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Blood, Vol. 96 No. 3 (August 1), 2000:
pp. 973-978
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
A topological study of the human  -glutamyl carboxylase
Jianke Tie,
Sheue-Mei Wu,
Dayun Jin,
Christopher V. Nicchitta, and
Darrel W. Stafford
From the Department of Biology, Center for Thrombosis and
Homeostasis, University of North Carolina at Chapel Hill, Chapel Hill,
NC, and the Department of Cell Biology, Duke University Medical Center,
Durham, NC.
 |
Abstract |
 -Glutamyl carboxylase (GC), a polytopic membrane
protein found in the endoplasmic reticulum (ER), catalyzes vitamin
K-dependent posttranslational modification of glutamate to
-carboxyl glutamate. In an attempt to delineate the structure of
this important enzyme, in vitro translation and in vivo mapping were
used to study its membrane topology. Using terminus-tagged full-length
carboxylase, expressed in 293 cells, it was demonstrated that the
amino-terminus of the GC is on the cytoplasmic side of the ER,
while the carboxyl-terminus is on the lumenal side.
In addition, a series of fusions were made to encode each
predicted transmembrane domain (TMD) followed by a leader
peptidase (Lep) reporter tag, as analyzed by the computer algorithm TOPPRED II. Following in vitro translation of
each fusion in the presence of canine microsomes, the topological
orientation of the Lep tag was determined by proteinase K
digestion and endoglycosidase H (Endo H) cleavage. From the topological
orientation of the Lep tag in each fusion, the GC spans
the ER membrane at least 5 times, with its N-terminus in the cytoplasm
and its C-terminus in the lumen.
(Blood. 2000;96:973-978)
© 2000 by The American Society of Hematology.
| |
Introduction |
-Glutamyl carboxylase (GC) is a polytopic resident
endoplasmic reticulum (ER) membrane protein1 that uses
carbon dioxide, oxygen, and vitamin K hydroquinone to catalyze the
carboxylation of a specific group of glutamate residues in the
vitamin K-dependent proteins.2 This
posttranslational modification is essential for the biological
activities of vitamin K-dependent proteins in regulating blood
coagulation, bone metabolism, and cell growth.
Despite purification3 and cloning4 of the
carboxylase, our understanding of carboxylation is solely dependent on
functional studies. For example, regions responsible for the active
site and propeptide binding5-10 were accomplished by
affinity labeling and site-specific mutagenesis studies.
Inactivation of the carboxylase by sulfhydryl-reactive
reagents11 indicates the presence of an essential
cysteine(s); however, the topological orientations of the cysteines and
their mechanistic functions remain to be identified.
In this study, we used in vitro translation/cotranslocation in an
attempt to determine the membrane topology of the carboxylase. This
method has been successfully used to identify the topogenic sequences
of several integral membrane proteins.12-17 In addition, we
determined the in vivo topological orientation of the amino- and
carboxyl-termini of the carboxylase using an intact, fully active
recombinant carboxylase.
We constructed a series of fusion molecules based on the TOPPRED II
computer analysis of the carboxylase sequence. Fusions containing
single putative transmembrane domains (TMDs) followed by the leader
peptidase (Lep) reporter tag were used for the in vitro
translation/cotranslocation study. Based on the signal-anchor potential
of each putative TMD, we predict that human GC spans the membrane at
least 5 times, with its N-terminus in the cytoplasm and its C-terminus
in the lumen of the ER.
| |
Materials and methods |
Materials
The following materials were used: XL1 Blue bacterial
strain and pSPUTK (Stratagene; San Diego, CA); the BacVector-3000 DNA kit (Novagen, Madison, WI); the in vitro transcription kit (Ambion, Austin, TX); rabbit reticulocyte lysate, pCl-neo expression vector, amino acid master mixture, proteinase K, and ribonuclease inhibitor (RNasin) (Promega Company, Madison, WI); 35S-methionine and
the enhanced chemoluminescence (ECL) Western blotting detection kit
(Amersham Life Sciences, Arlington Heights, IL); phenylmethylsulfonyl
fluoride (PMSF) and mouse anti-FLAG M2 monoclonal antibody (mAb) (Sigma
Chemical Co, St Louis, MO); endoglycosidase H (Endo H) (Boehringer
Mannheim, Indianapolis, IN); biotinylated protein size markers (Bio-Rad
Laboratories, Hercules, CA); avidin-HRP conjugate (Pierce, Rockford,
IL); restriction endonucleases (New England BioLab, Beverly, MA); and
HRP-conjugated secondary antibodies (Jackson Laboratories, West Grove,
PA). Professor von Heijne (Stockholm University, Stockholm, Sweden)
kindly provided rabbit anti-Lep antiserum and pING Lep-cDNA
(complementary DNA).
DNA manipulation
A 735-nucleotide (nt) DNA fragment coding for the reporter tag, a
peptide containing the C-terminal, 245 amino acid residues of the P2
domain of Lep, including a potential N-glycosylation acceptor site
(Asn214), was generated by polymerase chain reaction (PCR)
using pING-cDNA as the template. The PCR-amplified DNA fragment was
cloned into the pSPUTK vector at the BamHI/ClaI
(Bacillus amyloliquefaciens H/Caryophanon latum) sites to yield
pSPUTK-Lep, which served as the recipient of all
recombinant human (hGC) fragments.
Site-specific mutagenesis18 was performed to remove the
endogenous NcoI (Nocardia corallina) site at 765 nt
of the hGC cDNA. The resulting plasmid was used as a PCR template to
generate various hGC fragments. A 5'-NcoI site and a
3'-BamHI site were introduced into each PCR-amplified hGC
fragment. The PCR-amplified fragment containing a single TMD was cloned
into NcoI/BamHI-cleaved pSPUTK-Lep. The
5'-NcoI site was immediately 3' to the SP6 promoter
and served as the in-frame initiation codon. The 3'-BamHI
site introduced 2 amino acid residues (glycine [G] and serine [S])
between the hGC fragment and the Lep tag.
We used site-specific mutagenesis18 to introduce
appropriate restriction sites to accommodate the synthetic
oligonucleotides encoding the FLAG tag. A 10-amino acid peptide
(MDYKDDDDKG), including the FLAG epitope, was introduced to the
amino-terminus of the full length of hGC to make FLAG-hGC, and an
8-amino acid peptide (DYKDDDDK) was attached to the carboxyl-terminus
of the full length of hGC to make hGC-FLAG. The FLAG-tagged hGC cDNA
was subcloned into the EcoRI (Escherichia coli RY13)
site of the expression vector pCl-neo under control of the
cytomegalovirus (CMV) promoter.
In vitro transcription and translation
Prior to in vitro transcription, the recombinant pSPUTKs were
linearized at the ClaI site immediately 3' to the Lep
tag. Capped messenger RNA (mRNA) (Ambion) was synthesized according to
the manufacturer's instructions. Dog pancreatic microsomal membranes were prepared as described.19 In vitro translation using
rabbit reticulocyte lysate and cotranslocation using canine pancreatic microsomes were performed according to Shelness et al.15
The reactions were performed at 25°C for 30 minutes in a final
volume of 20 µL containing 10 µL rabbit reticulocyte lysate; 0.5 µL RNasin (40 U/µL); 0.4 µL of a 1 mmol/L amino acid mixture
without methionine; 1.6 µL 35S-methionine
(3.7 × 1013 Bq [1000 Ci/mmol]) at 370 MBq (10 mCi/mL); and 1 µg capped mRNA with or without 1 equivalent19 of canine pancreatic microsomes.
For further enzymatic cleavage, the translation products were chilled
on ice and mixed with 30 µL incubation buffer containing 110 mmol/L
potassium acetate, 2.5 mmol/L magnesium acetate, and 25 mmol/L K-HEPES
(potassium 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid) (pH 7.4). For deglycosylation, 1 µL Endo H (1 mU/µL) and 1 µL 5% CHAPS (3-[(3-cholamidopropyl)di-methylammonio]-1-propane sulfonate) were added to a 10-µL aliquot of the diluted translation products and incubated for 1 hour at 37°C. For protease digestion, 1 µL proteinase K (1 mg/mL) was added to a 10-µL aliquot
of the diluted translation product in the presence or absence of 0.5% CHAPS. The reactions were carried out on ice for 30 minutes and terminated with 3 mmol/L PMSF. Before being subjected to sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis, the
samples were precipitated with 2 volumes of saturated (NH4)2SO4, washed
with 5% trichloroacetic acid, and redissolved in 10 µL SDS-PAGE
sample buffer.
Expression of FLAG-tagged carboxylase
Full-length carboxylase molecules with a FLAG tag at either the N-
or C-termini were expressed in 293 cells. Transfection was performed
using calcium phosphate precipitation,20 and the cells were
selected with geneticin. Microsomes from cells expressing FLAG-hGC or
hGC-FLAG were prepared as described.21 Protease digestion
of the microsomes was performed as described above. After protease
digestion, the microsomes were washed 3 times with normal saline
buffer, and the pellet was resuspended and equally divided into 2 aliquots. One aliquot was directly subjected to SDS-PAGE without
further treatment. The other was denatured in 1× denaturation buffer
(0.5% SDS and 1% 2-mercaptoethanol) for 30 minutes at 45°C and
then digested with Endo H for 2 hours at 37° before SDS-PAGE.
SDS-PAGE and Western blot analysis
SDS-PAGE analysis was performed according to Laemmli22
under reducing conditions. Samples from the in vitro translation were
subjected to 12% SDS-PAGE, and the full-length recombinant carboxylase
from hEK-293 cells were subjected to 4%-20% SDS-PAGE. After
electrophoresis, the proteins were transferred to a polyvinylidene difluoride membrane, and autoradiography was performed (Molecular Dynamics Storm 840 PhosphoImager; Molecular Dynamics, Sunnyvale, CA).
For Western blot analysis, the proteins were probed with polyclonal
rabbit anti-Lep antiserum or mouse anti-FLAG M2 mAb followed by an
HRP-conjugated secondary antibody, and the proteins were detected using
the ECL kit. The relative molecular weight of each peptide was
estimated according to Weber and Osborn,23 using
biotinylated protein as a standard.
| |
Results |
Prediction of membrane topology of human carboxylase using
TOPPRED II
The vitamin K-dependent carboxylase is a 758-amino acid ER
integral membrane protein. Figure 1A
depicts the hydropathy plot of human carboxylase. The putative TMDs of
the carboxylase were determined using the computer algorithm TOPPRED
II24 with a default window of 21 amino acid residues. We
predicted 7 TMDs, with the amino-terminus located in the cytoplasm and
the carboxyl-terminus located in the lumen of the ER. For clarity, we
denoted the TOPPRED II-predicted TMDs with parentheses until they were
experimentally verified, after which we removed the parentheses.
(Parenthetical coding is noted in the following text.)
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| Fig 1.
Topological analysis of the human
-glutamyl carboxylase.
(A) TOPPRED II analysis of human GC. The hydropathy plot generated by
TOPPRED II with a default window of 21 is shown. We predicted 7 TMDs at
amino acid residues 60-80 (TM1), 115-135 (TM2), 138-158 (TM3), 197-217 (TM4), 252-272 (TM5), 293-313 (TM6), and 361-381 (TM7). (B) Schematic
representation of the GC and hGC-Lep constructs used to experimentally
determine the topology. The solid bars indicate the TM segments
predicted by TOPPRED II, and Y indicates potential N-linked
glycosylation sites. The P2 domain of Lep was fused to the C-terminus
of the hGC fragments in all constructs.
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Topological orientation of amino- and carboxyl-termini of
carboxylase in vivo
To determine the topological orientation of the amino- and
carboxyl-termini of human carboxylase, we constructed 2 full-length recombinant human carboxylase molecules tagged with a FLAG epitope at
either end: FLAG-hGC and hGC-FLAG. hEK-293 cells expressing either
construct displayed kinetic parameters characteristic of the native
carboxylase isolated from bovine liver. By characterizing FLAG-antibody-purified GC from these 293 cells, we obtained results that demonstrate the equivalence of tagged and native carboxylases (data not shown). This indicates correct folding and topological orientation of the recombinant enzyme. Microsomes from the 293 cells
expressing these constructs were subjected to proteinase K digestion in
the presence or absence of CHAPS and subjected to SDS-PAGE analysis.
The proteins were transferred to a PVDF membrane and probed with the
anti-FLAG M2 mAb.
As shown in Figure 2, without proteinase K
treatment, a 95-kd protein band of the size expected for the
full-length carboxylase was demonstrated in both hGC-FLAG (lane 1) and
FLAG-hGC (lane 5). Proteinase K digestion of the hGC-FLAG (lane 2)
revealed a 60-kd fragment, indicating the lumenal location of the FLAG
tag and therefore the carboxyl-terminus of the carboxylase.
In contrast, FLAG-hGC (lane 6) did not show a proteinase
K-resistant fragment except for the residual undigested full-length
carboxylase, which indicates the cytoplasmic location of the FLAG tag
and therefore the amino-terminus of the carboxylase. When 0.5% CHAPS
was included in the proteinase K digestion, hGC-FLAG (lane 4) lost
its 60-kd protease resistant fragment, further
demonstrating the lumenal location of the FLAG tag. Still further proof
of the lumenal location of the 60-kd fragment is provided by its shift
in electrophoretic mobility after Endo H treatment (lane 3). These data
are in agreement with the TOPPRED II prediction stating that the
N-terminus of the carboxylase is in the cytoplasm, and the C-terminus
is in the lumen of the ER. In addition, it provides evidence for the presence of an odd number of transmembrane domains.
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| Fig 2.
Localization of the amino- and carboxyl-termini of the
carboxylase.
Microsomes prepared from 293 cells expressing hGC-FLAG or FLAG-hGC were
subjected to proteinase K digestion in the presence (+) or absence
(-) of 0.5% CHAPS. Part of the protease digestion sample in the
absence of CHAPS was further digested by Endo H. The anti-FLAG M2 mAb
was used for Western blot analysis.
|
|
Determination of the signal-anchor potential of the putative
transmembrane sequences by in vitro translation/cotranslocation
Based on the TOPPRED II-predicted topology of hGC, the fusions were
made to contain a single putative TMD followed by the reporter tag Lep,
the P2 domain of the leader peptidase (amino acid residues 80-324)
(Figure 1B). We chose the Lep tag because it is a well-characterized
E Coli inner membrane protein, and it has been successfully
used to study the assembly of integral membrane
proteins.17,25 Furthermore, it has been demonstrated that
during in vitro translation, the Lep tag can be efficiently translocated into the lumen of canine pancreatic microsomes and glycosylated at Asn214, the potential
glycosylation site.26,27 Therefore, the glycosylation state
and the protease resistance of the Lep tag allow the determination its
topological orientation and, therefore, the signal-anchor potential of
a given amino acid sequence.
We made this series of constructs (Figure 1B) based on the assumption
that if the Lep tag became glycosylated and resistant to proteinase K,
then the given putative TMD functioned in vitro as a start-transfer
sequence, and therefore it is likely to be an authentic transmembrane
sequence in vivo.28-30 Figure 3
is the autoradiograph of an SDS-PAGE analysis of the single-TMD
fusions. As expected, without canine rough microsomes
(RM ) all fusions yielded a single protein band, with the
molecular weight expected of a nonglycosylated product (lanes 1, 6, 11, 16, 21, 26, and 31). In the presence of canine rough microsomes (RM+), fusions 158-230/(TM4) (transmembrane domain 4) and
220-276/(TM5) (lanes 17 and 22) yielded protein bands indistinguishable
from those of their RM-minus counterparts (lanes 16 and 21). Fusions 1-100/(TM1), 82-136/(TM2), 136-160/(TM3), and 279-320/(TM6), however, yielded additional bands of higher molecular weight than those of their
RM-minus counterparts (lanes 2, 7, 12, and 27). Interestingly, fusion
320-410/(TM7) yielded 2 additional bands compared to its RM-minus
counterpart, and these bands were also of higher molecular weight than
those of the RM-minus counterparts.
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| Fig 3.
Analysis of fusions containing a single predicted
transmembrane domain.
Phosphorimage of the 7 constructs containing a single TMD analyzed by
SDS-PAGE is shown. In vitro translations were done in the presence
(+) or absence (-) of canine pancreatic rough microsomes (RMs).
Endo H treatment was done in the presence (+) of 0.5%
CHAPS, while proteinase K treatment was done in the presence (+) or
absence (-) of 0.5% CHAPS. Glycosylated products are indicated by
circles, while proteinase K-resistant fragments are indicated by
asterisks.
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The extra additional band in fusion 320-410/(TM7) is likely due to the
endogenous N-glycosylation site, Asn389, of the human
carboxylase (lane 32). Endo-H digestion abolished all of the additional
higher molecular weight bands (lanes 3, 8, 13, 28, and 33) indicating
their glycosylated states. Fusions 158-230/(TM4) and 220-276/(TM5)
yielded products indistinguishable from those of their RM-minus
counterparts, and the electrophoretic mobility of their products
remained unchanged after Endo-H treatment (lanes 18 and 23). In
summary, these data indicate that TM1, TM2, TM3, TM6, and TM7 function
as type II signal-anchor sequences in vitro, while (TM4) and (TM5) do
not. The odd number of signal-anchor sequences determined in vitro
agrees with the conclusion drawn from our in vivo studies described above.
We encountered relatively low glycosylation efficiency in our in vitro
translation/cotranslocation system. Similar observations have been
reported16,31,32 and attributed to the possible inefficiencies in both translocation and glycosylation. In our hands,
most but not all of the nonglycosylated fusions were susceptible to
proteinase K treatment (Figure 3, lanes 4, 9, 14, 29, and 34) and were
removable from the membrane by alkaline extraction (data not shown).
This indicates that inadequate membrane insertion of the fusion is the
major cause of the apparent low glycosylation efficiency.
To confirm results obtained from the glycosylation state analysis of
the fusions, we examined the proteinase K susceptibilities of each
fusion. In Figure 3, fusions 158-230/(TM4) and 220-276/(TM5) were
degraded by proteinase K whether CHAPS was present or not (lanes 19, 20, 24, and 25). These results indicate that these 2 fusions remain on
the cytoplasmic side of the ER and therefore were not protected from
proteinase K cleavage; thus (TM4) and (TM5) lack type II signal-anchor
potential. In contrast, the remainder of the fusion constructs yielded
protease K-resistant fragments (lanes 4, 9, 14, 29, and 34), thereby
indicating the lumenal locations of their Lep tags. Since these
putative TMDs possess type II signal-anchor activities in
vitro, it is likely that they are authentic TMDs in vivo.
The size of the fusions and their proteinase K-resistant fragments was
primarily determined by the size of the Lep tag, a 245-amino acid
sequence, but the size was influenced by the number of amino acids
surrounding the potential signal-anchor sequence. For example, TM1
functions in vitro as a type II signal-anchor sequence in the fusion
1-100/TM1, which has about 60 amino acids preceding the TMD. This
fusion became significantly smaller after proteinase K digestion due to
the proteinase K sensitivity of the first 60 amino acids residing on
the cytoplasmic side of the ER membrane. Similarly, fusions 82-136/TM2
and 320-410/TM7, with approximately 30 and 40 amino acids preceding the
TM2 and TM7 segments, respectively, also became noticeably smaller
after proteinase K digestion (lanes 9 and 34). In contrast, fusion
136-160/TM3, with only 2 amino acids preceding the TM3 segment,
appeared unchanged in size after proteinase K digestion (lane 14).
Fusion 279-320/TM6, with 14 hydrophobic amino acids preceding TM6,
exhibited unchanged electrophoretic mobility after proteinase K
treatment (lane 29). Permeabilization of the microsomes with CHAPS
prior to proteinase K treatment abolished all protease-resistant
fragments from these fusions (lane 5, 10, 15, 30, and 35), indicating
the lumenal location of the protected fusion fragment.
Consistent with the glycosylation state studies described above,
Western blot analysis demonstrated that all the proteinase K-resistant
fragments contained the Lep tag (data not shown), indicating the
lumenal location of the Lep tag and therefore, the type II
signal-anchor activity of these putative TMDs. These results indicate
that TM1, TM2, TM3, TM6, and TM7 function as signal-anchor sequences in
vitro and are likely to function as authentic TMDs in vivo. In
contrast, (TM4) and (TM5) lack the type II signal-anchor activity in
our in vitro system of single TMD construct.
To further clarify the signal-anchor potential of (TM4) and (TM5),
experiments were performed with fusions containing adjacent-paired putative TMDs: 136-230/TM3-(TM4), 158-276/(TM4)-(TM5), and
220-320/(TM5)-TM6 (Figure 1B). The phosphorimage
analysis of the SDS-PAGE results of these fusions is shown in Figure
4. As expected, without canine microsomes
(RM-minus), all 3 fusions revealed a single protein band, with the
relative molecular weight of that estimated for a nonglycosylated
product (lanes 1, 6, and 11). In the presence of canine microsomes
(RM-plus), fusions 136-230/TM3-(TM4) and 220-320/(TM5)-TM6 contained an
additional minor protein band with a relative molecular weight slightly
greater than that estimated for a nonglycosylated product (lanes 2 and
12). Endo-H treatment selectively abolished these additional protein
bands indicating their glycosylated states (lanes 3 and 13). Proteinase
K digestion of these 2 fusions (lanes 4 and 14) revealed
protease-resistant fragments. Permeabilization of the microsomes with
CHAPS (lanes 5 and 15) during proteinase K treatment eradicated the
protected fusion fragments, which indicate their lumenal locations. In
contrast, in the presence of canine microsomes, fusion
158-276/(TM4)-(TM5) yielded a product identical to its RM-minus
counterpart (lane 7), and its translation product was not affected by
Endo H treatment (lane 8).
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| Fig 4.
Analysis of fusions containing adjacent pairs of
predicted transmembrane domain.
Phosphorimage of the 3 constructs containing adjacent pairs of TMDs
analyzed by SDS-PAGE is shown. In vitro translations were done in the
presence (+) or absence (-) of canine pancreatic RMs. Endo H
treatment was done in the presence (+) of 0.5% CHAPS, while
proteinase K treatment was done in the presence (+) or absence (-) of
0.5% CHAPS. Glycosylated products are indicated by circles, while
proteinase K resistant fragments are indicated by asterisks.
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In summary, fusions 136-230/TM3-(TM4) and 220-320/(TM5)-TM6 each
contain one signal-anchor sequence in directing the Lep tag into the
lumen of the ER. On the other hand, fusion 58-276/(TM4)-(TM5) either
lacks or contains 2 signal-anchor sequences that cause the retention of
the Lep tag on the cytoplasmic side of the ER. Combined with the
results obtained from the single-TMD fusion, these data support the
conclusion that TM1, TM2, TM3, TM6, and TM7 are functional type II
signal-anchor sequences in vitro and therefore are likely to be
authentic transmembrane sequences in vivo. In contrast, (TM4) and (TM5)
do not function as a type II signal-anchor sequence in vitro; however,
we cannot definitively rule out the possibility that they are authentic
transmembrane sequences in vivo.
| |
Discussion |
In an attempt to obtain a basic understanding of the structure of
the carboxylase, we used the in vitro transcription/cotranslation system to evaluate the putative TMDs predicted by TOPPRED II. This
method has been proved accurate in identifying authentic transmembrane
sequences in several integral membrane proteins.12-17 Thus,
in the absence of concrete structural data, it has become a useful
biochemical tool for identifying topogenic sequences of an integral
membrane protein. In this study, we adopted the hypothesis that a
signal-anchor sequence either functions as a start-transfer or a
stop-transfer sequence depending on its order in the polytopic coding
sequence.29,33 We made a series of single-TMD fusions with
a carboxyl reporter tag, Lep. We postulated that if the Lep tag became
glycosylated and resistant to proteinase K cleavage, the given putative
TMD would be able to function, in vitro, as a type II signal-anchor
sequence, that is a start-transfer, and therefore is likely to be an
authentic transmembrane sequence in vivo.28-30
In Figure 5A,B, the membrane topology of
the human GC derived from TOPPRED II is compared with that derived from
our experimental data. In principle, our experimental data agree well
with the TOPPRED II predictions; that is, the carboxylase contains an
odd number of TMDs, a cytoplasmic amino-terminus, and a lumenal
carboxyl-terminus. The only discrepancy between the TOPPRED II
prediction (Figure 5A) and our results is that our data do not support
(TM4) and (TM5) as authentic TMDs. Because the carboxylase contains an
odd-number of TMDs, both (TM4) and (TM5) either are or are not TMDs.
According to the hydropathy plot, (TM4) barely reaches the threshold to be defined as a transmembrane sequence. Furthermore, if (TM4) and (TM5)
were both authentic TMDs, an intervening loop between (TM4) and (TM5),
with a net charge of minus 2, would be placed in the cytoplasm, a
situation violating the positive inside rule.17,24
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| Fig 5.
The membrane topology of human GC.
(A) TOPPRED II-predicted topology. (B) Proposed membrane topology of
hGC, based on our results.
|
|
Based on our model, the configuration of the carboxylase (Figure 5B)
begins with a cytoplasmic amino-terminus, followed by 5 TMDs, and ends
with a lumenal carboxyl-terminus. Because all 5 TMDs are in the first
400 residues of the carboxylase, this region contains the cytoplasmic
amino-terminus, 2 cytoplasmic and 2 lumenal loops, thereby leaving the
large hydrophilic carboxyl-terminus half uninterrupted in the lumen.
Most of the carboxylase molecule (650 residues) is in the lumen
(residues 81-114, 159-292, and 381-758), while only 108 residues
(residues 1-59, 136-137, and 314-360) are in the cytoplasm. The net
charge distribution is +3, +1, and +9 in the cytoplasmic segments and
 1, 2, and 7 in the lumenal segments, which agrees with the
experimental rules devised for charge distribution.17,24
Although the theoretical deduction supports our model, the
experimental approach only allows us to identify regions that will
serve as a signal-anchor sequence in vitro.
Because the original sequential start-stop transfer model was proposed
for polytopic transmembrane proteins,28 several factors have been demonstrated to affect the orientation of a signal-anchor sequence in the membrane. It has been reported that in addition to the
flanking charged residues, the folding state of the amino-terminal segment, as well as the length and hydrophobicity of the signal-anchor sequence, are all important factors in determining its orientation in
the membrane.34-38 It is possible that (TM4) and (TM5) both function as type I signal-anchor sequences and assume an N(out)-C(in) orientation in our TMD fusions. In that case, fusions 158-230/(TM4) and
220-276/(TM5) would have a small proteinase K-resistant fragment which
contained the lumenal amino-terminus and the signal-anchor sequence.
However, small peptides of that size are beyond the separation limit of
most traditional electrophoresis systems. If we assume that (TM4) and
(TM5) are authentic signal-anchor sequences, data from the 2-TM fusions
would suggest that fusion 58-276/(TM4)-(TM5) would have an N(in)-C(in)
orientation, while fusions 136-230/TM3-(TM4) and 220-320/(TM5)-TM6
would have an N(out)-C(out) orientation. However, the size of their
proteinase K-resistant fragments strongly argues against the validity
of this assumption. Structures from distant or flanking regions of the
TM have been reported to affect the topological activity of a
given TM.39-42 Therefore, it is possible that (TM4) and
(TM5) are authentic TM sequences in the carboxylase, but their
topogenic activities were suppressed in our in vitro system
because other transmembrane sequences were not present. In the
absence of high resolution structural data, any such interpretation
remains a matter of conjecture.
Little is known about the structure-function relationship of the
carboxylase, and by studying its membrane topology, we hope to gain
insights into its functional and mechanistic properties. It has been
reported that the enzymatic activity of the carboxylase resides in the
lumen of the ER.1,43 Mutations at charged clusters 217/218,
234/235, and 359/361 have been reported to affect carboxylase activity.10 Using our 5-TMD model, we found
that mutations 217/218 and 234/235 reside in the lumen of the ER.
However, all 3 charged clusters are in the cytoplasmic region of the
7-TMD model. Comparing the cDNA sequences of human, bovine, and rat GC
reveals that most sequence variations occur in the cytoplasmic segments
of the carboxylase, while highly conserved sequences are found in the
lumenal segments, as would be expected for segments of an active site.
We previously reported a propeptide binding site near residues
438-507,9 while Yamada et al8 reported a
propeptide binding site between residues 184 and 225. According to the
TOPPRED II 7-TMD model, residues 438-507 reside in the lumen of the ER,
agreeing with our propeptide binding data but contradicting the Yamada et al data, which would have residues 184-197 in the lumen, residues 197-217 in the membrane, and residues 217-225 in the
cytoplasm.8 However, if our 5-TMD model is used, both
regions are located in the lumen of the ER. Because 2 linearly distant
regions can be conformationally near one another, it is possible that
the conditions used in the chemical cross-linking reaction to identify the propeptide binding site may have dictated the region where cross-linking occurred, thus explaining the apparent discrepancy.
One other structural feature important for understanding the
carboxylase's function is the location of disulfide bonds and a free
cysteine(s). Based on a series of simple step-by-step organic chemistry
reactions, Dowd44 formulates an elegant mechanistic model
for the carboxylation reaction. Using the published observation that
sulfhydryl-reactive reagents inactivate carboxylase
activity,11 he proposed that 2 essential cysteines are
directly involved in the catalytic event. According to the 7-TMD and
the 5-TMD models, both human and bovine carboxylases have 5 cysteines
(C99, C288, C450, C598, and C700) in the lumen of the ER. However, it
is unlikely that C700 is one of the critical cysteines proposed in
Dowd's hypothesis because it is replaced by an arginine in the rat
carboxylase. In addition, replacement of C700 with alanine does not
affect carboxylase activity (V.M., unpublished data, May, 1999). We
have previously demonstrated that the amino 30-kd and the carboxyl 60-kd tryptic fragments are linked by a disulfide bond(s) in the carboxylase. Because there is no apparent intermolecular disulfide linkage present in the purified carboxylase and because the enzyme is
sensitive to sulfhydryl-reactive reagents, we conclude that there must
be 2 free cysteines in the lumen of the ER. This agrees with Dowd's
hypothesis that vitamin K hydroquinone and carbon dioxide
(CO2) are coordinated with 2 individual cysteines that are
directly involved in the catalytic event.
In summary, human GC spans the membrane at least 5 times, with its
N-terminus in the cytoplasm and its C-terminus in the lumen of the ER.
Because the method we used in this study does not allow us to
unequivocally rule out that (TM4) and (TM5) function as authentic
transmembrane sequences in vivo, their definite topogenic activities require further investigation.
| |
Acknowledgments |
We thank Dr von Heijne for kindly providing anti-Lep antibody and the
Lep-containing plasmids, Pen-Jin Lin for technical support, and Dr
David L. Straight for critically reviewing the manuscript.
| |
Footnotes |
Submitted February 10, 2000; accepted March 30, 2000.
Supported by grant HL48318-05 from the National Institutes of
Health, Bethesda, MD.
Reprints: Darrel W. Stafford, Department of
Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC
27599-3280; e-mail: dws{at}emailunc.edu.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
| |
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Abstract
Introduction