From the
Dopamine
Dopamine
During the last 10 years,
efforts have been made to understand the structure of the soluble and
membrane-bound form of DBH and to elucidate the mechanism by which the
enzyme is anchored to the membrane. The amino acid sequences deduced
from the cDNAs coding for human
(8, 9) ,
bovine
(10, 11, 12, 13, 14) , and
rat
(15) DBH are highly similar and reveal the presence of an
amino-terminal signal peptide as the only hydrophobic domain of the
protein. Expression of a full-length bovine DBH cDNA in rat
pheochromocytoma PC12 cells demonstrated that the two forms are encoded
by the same mRNA
(16) . The structural differences between the
soluble and the membrane-bound DBH are not entirely understood. It is
assumed that the soluble form is composed of a single 73-kDa subunit as
analyzed by SDS-polyacrylamide gel electrophoresis, whereas the
membrane-bound form exhibits, in addition to the latter band, a 77-kDa
subunit. The comparison of the amino-terminal sequence of the purified
soluble form of bovine DBH
(17, 18) with that deduced
from the cDNA clone establishes that in the 73-kDa subunit the signal
peptide is always cleaved. In contrast, the amino-terminal sequence of
membraneous DBH indicates that 20-30% of the molecules, depending
on the authors, have retained the signal
peptide
(10, 19) . More precisely, most of the 77-kDa
subunits but none of the 73-kDa subunits of membraneous DBH contain an
uncleaved signal sequence
(10) . All of these results suggest
that the signal peptide is not systematically cleaved and may thereby
anchor the enzyme to the membrane. The current model proposes that
soluble DBH is a homotetramer of the 73-kDa processed subunit, whereas
membraneous DBH would be a heterotetramer of the 73- and 77-kDa
subunits. However, some data indicate a possibly more complex
situation. There are controversial reports of the 77-kDa subunit in the
purified soluble bovine DBH, as analyzed by SDS-polyacrylamide gel
electrophoresis
(16, 18, 19, 20) .
Additionally, deglycosylation of both forms of purified bovine DBH
resulted in a single band of lower molecular weight
(21) ,
although this finding was not confirmed by Bon et
al.(22) . More importantly, the expression in
Drosophila Schneider 2 cells of a bovine DBH cDNA lacking its
own signal sequence led to the synthesis of both soluble and
membraneous forms
(23) . This latter result demonstrates that DBH
can be membrane-bound independently of the presence of the signal
peptide.
At present, several features of DBH expression remain
unresolved. Mainly, the structural differences between the 73- and the
77-kDa subunits are unclear. According to the above mentioned reports,
this size heterogeneity may result from alternative translation
initiation, differential carbohydrate composition, or partial cleavage
of the signal peptide. Moreover, the correlation between the two
subunits and the soluble and membrane-bound forms of DBH is not
unambiguously established. Finally, the mechanisms by which the protein
binds the membrane are still unknown.
In this work, we analyzed the
human DBH in an attempt to resolve these matters. The expression
pattern of a full-length human DBH cDNA was compared with that of
different mutated or truncated cDNAs in two mammalian cell lines using
the vaccinia virus expression system.
The pgpt-DBH-t construct (t for truncated)
contains a human DBH cDNA (8), starting at nucleotide +4 of the
coding sequence. The full-length cDNA construct (pgpt-DBH-f) was
generated by adding the first ATG and the Kozak consensus
(27) to pgpt-DBH-t plasmid. This was done by replacing the first
54 nucleotides by a double-stranded 59-mer oligonucleotide, thereby
forming the complete sequence of the human DBH cDNA.
Mutations in
the NH
The soluble and membraneous as well as the washing
fractions were analyzed by Western blotting and enzyme activity assays.
Dopamine
The
human DBH cDNA first reported by Lamouroux et al.(8) lacks the first initiation ATG codon. Consequently, the
complete cDNA sequence was generated, and the full-length cDNA (DBH-f)
as well as the truncated cDNA (DBH-t) were each inserted into a
vaccinia virus by homologous recombination. In the case of the DBH-t
construct, translation should start at the second ATG, which is
surrounded by an appropriate Kozak consensus
(27) . The resulting
protein is expected to be produced with a signal sequence of 25 amino
acids, whereas the native signal sequence is 39 amino acids long.
Whole cell extracts of AtT 20 and RK 13 cells infected with
recombinant virus particles were analyzed by immunoblotting and assayed
for DBH activity. The protein produced by the truncated cDNA migrated
as a single homogeneous band with an apparent molecular mass of 73 kDa
(Fig. 1B, lane3), while the
full-length cDNA generated two distinct subunits of 73 and 77 kDa
(lane4). Both recombinant proteins were slightly
shifted downward as compared with the human pheochromocytoma membrane
and soluble DBH positive controls (lanes2 and
5). This migration heterogeneity was due to species-specific
differences of glycosylation (demonstrated below; see Fig. 4).
These expression experiments confirm that the 73- and 77-kDa subunits
are encoded by the same cDNA.
After
deglycosylation, DBH expressed by the full-length cDNA (lane5) is indistinguishable from that of the endogeneous DBH
(lane4), indicating that the recombinant protein is
correctly matured.
In this study, the biochemical characteristics of the human
DBH protein were analyzed by expressing different recombinant
DBH-vaccinia virus clones in two cell lines. Expression of the
full-length human DBH cDNA (DBH-f) resulted in the production of an
active protein composed of 77- and 73-kDa monomers, electrophoretically
indistinguishable from the native protein
(22, 30) . This
demonstrates that, as in the case of bovine DBH
(16) , the two
monomers arise from a single mRNA. A number of mechanisms could account
for the synthesis of two subunits: differential glycosylation,
initiation of translation at different ATG, and/or optional cleavage of
the signal peptide.
Glycosidase treatment of cell extracts
containing recombinant DBH-f maintained the presence of the two bands
with an increase in the electrophoretic mobilities. This indicates that
the human DBH molecular weight heterogeneity is not due to differences
in carbohydrate composition, as it has been reported for bovine
DBH
(21) .
The use of alternative ATG codons was examined by
constructing two recombinant mutated clones, in which DBH-f codons Met
2 or Met 2 and 3 were changed to Val codons. The expression pattern of
the mutants was indistinguishable from that of the wild type (apparent
molecular weight of the two monomer products, specific activity,
kinetics of expression, and proportion of membrane-bound and soluble
forms of the enzyme). These data clearly indicate that the first ATG is
the only effective initiation site and, more specifically, that the
73-kDa subunit does not result from an alternative initiation at the
second or third ATG.
A recombinant DBH clone lacking the first ATG
(DBH-t) was constructed. In this case, the second ATG serves as
initiator codon, and the signal peptide is reduced from 39 to 25
residues. The expression of such a cDNA in cells led to the synthesis
of a single 73-kDa subunit, comigrating with the soluble DBH control.
The resolution of the acrylamide gels was sufficient to exclude the
possibility that an intermediate form migrating between 73 and 77 kDa
went undetected. Deglycosylation of DBH-t protein confirmed that there
was only a single type of subunit produced.
All together, these
results offer compelling evidence that the DBH doublet arises from
optional cleavage of the signal peptide, where the 73-kDa form is the
completely processed DBH form and the 77-kDa form retains the signal
peptide.
Moreover, our results show that when the signal peptide is
shortened, its cleavage is rendered compulsory. This points to the
important part played by the amino-terminal 14 amino acids in the
regulation of the cleavage of the signal peptide.
The existence of
DBH subunits that have conserved the signal peptide has been previously
well established in species other than human. Amino-terminal sequence
analysis of the bovine DBH
(10, 19) demonstrated that
the signal peptide is not always cleaved. Likewise, Feng et
al.(34) reported that the full-length rat DBH mRNA,
translated in a cell-free system in the presence of pancreatic
microsomal membranes, produces a protein that retains the
amino-terminal signal sequence. Fig. 7compares the
amino-terminal peptide sequences of human, bovine, and rat DBH. They
are all composed of the three domains typical of a signal
peptide
(35, 36) : an NH
DBH exists both in soluble and membrane-bound forms. We used the
DBH-t and DBH-f constructs to investigate the role of the signal
peptide in anchoring the protein to the membrane. Fractionation of the
extracts from cells expressing the complete DBH cDNA indicates that all
of the 77-kDa subunit is associated with the membrane. It was not
detected in the soluble fraction. This result is consistent with
sequence analysis of bovine DBH showing that only the membraneous DBH
contains the signal sequence
(10, 19) . It is therefore
very likely that the uncleaved signal peptide is involved in attaching
the protein to the membrane. However, when the signal peptide is
completely cleaved, as in the case of the DBH-t construct,
approximately one-third of the DBH was still found associated with the
membrane fraction. This indicates that a second anchoring mechanism
must exist independent of the signal peptide. Similarly, Gibson et
al.(23) showed that the in vivo expression of a
bovine DBH devoid of its signal peptide generates both membrane-bound
and soluble forms of the enzyme.
A comparison of DBH-f and DBH-t
activities indicates that the Triton-extractible fraction was
significantly increased when the signal peptide was not cleaved
(DBH-f), with regard to the short construct (DBH-t). Therefore, the two
modes of membrane attachment co-exist and seem to be additive.
According to our enzymatic activity measurements, 20% of total DBH
(i.e. 37% of the membrane-bound fraction) would be anchored in
the membrane by an uncleaved signal peptide, whereas the second
attachment mechanism would participate for about 38%. This latter
estimation is consistent with the 40% value found by Gibson et
al.(23) for bovine DBH expressed in Drosophila cells. On the other hand, amino-terminal sequence analysis of
purified bovine membraneous DBH indicates that approximately
20-30% of membrane-associated subunits contain uncleaved signal
sequence
(10, 19) .
Several questions still remain
about the nature of the second anchoring mechanism, about the
relationship between the two modes of attachment, and finally about the
regulation of the ratio of soluble to membraneous DBH forms.
There
have been several studies aimed at elucidating the mechanism of DBH
membrane attachment. The most obvious possibilities, covalently bound
lipids (myristylation, palmitylation, glycosyl-phosphatidylinositol
attachment), the presence of an hydrophobic domain other than the
signal peptide, and a putative membrane anchor protein, have been ruled
out
(22, 39, 40, 41) . However,
phosphatidylserine (PS) has been suggested to bind DBH to membranes
(19, 42, 43). This binding is noncovalent, dependent on pH and on
vesicle PS availability, and remains irreversible in the absence of
detergent. This latter fact implies that the proportion of PS
membrane-bound DBH should be directly dependent upon the concentration
of PS in the membrane and more precisely in the inner leaflet of the
chromaffin granule membrane. The percent of DBH anchored by a mechanism
other than the signal sequence is approximately the same (around 40%),
whether or not the host cells possess secretory vesicles (AtT 20 and RK
13 cells in this study; Schneider 2 Drosophila cells in Gibson
et al.(23) ). This might reflect the cotranslational
capture of DBH by every available membrane in these heavy expression
systems, resulting in an overestimation of membrane-bound forms
compared to the situation in vivo. The determination of the
precise localization of the membrane-bound DBH in all of these
expression systems would therefore be valuable.
If we presume that
phosphatidylserine is responsible for the alternate and irreversible
anchoring mechanism, a simple model explaining the necessity for as
well as the relationships between the two modes of attachment can be
proposed. The PS-bound DBH may constitute a stock of membraneous DBH,
whereas the signal peptide-attached DBH would be a supply of the
soluble form. The phosphatidylserine sites would be saturated with
soluble DBH to constitute a reserve of membraneous active DBH. On the
other hand, the amount of soluble DBH would be controlled by a sharp
regulation of the cleavage of the signal peptide. This hypothesis
implies the presence of a signal peptidase or a specific endopeptidase
inside the vesicles. Additionally, the signal peptide might also be
cleaved in the endoplasmic reticulum as argued by two lines of
evidence. A precursor-product relationship has been established between
the 77- and the 73-kDa subunit of rat DBH
(44, 45) . The
conversion seems to occur in the secretory pathway, prior to the exit
from the trans-Golgi apparatus
(46, 47) . Moreover, the
73-kDa DBH polypeptide is produced in mammalian cells that do not
possess synaptic-like vesicles (RK 13 cells). The proportion of
molecules cleaved at this step may be random, depending on the
accessibility of the cleavage site. It should be noticed that sequence
analysis has identified two amino termini indicating two cleavage
sites, separated by 3 amino acids, in the purified soluble bovine
DBH
(17, 18) . This amino-terminal heterogeneity could
reflect the different compartments where the signal peptide is cleaved.
Finally, the physiological importance of the presence of both
soluble and membrane-bound DBH is still unclear. The soluble form is
released by exocytosis into various body fluids (plasma, lymph, saliva,
and cerebral spinal fluid), whereas the membraneous DBH is recycled
back into the cell. A possibility for the functional significance of
the existence of the three interacting forms of DBH (two membraneous
and one soluble) would be to exert a precise regulation of the amount
of circulating DBH by controlling at once its source and its
resorption.
We thank J.P. Henry for providing human anti-DBH
antibody. We are grateful to H. Stunnenberg and J. Schmitt for the
pgpt-ATA 18 vector and the wild-type vaccinia virus and for helpful
advice for constructing the recombinant viruses. We also acknowledge P.
Ravassard for assistance in comparing amino acid sequences and all our
colleagues for critically reading the manuscript.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-hydroxylase (DBH) is found in neurosecretory
vesicles in both membrane-bound and soluble forms. We expressed various
human DBH cDNAs in two mammalian cell lines, using the vaccinia virus
expression system. The expression of a full-length DBH cDNA (DBH-f)
reproduced the native DBH electrophoretic pattern and led to the
synthesis of an active enzyme composed of two subunits of 77 and 73
kDa. In contrast, a truncated cDNA lacking the first ATG (DBH-t)
generated a single band of 73 kDa. Analysis of mutated recombinant
clones demonstrates that the two polypeptides do not result from the
use of an alternative translation initiator codon. These results,
combined with deglycosylation experiments, allow us to attribute the
double band pattern to an optional cleavage of the signal peptide. When
the NH
-terminal extremity is shortened, cleavage becomes
obligatory, underlining the role of the first 14 amino acids in the
regulation of the cleavage of the signal peptide. Subcellular analysis
of recombinant DBH-t and DBH-f proteins indicates that DBH is anchored
to the membrane by two distinct mechanisms; one of them is due to the
non-removal of the signal peptide, whereas the second one is
independent of the presence of the signal sequence. Moreover,
quantification of the fractionation experiments suggests that the two
modes of membrane attachment are additive.
-hydroxylase (DBH)
(
)
(EC
1.14.17.1) catalyzes the conversion of dopamine to norepinephrine, a
step in the biosynthetic pathway of catecholamine neurotransmitters
(1). The enzyme is found inside the chromaffin granules of the adrenal
medullary cells
(2) and in the neurosecretory vesicles of the
noradrenergic neurons of the peripheral and central nervous
systems
(3, 4) . The biochemical properties of DBH are
well established, mostly with the bovine enzyme (for a review, see Ref.
5). DBH is a copper-containing glycoprotein. The enzyme is known to
exist as both a soluble and a membrane-bound protein in similar amounts
according to activity measurements
(6) . Both forms are tetramers
composed of two disulfide-linked dimers, with the dimers held together
by noncovalent interactions
(7) .
Plasmid Constructions
The vaccinia
virus recombination vector (pgpt-ATA-18) used in this study is derived
from pATA-18 vector
(24) , into which a bacterial guanine
phosphorybosyl transferase (gpt) gene driven by the vaccinia
early/intermediate I3 promoter has been inserted
(25) . The
expression of the gpt gene allows efficient dominant selection of
recombinant viruses
(26) . The various DBH cDNAs were introduced
downstream of the mutated vaccinia 11K late promoter at appropriate
sites in the polylinker.
-terminal coding region of pgpt-DBH-f (full-length
cDNA) were introduced by polymerase chain reaction (PCR) as described
by Higuchi
(28) . Two primary PCR reactions were performed with
each of the complementary oligonucleotides containing a mutation and a
5`- or 3`-flanking oligonucleotide. The resulting PCR products were
then combined and reamplified with the outside primers. The final
secondary PCR products were subcloned into pgpt-DBH-f plasmid. Two
mutants were generated: pgpt-DBH-m1, in which the second methionine
codon is changed to a valine codon, and pgpt-DBH-m2, in which both the
second and the third methionine codons are changed to valine codons.
The mutagenic and the flanking primers were, respectively, a 20-mer
(m1, 5`-CCCAGCGTGCGGGAGGCAGC-3`), a 33-mer (m2,
5`-CCCAGCGTGCGGGAGGCAGCCTTCGTGTACAGC-3`), a 22-mer (p-ATA-18,
5`-GTACGCTAGTCACAATCACCAC-3`), and a 20-mer (O2,
5`-ATAGGCAGTGTCCCCATCGG-3`). All constructions were confirmed by
sequencing.
Generation of Recombinant
Viruses
Recombinant vaccinia viruses were obtained by
following the protocol of Stunnenberg and Schmitt
(25) . Briefly,
a human 143 cell line (TK) infected with wild-type
vaccinia virus at a multiplicity of 0.1 plaque-forming unit per cell
was transfected with pgpt-DBH precipitated with calcium phosphate. DBH
cDNA was inserted into the virus by homologous recombination in the
thymidine kinase gene. Recombinant viruses expressing DBH were selected
by three consecutive rounds of plaque purification in the presence of
mycophenolic acid, xanthine, and hypoxanthine (gpt-selective medium).
Expression of Dopamine
RK 13 and AtT 20 cells were grown in Dulbecco's
modified Eagle's medium (Life Technologies, Inc.) and
Dulbecco's modified Eagle's medium:F12 (1:1), respectively,
supplemented with 10% fetal calf serum. For infection with recombinant
vaccinia virus, cells were plated the day before infection at 3 -Hydroxylase in Mammalian
Cells
10
cells/100-mm tissue culture dish. Virus was added at a
multiplicity of infection of 0.5 plaque-forming unit per cell. The
infected cells were harvested after incubation for 14 h at 37 °C.
Subcellular Fractionation of AtT 20 and RK 13
Cells
Infected cell pellets were resuspended in 150 µl
of 10 mM phosphate buffer, pH 7.0, 1 mM
phenylmethylsulfonyl fluoride, and lysed by repeated freeze/thaw steps.
The cell suspensions were centrifuged for 10 min at 100,000
g, and the supernatants (soluble fraction) containing
cytoplasmic and lumenal proteins were collected. The pellets were
washed three times with the same buffer. They were then resuspended in
150 µl of 10 mM phosphate buffer, pH 7.0, containing 0.5%
Triton X-100 and centrifuged for 10 min at 100,000
g.
The resulting supernatants were designated the solubilized membraneous
fractions.
-hydroxylase activity was assayed in whole cell
extracts and subcellular fractions as previously described by Vayer
et al.(29) .
N-Glycanase Digestion
Samples to be
assayed for DBH deglycosylation were adjusted to 0.5% SDS and 0.1
M 2-mercaptoethanol and boiled for 3 min. The samples were
then diluted with 0.2 M sodium phosphate buffer, pH 8.0, and
digested with 10 units/ml N-glycanase (Genzyme) at 37 °C
for 20 h.
Western Blot Analysis
Proteins in whole
cell extracts or the subcellular fractions were separated by
5-12% polyacrylamide gel electrophoresis in the presence of SDS
and transferred to nitrocellulose. Blots were incubated with a 500-fold
dilution of a rabbit antiserum to human dopamine
-hydroxylase
(30) . Bound antibody was detected either with
alkaline phosphatase-conjugated anti-rabbit IgG antibody or with
I-labeled protein A. The autoradiograms were quantified
by densitometry (Biocom). The soluble and membraneous DBH used as
positive controls were prepared from human pheochromocytoma, as
described by Roisin et al. (31).
Expression of Recombinant DBH Using Vaccinia
Virus
The structural and functional properties of human DBH
were analyzed using the vaccinia virus expression system. This viral
vector is an efficient system for the expression of foreign genes in a
wide range of host cells. Two different mammalian cell lines (AtT 20
and RK 13) were used. The mouse pituitary AtT 20 cells were chosen
because they contain secretory vesicles, as do the chromaffin cells
that naturally synthesize DBH. A comparative study was done with the
rabbit kidney RK 13 cells, which are devoid of such vesicles.
Figure 1:
Expression of human DBH cDNAs by
recombinant vaccinia virus. A, the 5`-nucleotide sequence of
human DBH cDNA (8, 9) is shown. Arrows indicate the
5`-extremities of the full-length (DBH-f) and the truncated (DBH-t)
cDNA, respectively. The first and the second ATG codons are shown in
boldfaceletters. The corresponding predicted amino
acid sequences are represented below. B, AtT 20 cells
were infected with the different vaccinia virus constructs, incubated
for 14 h, and harvested. Whole cell extracts were separated by
5-12% polyacrylamide gel electrophoresis in the presence of SDS,
blotted, and reacted with antiserum to human DBH. Lane1, wild-type vaccinia virus-infected cell extracts;
lane2, human pheochromocytoma membrane extracts;
lane3, DBH-t recombinant virus-infected cell
extracts; lane4, DBH-f recombinant virus-infected
cell extracts; lane5, human pheochromocytoma soluble
extracts.
Figure 4:
Analysis of N-glycanase treatment
of DBH by immunoblotting. Soluble and membrane human pheochromocytoma
extracts and recombinant DBH-t- and DBH-f-infected AtT 20 cell extracts
were incubated with N-glycanase at 37 °C for 20 h.
Lanes1 and 2, untreated extracts of
membrane pheochromocytoma (0.4 and 2 µg); lane3,
untreated DBH-f-infected cell extracts; lane4,
N-glycanase-treated membrane pheochromocytoma extracts (2
µg); lane5, N-glycanase-treated
DBH-f-infected cell extracts; lane6,
N-glycanase-treated DBH-t-infected cells extracts; lane7, N-glycanase-treated soluble pheochromocytoma
extracts; lane8, untreated DBH-t-infected cell
extracts; lane9, untreated soluble pheochromocytoma
extracts.
The DBH-t and DBH-f extracts from both
cell lines were enzymatically active (Fig. 3A). The
activity of the enzyme produced by the DBH-f construct was three or
four times lower than that obtained with DBH-t, although it was
1.5-fold more immunoreactive as measured by densitometry
(Fig. 3B). These findings were consistently observed for
at least three independent clones for each construct. The reason for
this difference in specific activity is unclear, but might be due to
different folding of the proteins.
Figure 3:
Analysis of recombinant DBH produced in RK
13 and AtT 20 cells. RK 13 (blackbars) and AtT 20
(hatchedbars) cells were infected with DBH-t, DBH-f,
DBH-m1, and DBH-m2 vaccinia constructs. Whole cell extracts were
assayed for DBH enzymatic activity (histogramA) and
subjected to Western blot analysis (histogramB).
Determinations were done in duplicate. DBH immunoreactivity was
quantified using a Biocom densitometer. One typical experiment is
shown.
Investigation of an Alternative Translation
Initiation Codon
The 5`-end of the coding region for the
human DBH gene contains three in-phase ATG codons
(9) . The two
subunits may therefore be generated by the initiation of translation at
different ATGs. Indeed, the two upstream codons have a favorable
context for the initiation of translation
(27) , whereas the more
downstream ATG has a weak Kozak consensus but is nevertheless conserved
in rat and bovine DBH (Fig. 7). We used site-directed mutagenesis
to change the second and third methionine codons to valine residues.
Western blots of the proteins extracted from cells expressing the
mutated clones DBH-m1 and DBH-m2 gave two immunoreactive bands
(Fig. 2, lanes2 and 3) with
electrophoretic mobilities indistinguishable from those produced by the
DBH-f construct (lane5). The valine substitutions
affected neither the enzymatic activity of DBH nor the intensity of the
doublet band (Fig. 3, A and B). Thus, the first
ATG appears to be the only physiological initiator codon used for the
translation of the two polypeptides.
Figure 7:
The NH-terminal regions of
bovine, rat and human DBH. Sequences are aligned to give the maximum
homologies. Amino acid sequences are taken from the following
references: bovine DBH (10-14), rat DBH (15), and human DBH (8,
9). Black and shadedboxes represent
identical and similar residues, respectively. The positively charged
(n) and the hydrophobic (h) regions (35, 36) are underlined.
Arrows indicate the putative signal peptide cleavage sites of
bovine DBH (10, 17, 18, 37).
Figure 2:
Western blot analysis of mutated
recombinant DBH. In recombinant DBH-m1, the second Met codon has been
changed to a Val codon, and in recombinant DBH-m2 the second and the
third Met have been changed to Val codons (underlined,
boldfaceletters). AtT 20 cells were infected with
DBH-t (lane1), DBH-m1 (lane2),
DBH-m2 (lane3), and DBH-f (lane5). Human pheochromocytoma membrane extracts were used as
control (lane4).
Deglycosylation of DBH
DBH is a
glycoprotein bearing asparagine-linked oligosaccharides consisting of
both high mannose and biantennary complex
oligosaccharides
(32, 33) . To determine whether the two
bands of human DBH were due to a single polypeptide with two different
glycosylation states, as has been proposed for bovine DBH
(21) ,
soluble and membrane DBH controls and recombinant DBH-t and DBH-f were
treated with N-glycanase. In all cases, the apparent molecular
weight of each protein decreased, but the migration pattern was
maintained for each treated sample (Fig. 4, lanes4-7). This clearly indicates that the difference
between the two subunits is not due to the sugar moiety.
Subcellular Localization of Recombinant
DBH
We investigated the subcellular localization of both
DBH-t and DBH-f in AtT 20 and RK 13 cells by fractionation. Both 77-
and 73-kDa subunits of DBH-f were found by Western blot (Fig. 5)
in the membrane fraction (lane5). In contrast, the
soluble fraction contained only the 73-kDa subunit (lane4). DBH-t gave a 73-kDa band in both the soluble and
membrane fractions (lanes3 and 4). No
immunoreactivity was detected in the washing fractions (data not
shown).
Figure 5:
Western blot analysis of AtT 20
subcellular fractions. AtT 20 cells were infected with recombinant
DBH-t and DBH-f vaccinia constructs. The cell extracts were
fractionated as described under ``Materials and Methods'' and
analyzed by Western blot. Lane1, human
pheochromocytoma soluble extracts; lane2, soluble
fraction of DBH-t-infected cells; lane3, membrane
fraction of DBH-t-infected cells; lane4, soluble
fraction of DBH-f-infected cells; lane5, membrane
fraction of DBH-f-infected cells; lane6, human
pheochromocytoma membrane extracts.
The ratio between the membrane-bound and soluble DBH was
obtained by enzymatic activity measurements as well as protein
quantification by densitometry. The proportion of DBH in the membrane
fraction (relative to total DBH) was determined for each construct in
the AtT 20 and RK 13 cell lines (Fig. 6). Both the enzymatic
activity (56 ± 15%) and protein content (62 ± 16%) of
membrane-bound DBH-f were significantly higher than those of the
membrane-bound DBH-t (38.5 ± 9% and 44 ± 10%,
respectively) in the AtT 20 cell line. The results obtained with the
fractionated RK 13 extracts were similar (the enzymatic activity of the
DBH-f membrane fraction was 57.5 ± 13% of the total DBH, and the
amount of protein was 64 ± 15%, whereas for the DBH-t membrane
fraction the corresponding values were 37 ± 8% and 42 ±
7%, respectively).
Figure 6:
Distribution of DBH in subcellular
fractions. Soluble and membrane-bound fractions of RK 13
(black) and AtT 20 (hatched) cells infected with
DBH-t and DBH-f vaccinia constructs were assayed for DBH enzymatic
activity and immunoreactivity. HistogramA shows the
enzymatic activity of the membrane-bound fraction (expressed as a
percent of total DBH enzymatic activity). HistogramB shows the corresponding relative amounts of DBH in the
membrane-bound fraction, quantified by densitometry of autoradiograms.
Data are mean ± S.E. (bars) values of four separate
experiments. Statistical comparison of mDBH-t ratio versus mDBH-f ratio was made using Student's t test; p < 0.01
The DBH-m1 and DBH-m2 constructs were similarly
analyzed. The ratio of membrane-bound and soluble forms were not
different to that of DBH-f (data not shown).
-terminal region,
which is positively charged (n-region), a central hydrophobic part
(h-region), and finally, a more polar carboxyl-terminal domain
(c-region) with potential signal peptidase cleavage sites. Two of these
cleavage sites appear to be functional in bovine
DBH
(10, 17, 18, 37) . However, this
signal peptide is atypical in that it also exhibits features of
uncleaved signal peptides of class II proteins
(38) . Both n- and
h-regions are much longer than their counterparts in cleaved signal
peptides. The n-region is 10-20 amino acids long, depending on
the species, rather than 1-5 amino acids in length. Similarly,
the hydrophobic h-region is 20 residues long compared with the
7-15 amino acids in the classical model. These uncommon
characteristics could explain why the signal peptide is not always
removed. The behavior of the DBH-t construct clearly indicates that a
truncated signal peptide promotes an efficient cleavage. Thus, a
feature allowing the cleavage to be optional may be localized in the
n-region. The amino-terminal extremities of the signal peptide in the
three species differ both in length and in amino acids composition. The
only noticeable shared characteristic is the presence of a conserved
PXPS motif. The presence of these
-turn-promoting
residues could modify the folding of this long signal peptide,
rendering the cleavage site less accessible or susceptible to cleavage.
-hydroxylase; gpt, xanthine
guanine phosphorybosyl transferase; PS, phosphatidylserine; n-region,
NH
-terminal region of a signal peptide, which is positively
charged; h-region, a central hydrophobic part of a signal peptide;
c-region, a more polar carboxyl-terminal domain of a signal peptide;
PCR, polymerase chain reaction.
-Hydroxylase Membrane Attachment, Ph.D. thesis, Georgetown University
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.