass the putative cleavage site between 486 and
516. The expression constructs were transiently transfected into COS-7
cells where it was demonstrated that each transfectant had very similar
levels of HDC activity (Fig.
3A, upper
panel).

View larger version (15K):
[in this window]
[in a new window]
|
Fig. 3.
A, COS-7 cells were transiently
transfected with pEP7-HDC1/516HA, pEP7-HDC1/505HA, pEP7-HDC1/498HA, or
pEP7-HDC1/486HA. ve refers to cell transfected with the
pEP7-HA empty vector. Lysates were analyzed for enzymatic activity
(top panel, mean ± S.D.) or fractionated by denaturing
SDS-PAGE and analyzed for expression of HDC isoforms by immunoblotting
with an anti-HDC antibody (lower panel). B, COS-7
cells were transiently transfected with pEP7-HDC1/516HA and
pEP7-HDC1/486HA. ve refers to cell transfected with the
pEP7-HA empty vector. Cell lysates were fractionated on native
(upper panel) or denaturing SDS (lower
panel)-polyacrylamide gels for immunoblotting with the anti-HDC
antibody.
|
|
From immunoblots with the anti-HDC antibody (Fig. 3A,
lower panel) it was apparent that the primary translation
products of the HDC1/516HA and HDC1/505HA proteins (lanes 5 and 4, respectively) were still being processed to generate
the smaller 55- and 36-kDa isoforms (arrows). In both cases
the 55-kDa isoform was visible just under the primary translation
product. Additional carboxyl-terminal truncations to produce the
HDC1/498HA and HDC1/486HA proteins, on the other hand, seemed to
abolish the ~55-kDa cleavage site (lanes 3 and
2, respectively). These truncated proteins were unable to
produce the endogenous ~55-kDa isoform but were just as catalytically active as isoforms that underwent processing. Indeed, on native polyacrylamide gels, where HDC monomers could not be detected, the
HDC1/486HA protein was as capable of forming presumptive dimers as the
HDC1/516HA isoform (Fig. 3B, upper panel).
These data suggested that amino acid sequences located specifically
between amino acids 498 and 505 are important for 55-kDa HDC processing
but that the specific production of the ~55-kDa isoform is not
essential for catalysis. To test this hypothesis we generated a number
of mutations in the HDC1/516HA protein just upstream from 505. The
first mutant, which carried mutations in amino acids 502, 503, and 504 (HDC1/516HA
502/3/4), was transiently expressed into COS-7 cells and
lysates analyzed for enzymatic activity and production of the 55-kDa
isoform (Fig. 4A). As shown in
the upper panel, this mutation had no affect on enzymatic
activity. Nevertheless it was apparent in anti-HDC immunoblots that the SKD502/503/504PNS mutation compromised the processing of HDC1/516HA, because the 55-kDa isoforms was no longer being detected (Fig. 4A, lower panel). A second protein, HDC1/516HA
492/3/4, carrying the mutation LIP
492/493/494HAS, had no affect
on HDC processing or enzymatic activity (data not shown).

View larger version (9K):
[in this window]
[in a new window]
|
Fig. 4.
COS-7 cells were transiently transfected with
pEP7-HDC1/516HA and pEP7-HDC1/516HA 502/3/4
(A) or pEP7-HDC1/656HA and pEP7-HDC1/656HA
502/3/4 (B) as indicated.
ve refers to cell transfected with the pEP7-HA empty
vector. Lysates were analyzed for enzymatic activity (top
panels, mean ± S.D.) or fractionated by denaturing SDS-PAGE
and analyzed for expression of HDC isoforms by immunoblotting with an
anti-HDC antibody (lower panels).
|
|
Similar results were obtained when the full-length HDC1/656HA was
mutated at amino acids 502, 503, and 504 (HDC1/656HA
502/3/4). In
Fig. 4B, lower panel, it is apparent that the
major 55-kDa HDC isoform was no longer detected (lane 3).
This mutation had no significant affect on enzymatic activity (Fig.
4B, upper panel). Interestingly, these mutations
had little effect on formation of the 36-kDa carboxyl-truncated
isoform, indicating that formation of this isoform is not sequentially
dependent on formation of the ~55-kDa isoform.
Differential Expression of HDC Isoforms Allows for the
Physiological Regulation of HDC Activity--
Our results indicated
that the ~74-kDa primary translation product generates a 55-kDa
carboxyl-truncated isoform that is enzymatically active. Nevertheless,
HDC activity is not solely dependent on production of this isoform, or
at least not to the extent that has previously been proposed. Instead,
multiple active isoforms appear to be generated. We were interested in
seeing whether this might be important in the physiological regulation
of histamine biosynthesis. Little is known about the regulation of HDC
activity in our previous in vivo model, the fetal liver.
Instead, histamine biosynthesis by enterochromaffin-like cells of the
gastric mucosa is known to represent a major regulatory step in the
control of gastric acid secretion (19-21). Furthermore, it has been
demonstrated that regulation of HDC activity in gastric
enterochromaffin-like cells can occur independently of mRNA
expression (22). To determine whether the production of multiple active
HDC isoforms is important in gastric histamine production, rats that
had been fasted for 24 h were re-fed. This led to a significant
increase in HDC activity (Fig. 5,
top panel, p < 0.05, n = three pairs). When whole stomach lysates were fractionated for anti-HDC
immunoblotting it was apparent that this treatment was increasing the
expression of only isoforms that were greater than ~55 kDa in size
(Fig. 5, lower panel, see arrows). The expression
and regulation of multiple HDC isoforms appears to be important
therefore for the physiological regulation of HDC activity.

View larger version (17K):
[in this window]
[in a new window]
|
Fig. 5.
Stomach extracts were derived from fasted or
fed rats as described under "Experimental Procedures."
Extracts were analyzed for enzymatic activity (top panel,
mean ± S.D., n = three pairs, * shows significant
difference compared with fasted controls, p < 0.05 by
Student's t test) or fractionated by denaturing SDS-PAGE
for immunoblotting with the anti-HDC antibody (lower panel).
Arrows on the right hand side indicate isoforms
whose expression was changed by re-feeding.
|
|
Activation of Protein Kinase C and Protein Kinase A Pathways,
Respectively, Allows for the Regulated Stabilization or Regulated
Processing of HDC Isoforms--
Our data from rat stomach extracts
provided supportive evidence that at least one of the higher molecular
weight HDC isoforms is capable of contributing toward activity
in a whole animal model and indicated that the ratio of expressed
isoforms is not fixed within a particular organ. Instead, the
physiological expression of different isoform could be differentially
regulated. We wished to explore the molecular basis for this pattern of
expression, and it was immediately apparent from our re-feeding
experiments that the differentially regulated isoforms were, based on a
carboxyl-terminal patterns of processing, all expected to contain parts
of the degradation-promoting PEST domain. This suggested regulation at
the level of protein stabilization; however it has never been
specifically demonstrated that PEST domain-containing isoforms could be
regulated differently from carboxyl-truncated PEST-deficient ones. To
determine whether this is the case, and to characterize the cellular
signaling pathways that might mediate such a pattern of regulation,
COS-7 cells were transiently transfected to express HDC1/656HA, which
contains the full PEST domain, and HDC1/486HA, which does not. Cells
were metabolically labeled for 2 h and chased for 8 h in the
presence or absence of 0.1 µM phorbol 12-myristate
13-acetate, 100 µM forskolin, or 1 µM
thapsigargin, which, respectively, activate protein kinase C, protein
kinase A, and intracellular calcium release. Of these compounds only
activation of protein kinase C pathways resulted in the stabilization
of the PEST domain-containing HDC1/656HA isoform (Fig.
6A, upper panel).
This stabilization could be mimicked by the addition of the proteasome
inhibitor lactacystin. The PEST-deficient HDC1/486HA isoform was not
stabilized by either of these two treatments (Fig. 6A,
lower panel).

View larger version (19K):
[in this window]
[in a new window]
|
Fig. 6.
A, COS-7 cells were transiently
transfected with pEP7-HDC1/656HA (upper panel) or
pEP7-HDC1/486HA (lower panel). ve refers to
cell transfected with the pEP7-HA empty vector. Cells were
pulse-labeled with [35S]methionine and then chased for an
8-h period in the presence or absence of 0.1 µM phorbol
12-myristate 13-acetate or 10 µM lactacystin as
indicated. Cells were harvested, and labeled proteins were
immunoprecipitated using an anti-HA antibody. Immunoprecipitated
proteins were fractionated by denaturing SDS-PAGE. The results shown
were confirmed in two additional experiments. B, COS-7 cell
were transiently transfected with pEP7-HDC1/656HA in the presence or
absence of 100 µM forskolin (forsk.) as
indicated. ve refers to cell transfected with the pEP7-HA
empty vector. Cell lysates were fractionated by denaturing SDS-PAGE for
immunoblotting with the anti-HDC antibody. Arrows on the
right hand side indicate isoforms whose expression was
altered after incubation with forskolin.
|
|
These are the first studies to definitively demonstrate that
PEST-containing HDC isoforms can be regulated differently from PEST-deficient ones. Furthermore, they indicated that isoform stabilization was specific for activation of protein kinase C pathways;
it was not observed when the cells were treated with forskolin or
thapsigargin (data not shown). Instead, it was noted that forskolin was
capable of suppressing the production of the ~63- and ~55-kDa
isoforms although the exact molecular mechanisms involved remain
unclear (Fig. 6B, see arrows on right hand
side). A slight decrease in enzyme activity was consistently noted
but was not significant over three independent experiments (11.7 ± 1.1 nmol/mg/h versus 9.5 ± 0.9 nmol/mg/h, mean ± S.D., n = 3).
The HDC protein contains two putative N-glycosylation sites
located at amino acids Asn-219 and Asn-305. We wished to
determine therefore whether isoform expression could be influenced by
post-translational glycosylation. Culturing cells in the presence of 10 µM tunicamycin, which inhibits
N-glycosylation, had no effect on patterns of isoform expression, suggesting that HDC is not regulated in this way (data not shown).
Isoforms Less Than 53.7 kDa in Size (i.e. Carboxyl-terminal
Truncations Beyond Amino Acid 477) Are Inactivate--
Thus far our
results indicated that protein processing is essential for enzyme
activity and that multiple isoforms, 55 kDa and greater, contribute
toward catalysis. However other shorter isoforms, such as the
carboxyl-truncated ~36-kDa isoform, were also generated. Although we
found no evidence in these studies for the regulation of these
isoforms, we nevertheless wished to determine their contribution to
overall levels of HDC catalysis. To study the functional significance
of generating these smaller HDC isoforms, a series of constructs were
generated that express proteins where the carboxyl terminus was further
truncated. The isoforms HDC1/477HA, HDC1/472HA, and HDC1/460HA were
developed to express carboxyl-truncated proteins that were 53.7, 53.2, and 51.7 kDa in size.
Constructs expressing these proteins, along with that expressing
HDC1/486HA, were transfected into COS-7 cells. Lysates were used in
enzyme assays and indicated that truncations to amino acids 472 and 460 was inactivating the enzyme (Fig.
7A, top panel). Identical results were obtained when the constructs were expressed in
reticulocyte cell lysate reactions or when COS-7 cells were transfected
to express constructs that completely lacked the HA tag (data not
shown). Carboxyl-truncated ~53-kDa rat HDC isoforms were inactive
therefore, both in vitro and in vivo. Further
carboxyl-truncations to generate proteins HDC1/339HA (~37 kDa) and
HDC1/242HA (~27 kDa) were also inactive (data not shown).

View larger version (10K):
[in this window]
[in a new window]
|
Fig. 7.
A, COS-7 cells were transiently
transfected with pEP7 HDC1/486HA, pEP7 HDC1/477HA, pEP7 HDC1/472HA, or
pEP7 HDC1/460HA. ve refers to cell transfected with the
pEP7-HA empty vector. Lysates were analyzed for enzymatic activity
(top panel, mean ± S.D.) or fractionated by SDS-PAGE
and analyzed for expression of HDC isoforms by immunoblotting with an
anti-HDC antibody (lower panels). B, COS-7 cells
were transiently transfected with pEP7-HDC1/516HA and pEP7-HDC1/516HA
308. ve refers to cell transfected with the pEP7-HA
empty vector. Lysates were analyzed for enzymatic activity (top
panel) or fractionated by denaturing SDS-PAGE and analyzed for
expression of HDC isoforms by immunoblotting with an anti-HDC antibody
(lower panels).
|
|
Lysates were subsequently fractionated on denaturing SDS gels (Fig.
7A, lower panel) and immunoblotted with an
anti-HDC antibody. These experiments initially suggested that the
removal of amino acids between 477 and 472 was leading to the loss of
the 36-kDa isoform, in parallel with loss of enzymatic activity. One
explanation for these data is that the inactive 36-kDa HDC isoform is
derived from larger active isoforms after the release of histamine and might therefore not be produced by inactive isoforms such as HDC1/460HA and HDC1/472HA. To test this hypothesis, COS-7 cells were transfected to express either HDC1/516HA or a second protein carrying a mutation in
lysine residue 308 that binds the essential pyridoxal phosphate co-factor (18, 23). Lysates were analyzed for enzymatic activity and
demonstrated that the mutant HDC1/516HA
308 was indeed inactive (Fig. 7B, upper panel). In anti-HDC immunoblots,
however, it was apparent that the mutant protein was still capable of
generating the 36-kDa isoform (Fig. 7B, lower
panel).
Enzyme Inactivation by Truncation Beyond Amino Acid 477 Is
Associated with Increased Degradation of HDC Isoforms--
Our results
suggested that inactivation of the enzyme by truncation between amino
acids 477 and 472 might also be associated with a decrease in the
steady state levels of the HDC1/472HA and HDC1/460HA proteins (Fig.
7A, lanes 3 and 2, respectively).
Indeed, increased degradation of the HDC1/460HA and HDC1/472 isoforms could explain why it was not possible to detect the processed 36-kDa
isoform in cell transfected with these proteins. Pulse-chase experiments (Fig. 8A) and
incubation of HDC1/472HA-expressing cells with the proteasome
inhibitor lactacystin (Fig. 8B), indicated that inactivation
and decreased steady state protein levels was associated with increased
degradation of HDC1/472HA by the proteasome.

View larger version (16K):
[in this window]
[in a new window]
|
Fig. 8.
A, COS-7 cells were transiently
transfected with pEP7-HDC1/472HA or pEP7-HDC1/486HA. Cells were
pulse-labeled in the presence of [35S]methionine for
2 h and then chased for 2-, 4-, and 6-h periods as indicated. The
results shown are representative of six independent experiments.
B, COS-7 cells were transfected with pEP7-HDC1/472HA and
cultured in the presence and absence of the proteasome inhibitor
lactacystin (Lact; 10 µM). ve
refers to cell transfected with the pEP7-HA empty vector. Fractionated
lysates were analyzed for expression of HA-tagged protein by
immunoblotting.
|
|
 |
DISCUSSION |
Within the wider family of mammalian L-amino acid
decarboxylases the histamine-producing decarboxylase, HDC, exhibits a
unique pattern of post-translational processing. Specifically, it is translated as an unstable protein that is proposed to undergo carboxyl-terminal processing post-translationally. Therefore, whereas
enzymes such as ornithine decarboxylase, DOPA decarboxylase, and
glutamic acid decarboxylase are all translated as active enzymes, it
has been widely reported that the active unit of cellular HDC activity
is a 100-110-kDa dimer consisting of two carboxyl-truncated ~53-55-kDa monomers. Recently, however, a number of additional processed isoforms have been described.
If the cell is capable of generating multiple isoforms, what
evidence then exists that a 53-55-kDa one is the preferred cellular isoform for histamine biosynthesis, as is suggested from other studies.
Much emphasis has been placed on studies that isolated active
100-110-kDa HDC complexes, but these purification procedures were
essentially performed on soluble protein fractions (2, 4). Although
this is standard practice in many purification protocols it has only
become apparent in recent years that HDC isoforms are differentially
localized within the cell (7-9). Under such circumstances where the
first step of an isolation procedure specifically selects against some
isoforms, it is questionable whether the purified isoform accurately
reflects the active form in the cell. Because carboxyl-truncated HDC
isoforms are now known to be more stable (14), it is hardly surprising
that the most stable and soluble isoform turned out to be the isoform
that was eventually isolated.
Emphasis has also been placed on results obtained for one particular
antiserum that has without doubt contributed significantly to our
current understanding of HDC. However, in rat basophilic RBL-2H3 cells
this antiserum recognized only 74- and 53-55-kDa isoforms and not 63-, 58-, or 36-kDa isoforms (9). In contrast, Fajardo et al.
(24), using the RBL-2H3 cell line but the same antibody used in
this study, were recently able to observe expression of the 63/4-kDa
isoform. Other studies that claimed to detect the 63/4kDa isoform in
fetal rat liver were initially described as flawed, yet here we show
that such an isoform is clearly expressed. Considerable confusion
exists therefore, with regard to the processing of the primary
translation product, as well as the relative activities of processed isoforms.
Here we demonstrate definitively that post-translational processing of
74-kDa HDC is required for catalytic activity. However, while an active
55-kDa isoform is certainly generated by carboxyl-terminal processing,
HDC catalysis is not solely dependent on this isoform, as would be
suggested from previous studies (5, 6). Instead, other processed
isoforms greater than ~55 kDa in size can contribute toward histamine
production. This conclusion has wide ranging implications with respect
to our overall understanding of HDC catalysis, as it suggests that the
enzyme is quite flexible. Therefore, while there is likely to be a core
domain in HDC whose structure is essential for catalysis, amino acid
sequences outside this domain are less important for function. Instead,
our studies here showed that the non-essential carboxyl-terminal domain
contains regulatory elements that allow for the differential expression of HDC isoforms. This is important in the rat stomach, where feeding triggers the release of histamine from enterochromaffin-like cells in
the gastric mucosa and leads to histamine H2 receptor mediated stimulation of gastric acid secretion (25). In physiological terms
therefore the pattern of regulated HDC isoform expression observed here
would allow for a transient spike in activity and histamine
biosynthesis to occur. This would mean that there is a constant
background synthesis of histamine but that production could be boosted
and rapidly replenished in anticipation of subsequent feeding. This
unique pattern of post-translational regulation could explain in a
physiological setting why it is so important to generate multiple
active isoforms.
We also showed that the expression of different isoforms can be
suppressed by the activation of protein kinase A pathways and
demonstrated for the first time that HDC isoform expression can be
regulated in this way. Although the exact physiological importance of
this is not yet clear, there have been reports of tissue-specific
decreases in HDC activity by cAMP, although the molecular basis for
this regulation was never identified (26, 27). A range of physiological
conditions therefore are likely to determine the ratio of
PEST-containing and PEST-deficient isoforms and consequently, the
duration of histamine synthetic pulses.
The fact that HDC catalysis involves multiple active isoforms might in
some respects have been anticipated from previous studies where
carboxyl-truncated 64-kDa HDC isoforms were transiently expressed in
COS-7 or infected Sf9 cells (7, 14). However, in these earlier
studies, no steps were taken to prevent the formation of other
isoforms, including the 55-kDa one, and the activity levels could have
been due in part to processing of 64-kDa HDC. Here, however, not only
do we provide the first definitive evidence that processed isoforms
apart from the 55-kDa one contribute toward catalysis, but we
additionally identify the TRDSKDL domain that is important for 55-kDa
processing. Human
-galactosidase is post-translationally processed
by cleavage at a similar RDS motif. The protease that performs this
maturation is known as "protective protein" and post-proteolytically acts to stabilize
-galactosidase and regulate its intralysosomal degradation (28, 29). These are the first data that
implicate a specific cellular protease with HDC processing in
vivo, and it is interesting that this protease is linked to the
lysosome. Is it possible therefore that the 55-kDa isoform is only
being produced so as to be chaperoned until degradation?
In support of other studies we found evidence for a minor 1-2-kDa
amino-terminal truncation (14). An example of this is shown in Fig.
7A where the minor band runs just underneath the primary
translation product. Future studies will need to address the functional
significance of such a step, particularly because amino truncation of
the ~55-kDa isoform would generate a ~53-kDa one, and might help
explain some of the inconsistencies observed in the literature. This is
highlighted in, but by no means limited to, studies on mouse
mastocytoma cells. Hammar and Hjerten (5) described production of a
~55-kDa isoform, whereas Yamamoto et al. (7, 29) described
the production of a carboxyl-truncated ~53-kDa isoform from the same
cell type. Although we cannot completely rule out slight variations in
the exact size estimations performed in other studies, our results on
this are clear, the ~53.7-kDa HDC1/477 isoform is the minimal unit of
carboxyl-truncated rat HDC that is capable of retaining enzymatic
activity. Carboxyl-truncated 53-kDa isoforms are inactive, both
in vitro and in vivo. A carboxyl-truncated 36-kDa
isoform is also inactive, which is of interest given recent speculation
concerning the functional importance of smaller HDC isoforms (30), but
leads to the question of why inactive HDC isoforms are generated as
part of normal in vivo HDC protein processing. It was not
apparent from our study why the removal of amino acids 472 to 477 was
inactivating the enzyme. There are no cysteine residues in this region
that could be critical for structure. Furthermore, inactivation was not
a gradual event as might be expected if the enzyme was gradually
unfolding. Instead, inactivation was immediate and absolute, suggesting
a more direct role for amino acids 472-477 in catalysis. Future
studies will address why these AANLV (472-477) residues are so
important for catalysis and stability.
Data presented here certainly do not discount the possibility that the
55-kDa isoform is the preferred isoform for histamine biosynthesis.
However, our data support the proposition that activity does not depend solely on this isoform, and we conclude that current evidence does not allow for cellular HDC activity to be attributed to
any specific isoform. Indeed, we highlight physiological conditions where it would be advantageous to express and regulate multiple active
isoforms. We feel that it is inappropriate, therefore, in light of
evidence and arguments presented here, to continue referring to the
55-kDa isoform as the active or mature isoform. The challenge remains
to devise experimental approaches to specifically address which of the
isoforms is responsible for histamine biosynthesis in vivo.
Our study suggests that multiple isoforms are likely to be involved.