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The Carboxyl-Terminal Region of Protein C Is Essential for Its Secretion
By Akira Katsumi, Tetsuhito Kojima, Takao Senda, Tomio Yamazaki, Hiroaki Tsukamoto, Isamu Sugiura,
Shigeru Kobayashi, Toshiyuki Miyata, Hideaki Umeyama, and Hidehiko Saito
We have previously reported a mutated protein C, designated protein C Nagoya (PCN), characterized by the deletion
of a single guanine residue (8857G). This frameshift mutation
results in the replacement of the carboxyl-terminal 39 amino
acids of wild-type protein C (G381-P419) by 81 abnormal
amino acids. This elongated mutant was not effectively
secreted, and was retained in the endoplasmic reticulum. To
determine why PCN is not secreted, we constructed a series
of mutants from which some or all of the 81 amino acids
were deleted. None of these shortened proteins were secreted from producing cells, indicating that the carboxylterminal extension is not mainly responsible for the intracellular retention of PCN, and that the 39 carboxyl-terminal
amino acids of wild-type protein C are required for secretion.
To determine which residues are essential for the secretion
of protein C, deletion mutants of the carboxyl-terminal
region (D401-P419) were prepared. Metabolic labeling
showed that mutants of protein C truncated before W417,
Q414, E411, or K410 were efficiently secreted. On the other
hand, the mutants truncated before D409 were retained and
degraded intracellularly. Immunofluorescence and immunoelectron microscopy showed that truncation before D409
blocks the movement from rough endoplasmic reticulum to
the Golgi apparatus. To understand the conformational
change in the carboxyl-terminal region, two models of
truncated activated protein C were constructed using energy
optimization and molecular dynamics with water molecules.
r 1998 by The American Society of Hematology.
P
and is associated with 78-kD glucose regulated protein (GRP78)/
Immunoglobulin heavy chain binding protein (BiP) and 94-kD
glucose regulated protein (GRP94).5 These data suggest that the
carboxyl-terminal portion of this protein is important for its
efficient secretion.
In the present study, we produced a series of deletion mutants
of both PCN and wtPC using site-directed mutagenesis to
identify amino acid sequences that contribute to the impaired
secretion of protein C. The results of pulse-labeling, immunofluorescence, and immunoelectron microscopy indicated that the
carboxyl-terminal region of protein C plays an essential role in
its intracellular transport. We constructed structural models of
the protease domains of truncated protein C and discussed the
role of the carboxyl-terminal region in the secretion of protein C.
ROTEIN C IS A VITAMIN K-dependent glycoprotein and
plasma serine protease precursor that acts as an anticoagulant and plays an important role in hemostasis.1 The native
human protein C molecule is a disulfide-linked heterodimer
composed of light and heavy chains. It is synthesized in the liver
as a 461-amino acid precursor protein that undergoes extensive
cotranslational and post-translational modification.2 Protein C
deficiency is an autosomally inherited disorder that is associated
with a high risk of recurrent venous thrombosis. The protein C
mutation database contains 160 unique mutations in the protein
C gene from a total of 315 unrelated probands.3 Most are
missense mutations, with the remainder consisting mainly of
nonsense mutations, splice-site abnormalities, frameshift mutations, and others.
We previously reported on a mutated protein C, designated
protein C Nagoya (PCN), characterized by the deletion of a
single guanine residue ( 8857 G) among four consecutive guanine
nucleotides [W380 (TGG)-G381 (GGT)].4 This results in a
frameshift mutation with replacement of the carboxyl-terminal
39 amino acids of wild-type protein C (wtPC) (G381-P419) by
81 abnormal amino acids. This elongated variant is mostly
retained and degraded within the endoplasmic reticulum (ER),
From the First Department of Internal Medicine and the First
Department of Anatomy, Nagoya University School of Medicine,
Nagoya, Japan; the School of Pharmaceutical Sciences, Kitasato
University, Tokyo, Japan; the Laboratory of Thrombosis Research,
National Cardiovascular Center Research Institute, Suita, Japan; and
the Aichi Blood Disease Research Foundation, Nagoya, Japan.
Submitted July 25, 1997; accepted January 2, 1998.
Supported in part by the Grants-in-Aid from the Ministry of
Education, Science, and Culture, Japan, and the Surveys and Research
on Specific Diseases from the Ministry of Health and Welfare, Japan.
Address reprint requests to Akira Katsumi, MD, Howard Hughes
Medical Institute Research Laboratories, Washington University School
of Medicine, 660 South Euclid Avenue, Box 8022, St Louis, MO 63110;
email: [email protected].
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734 solely to indicate
this fact.
r 1998 by The American Society of Hematology.
0006-4971/98/9110-0020$3.00/0
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MATERIALS AND METHODS
Construction of carboxyl-terminal deletion mutants. Complementary DNA (cDNA) fragments coding for wtPC and PCN were ligated
into the multicloning site of the pBluescript II KS (1) (Stratagene, La
Jolla, CA) as described previously,4 and designated pBPC and pBPCN,
respectively. The carboxyl-terminal deletion mutants of PCN and wtPC
were constructed by oligonucleotide-directed mutagenesis. The oligonucleotides used to construct the deletion mutants of PCN and wtPC are
shown in Table 1. The mutant bases are indicated by lower case letters.
The mutated fragments were constructed by polymerase chain reaction
(PCR) amplification with C1 and other antisense primers. Each pair of
PCR fragments were then subcloned in pBluescript II KS (1) by T-A
cloning.6 After digestion with ApaI and XbaI, the amplified fragments
were inserted in pBPCN or pBPC. The resulting mutations were
confirmed by the dideoxy chain termination sequencing method.7 The
mutated cDNAs were digested from pBluescript II KS (1) by EcoRI
and ligated into the pED vector.8 Mutated proteins were designated as
shown in Fig 1A.
Stable expression of recombinant protein C. CHO-DUKX-B11
cells9 were stably transfected with expression vectors using Lipofectin
reagent (GIBCO-BRL, Gaithersburg, MD) as previously described.5
Pulse-labeling and immunoprecipitations were performed as previously
described.5 Data are presented as the mean 6 standard deviation (SD)
for three observations.
Northern blot analysis. Northern blot analysis was performed as
previously described.10 In brief, total RNA (5 µg) from each clone was
separated by 1% formaldehyde-agarose gel electrophoresis in a minigel
system (ADVANCE Co, Ltd, Tokyo, Japan), transferred to a Zeta-Probe
Blood, Vol 91, No 10 (May 15), 1998: pp 3784-3791
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CARBOXYL-TERMINAL OF PROTEIN C
3785
Table 1. The Oligonucleotides Used to Construct the Deletion Mutants of PCN and wtPC
a. PCN Oligonucleotides
Proteins
Sense:
C1 (58-GTGTCTGAGAACATGCTGTGT-38, nucleotides 1238-1258 in pBPCN)
Antisense:
N380 (58-AAGGAGCCCACAGCCCTCta-38, nucleotides 1383-1364 in pBPCN)
N388 (58-CCGTttaTGTGAAGGAGCCCACA-38, nucleotides 1394-1372 in pBPCN)
N425 (58-ttaCCTGCAGGGAGGGTCGC-38, nucleotides 1501-1482 in pBPCN)
N444 (58-AGCAGtCaGGTGTGCTTGTT-38, nucleotides 1563-1544 in pBPCN)
PCN380
PCN388
PCN425
PCN444
b. wtPC Oligonucleotides
Proteins
Sense:
C1 (58-GTGTCTGAGAACATGCTGTGT-38, nucleotides 1238-1258 in pBPC)
Antisense:
C401 (58-CCCATGGATtCAGTCGAGGTA-38, nucleotides 1438-1418 in pBPC)
C403 (58-CTGATGTGCCCTtaGATCCAGT-38, nucleotides 1446-1425 in pBPC)
C406 (58-CTTGTCTCTctaGTGCCCATGG-38, nucleotides 1453-1432 in pBPC)
C407 (58-TCTCaGATGTGCCCATGGATCC-38, nucleotides 1449-1428 in pBPC)
C408 (58-GGCTTCCTTcTaTCTGATGTGC-38, nucleotides 1459-1438 in pBPC)
C409 (58-GGCTTCcTaGTCTCTGATGTG-38, nucleotides 1459-1439 in pBPC)
C410 (58-GGGCTTaCTTGTCTCTGATGT-38, nucleotides 1460-1440 in pBPC)
C413 (58-AGCTCTTTCTaGGGGGCTTCC-38, nucleotides 1472-1453 in pBPC)
C416 (58-GCTAAGGTGCtCAGCTCTTCT-38, nucleotides 1484-1464 in pBPC)
PC401
PC403
PC406
PC407
PC408
PC409
PC410
PC413
PC416
The oligonucleotides used to construct the deletion mutants of PCN and wtPC are shown. The mutant bases are indicated by lower case letters.
membrane (Bio-Rad, Hercules, CA), and then crosslinked using a UV
Stratalinker 1800 (Stratagene). The membrane was hybridized with
32P-labeled PC cDNA probe,4 and exposed to Kodak-XR5 film (Eastman Kodak, Rochester, NY) for 20 hours.
Measurements of protein C antigen and activity. The protein C
antigen level and anticoagulant activity were determined as previously
described.5 Statistical analysis of secreted protein C antigen and activity
level was performed between wtPC and other secreted proteins by
Mann-Whitney test. A P value of less than .05 was considered to be
significant.
Immunofluorescence microscopy and immunoelectron microscopy.
Immunofluorescence microscopy and immunoelectron microscopy were
performed as previously described.5
Molecular model of protein C. Molecular models of activated
protein C were constructed using the homology modeling methods, as
described previously.11 Briefly, coagulation factor Xa (Protein Data
Bank code: 1HCG)12,13 was used as the template protein for model
construction of the second EGF-like and protease domains of activated
Fig 1. (A) The alignment of carboxyl-terminal amino acids of
wtPC, PCN, and their deletion
mutants. The carboxyl-terminal
amino acids are indicated by
standard one-letter designations.
Hydrophobic regions of PCN are
underlined. The hydropathy plot
was performed with the LASERGENE Navigator (DNASTAR Inc,
Madison, WI) determined by the
methods of Kyte and Doolittle.34
(B) Northern blot analysis of the
stably transfected CHO cells. Total RNA (5 µg) from each cell line
was separated by 1% formaldehyde-agarose gel electrophoresis, transferred to a Zeta-Probe
membrane (Bio-Rad), and hybridized with 32P-labeled PC cDNA
probe.4
B
protein C. In the present study, the catalytic domain was modeled and
energetically optimized for the position 170-41211 and, hence, the wtPC
model was called PC412. To obtain the models of two deletion mutants
of protein C named PC408 and PC410, the carboxyl-terminal amino
acids corresponding to D409-A412 and E411-A412 were deleted,
respectively. To obtain the best structural deletion models, the full set of
protein C protease domain coordinates was subjected to energy
optimization and molecular dynamics (MD) calculation in a sphere of
76Å diameter packed with 5746 and 5738 water molecules for PC408
and PC410, respectively. Energy minimization and MD calculation
procedures were carried out using a program APRICOT14 with AMBER
united atom force field.15 Constraints by SHAKE algorithm16 were used
on bonds with hydrogens and lone pairs.
RESULTS
Pulse-labeling of the deletion mutants of PCN. The 81
carboxyl-terminal amino acids of PCN contain highly hydropho-
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3786
bic regions at residues 445-458 (Fig 1A). Previous study
showed that the carboxyl-terminal long hydrophobic amino
acids of pregnancy-specific glycoprotein (PSG) specified intracellular retention and stability.17 To investigate the effects of the
carboxyl-terminal hydrophobic region of PCN on its secretion,
we constructed four deletion mutants truncated at 444, 425, 388,
and 380, which is immediately before the frameshift mutation.
Chinese hamster ovary (CHO) cells stably transfected with the
different constructs were pulse-labeled with [35 S]Met and
chased for up to 6 hours. None of the truncated variants were
efficiently secreted from CHO cells (Fig 2A). The protein C
messenger RNA (mRNA) levels of each cell line were comparable (Fig 1B), indicating that impaired secretion of the mutant
proteins was not due to the depressed levels of transcripts.
These results showed that the carboxyl-terminal extension is not
predominantly responsible for the intracellular retention of
PCN, and that the 39 carboxyl-terminal amino acids of wtPC
are required for secretion.
Truncation of wtPC before D409 prevents its secretion. To
obtain information on the structural requirements necessary to
the secretion, we expressed a series of recombinant molecules
containing limited deletion of the sequence present in the
carboxyl-terminal of wtPC. The results showed that PC416,
PC413, PC410, and PC409 were efficiently secreted from the
cells. The secreted antigen levels of these mutants were
KATSUMI ET AL
significantly lower than that of wtPC (Table 2). The relative
activities of secreted protein C mutants were not significantly
different from wtPC (Table 2). On the other hand, PC408,
PC407, PC406, PC403, and PC401 appeared to undergo
intracellular retention and degradation. In all of these cases,
intracellular degradation was apparent by 6 hours after initiation
of the chase (Fig 2B).
Immunofluorescence microscopy. Intracellular localization
of selected mutants of protein C was examined by immunofluorescence microscopy (Fig 3). In transfected CHO cells expressing PC407 and PC408, cytoplasmic reticular staining was
observed, whereas a perinuclear spot was not observed (Fig 3a
and 3b). In the cells expressing PC409 (Fig 3c) and wtPC (Fig
3d), both perinuclear spots with prominent brightness and
cytoplasmic reticular staining were observed, which correspond
to the rough ER and the Golgi apparatus, respectively.5,18,19,20
No immunofluorescence was detected in mock-transfected
CHO cells (Fig 3e). Thus, PC407 and PC408 appear to be
retained in the rough ER, whereas PC409 exhibits normal
secretion kinetics.
Immunoelectron microscopy. Precise localization of PC407,
PC408, and PC409 was determined by postembedding immunoelectron microscopy using a 10 nm colloidal gold-conjugated
second antibody. In the CHO cells expressing PC407 and
PC408, gold particles were mainly distributed in the rough ER
Fig 2. (A) Pulse-chase analysis of PCN and its deletion mutants. Cells were labeled and chased as described previously.5 The data are
expressed as percent of the intracellular 35S-labeled protein C present at the zero time point. (d, intracellular protein C; s, secreted protein C).
Values represent the means 6 standard deviation of three independent experiments. (B) Pulse-chase analysis of wild-type protein C and its
deletion mutants. Cells were labeled and chased as described previously.5 The data are expressed as percent of the intracellular 35S-labeled
protein C present at the zero time point (d, intracellular protein C; s, secreted protein C; 3, total protein C, ie, the summation of secreted and
intracellular material). Values represent the means 6 standard deviation of three independent experiments.
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CARBOXYL-TERMINAL OF PROTEIN C
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Table 2. Expression Levels of Normal and Mutated Protein C
From Stably Transfected CHO Cells
Proteins
Intracellular Protein
C Ag (ng/1 3 106
cells/24 h) n 5 2
Secreted Protein
C Ag (ng/1 3 106
cells/24 h) n 5 3
Relative Activity
of Secreted Protein
C (%) n 5 3
PCN
PCN444
PCN425
PCN388
PCN380
wtPC
PC416
PC413
PC410
PC409
PC408
PC407
PC406
PC403
PC401
85.2 6 5.5
9.8 6 0.2
9.3 6 2.8
7.4 6 0.7
22.9 6 0.3
151.7 6 0.4
71.3 6 9.2
62.2 6 3.7
58.5 6 0.1
47.6 6 2.1
21.2 6 0.5
7.3 6 0.5
16.5 6 0.5
7.8 6 0.5
12.7 6 0.7
ND
ND
ND
ND
ND
707.2 6 21.3
557.5 6 73.4*
528.9 6 97.8*
454.7 6 82.6*
435.0 6 72.2*
ND
ND
ND
ND
ND
NE
NE
NE
NE
NE
94.3 6 0.8
88.4 6 5.1
96.5 6 10.0
92.5 6 1.0
93.2 6 5.8
NE
NE
NE
NE
NE
The antigen and relative activity of protein C were measured as
described previously.5 Statistical analysis of secreted protein C antigen and activity levels were performed between wtPC and other
secreted proteins by Mann-Whitney test.
*Denotes a P value of ,.05.
Abbreviations: ND, not detected; NE, not examined.
(Fig 4a and 4c), and no gold particles were localized to the
Golgi apparatus (Fig 4b and 4d). In the cells expressing PC409,
the Golgi apparatus was heavily labeled with gold particles (Fig
4f) in addition to the localization of particles in the rough ER
Fig 3. Immunofluorescence microscopic localization of different protein C variants transfected in the
CHO cells protocols are described under Materials
and Methods. In the cells expressing PC407 (a) and
PC408 (b), cytoplasmic reticular staining was observed. In the cells expressing PC409 (c) and wtPC
(d), a perinuclear spot with prominent brightness
(arrow heads) was observed in addition to the
cytoplasmic reticular staining. No immunofluorescence was detected in the mock-transfected CHO
cells (e). Scale bars, 10 mm.
(Fig 4e). Mock-transfected CHO cells did not show any specific
immunolabelling (Fig 4g and h). From these results, we
conclude that the transport of PC407 and PC408 from rough ER
to the Golgi apparatus is blocked and that these mutant proteins
are retained in the rough ER, whereas PC409 was transported
through the Golgi apparatus and secreted normally.
Molecular modeling and MD calculation of the catalytic
domain of two truncated protein C mutants. To distinguish the
structural differences between the secreted form PC410 and the
ER retention form PC408, we constructed models of two
deletion mutants of activated protein C. They were constructed
with water molecules to obtain the information about conformational stability at room temperature and to describe the fluctuation of the hydrophobic core regions during the MD calculation.
Figure 5A shows the full set of PC410 coordinates in a sphere of
76Å diameter packed with 5,738 water molecules. The overall
tertiary structural model of PC410 was superimposed with that
of wild-type PC412 except for the carboxyl-terminal two amino
acids deletion in root mean square deviation for main chain Ca
atoms of 1.80Å (data not shown). The PC410 model constructed
with water molecules predicts that the carboxyl-terminal region
of the protease domain has an alpha-helix with three turns from
Y399 to K410 (Fig 5B and 5C). This structural feature in the
carboxyl-terminal region is widely observed among serine
protease family proteins, which are 1TLD (bovine beta-trypsin),
1PFX (porcine factor IXa), 1TRN (human trypsin), 1PPB
(human alpha-thrombin), 1HCG (human coagulation factor
Xa), 1LMW (human urokinase-type plasminogen activator),
5CHA (bovine alpha-chymotrypsin A), 2PKA (porcine kal-
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3788
KATSUMI ET AL
Fig 4. Immunoelectron microscopic localization
of different protein C variants transfected in the CHO
cells. Transfected CHO cells were examined by
postembedding immunoelectron microscopy using
10 nm colloidal gold-conjugated second antibody. In
the cells expressing PC407 (a, b) and PC408 (c, d),
gold particles were mainly distributed in rough ER
(a, c), and no gold particles were localized to the
Golgi apparatus (b, d). In the cells expressing PC409,
gold particles were distributed in both rough ER (e)
and Golgi apparatus (f). The mock-transfected CHO
cells showed no specific immunolabelling (g, h).
Arrowheads, rough ER; G, Golgi apparatus; N,
nucleus. Scale bar, 100 nm for a, c, e and g; 50 nm for
b, d, f and h.
likrein A), 3EST (porcine pancreas elastase), 3RP2 (rat mast
cell protease), 1HNE (human leukocyte elastase), 1TON (rat
tonin), and 1FUJ (a neutrophil serine protease antigen of
Wegener’s granulomatosis antibodies) in the Protein Data Bank
(Table 3). The alpha-helix with three turns forms the hydrophobic core, and four hydrophobic interactions exist in this
hydrophobic core of these serine proteases. In the hydrophobic
core of PC410, three hydrophobic amino acid residues Y399,
I403, and I407 on the inner face of the alpha-helix interact
specifically with the side chains of I201, W205, I258, and L278
(Fig 5C). Table 3 shows four hydrophobic distances in this
hydrophobic core. The hydrophobic distances were defined to
be the nearest distance between two hydrophobic amino acid
residues. This hydrophobic core formation is important to
maintain the tertiary structure. Therefore, it is worth examining
if the carboxyl-terminal alpha-helix is conserved in the ER
retention form PC408. We constructed the model of PC408
using the same procedures. The resulting PC408 model was
superimposed with that of PC412 (data not shown). In the
comparison of PC408 with PC410, the root mean square
deviation for Ca atoms of the catalytic domain was 1.77Å. The
PC408 model showed the same four hydrophobic distances as
those in PC410 (Table 3). Trypsin also showed the same
hydrophobic distances that are positioned in the equivalent
places on the sequence alignment. Because hydrophobic distances are in a range from 3.4 to 6.4,21 all three residues of I407,
I403, and Y399 can still form the expected bonds with
hydrophobic amino acid residues in the catalytic domain in both
secretory form PC410 as well as ER retention form PC408,
indicating that the hydrophobic core is intact. Furthermore, the
amino acid deletion at the carboxyl-terminal region did not
expose any hydrophobic regions on either PC410 or PC408.
DISCUSSION
Several studies have shown the significance of the carboxylterminal region of protein C. Peptide inhibition studies indicate
that potential sites of the activated protein C factor Va interaction are located on the exposed portion of the carboxyl-terminal
alpha helix, K395-H404.22 The crystal structure of protein C
supported this finding.23 A database of naturally occurring
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CARBOXYL-TERMINAL OF PROTEIN C
mutations and their clinical phenotypes has been established,3
and analysis of these mutants of protein C showed that natural
type I mutants I403L and I403M occur in the carboxyl-terminal
helix H3 (L400-R408) within the hydrophobic patch, and the
likely perturbation of the helix would interfere with the proper
interaction of the serine protease domain with the second EGF
domain.24,25,26
Earlier results indicated the existence of a complex containing GRP78, GRP94, and PC.5 Whereas these two stress proteins
are strongly associated with PCN, only low amounts form a
complex with wtPC. Our interpretation of these data is that PCN
does not acquire its proper folding and is available for longer
3789
Table 3. Hydrophobic Distances (Å) Between Two Hydrophobic
Amino Acid Residues
I407Cg2-W205Cg
I403Cg2-I201Cd1
I403Cd2-L278Cd2
Y399Cd2-I258Cg1
PC408
(Å)
PC410
(Å)
PC412
(Å)
Trypsin*
(Å)
Average and
Standard Deviation
of 13 Serine
Proteases
3.6
3.85
5
3.47
3.44
3.58
4.94
3.52
3.74
3.7
4.94
4.86
4.46
3.71
4.97
5.15
3.76 6 0.41
4.2 6 0.68
3.97 6 0.36
3.74 6 0.24
Hydrophobic distances (Å) in the hydrophobic core in relation to the
alpha-helix with three rounds from Y399 to K410 in PC408, PC410,
PC412, bovine beta-trypsin, and average and standard deviation of 13
serine proteases, which are 1TLD (bovine beta-trypsin), 1PFX (porcine
factor IXa), 1TRN (human trypsin), 1PPB (human alpha-thrombin),
1HCG (human coagulation factor Xa), 1LMW (human urokinase-type
plasminogen activator), 5CHA (bovine alpha-chymotrypsin A), 2PKA
(porcine kallikrein A), 3EST (porcine pancreas elastase), 3RP2 (rat
mast cell protease), 1HNE (human leukocyte elastase), 1TON (rat
tonin), and 1FUJ (a neutrophil serine protease antigen of Wegener’s
granulomatosis antibodies) in the Protein Data Bank. The nearest
distance between two hydrophobic amino acid residues is defined as
the hydrophobic distance.
*Amino acids number is described by protein C numbering. The
alignment was shown previously.11
periods of time than wtPC to associate with these two ER
chaperones.
In the present study, to examine why PCN is not secreted, we
constructed a series of deletion mutants of the 81 abnormal
amino acids at the carboxyl-terminal of PCN. This rationale was
based on a previous study showing that mutant PSG with
carboxyl-terminal hydrophobic amino acids was retained in the
ER, and deletion of this region caused effective secretion. The
extra hydrophobic amino acids constituted an ER retention
signal.17 Moreover, several studies indicated that the carboxylterminal region of certain proteins is essential for secretion and
stability.27,28 In the present study, however, none of the truncated PCN proteins were secreted. Although the presence of 81
abnormal amino acids may contribute to the intracellular
retention, these results indicated that the loss of normal
carboxyl-terminal amino acids (G381-P419) is responsible for
;
Fig 5. (A) The model of the secreted form of truncated protein C,
PC410, in the presence of water molecules. The full set of protein C
protease domain coordinates was subjected to energy optimization
in a sphere of 76Å diameter packed with 5,738 water molecules to
obtain the best structural deletion models. The activated protein C
was shown by tube model (red) and the water molecules were shown
by blue. (B) The model of the secreted form, PC410. The average main
chain coordinates during 45 and 60 ps obtained from MD calculation
was shown by the tube model structure. The active site triad was
shown by the CPK shells (blue), and the carboxyl-terminal threerounds alpha-helix from Tyr399 to Lys410 was shown by yellow. The
side chains of Arg408, Asp409, and Lys 410 were shown by the ball
and stick model by red, green, and blue, respectively. (C) Hydrophobic
interaction of the carboxyl-terminal alpha-helix with the surrounding
residues. The carboxyl-terminal structure of PC410 was enlarged and
the residues involved in the hydrophobic interaction with the carboxylterminal alpha-helix from Tyr399 to Lys410 were shown by the ball
and stick model in yellow. This figure was taken from the backside of
Fig 5B.
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3790
the intracellular retention of PCN rather than the extension by
abnormal amino acids.
Deletion mutagenesis of wtPC shows that forms of protein C
differing in length by only one amino acid (K408 versus D409
residues) have very different fates. Pulse-labeling analysis
showed that the shorter proteins were degraded intracellularly.
Immunoelectron microscopy showed these molecules were not
transported from the ER to the Golgi apparatus. The longer
molecules, extended by one residue to include D409, were
mostly secreted. A part of these longer molecules was degraded
intracellularly, indicating that they did not show an entirely
normal secretion pattern (Fig 2b). These data showed that
changes in this carboxyl-terminal region may have significant
effects on the secretion of this protein.
Previous studies have reported that carboxyl-terminal amino
acids play important roles in the secretion of many proteins.17,29,30,31 Among vitamin K-dependent glycoproteins, the
carboxyl-terminal amino acid residues 403-415 of factor IX
(398-410 in protein C) were studied for their role in protein
secretion.32 The mutations of T415 (K410 in protein C) showed
only small (T415L, T415S and T415R) or moderate (T415G
and T415I) reductions both in the intracellular and secreted
factor IX levels. On the other hand, another group (Y404H,
Y404P, W407R, I408N, T412K and T412N) had high to
moderate levels of intracellular factor IX with no detectable or
only basal levels of secreted factor IX. The specific activities
observed for secreted mutant factor IX were similar to the
normal protein. Severe phenotypes observed for hemophilia B
patients with K411stop and W407stop (H406 and W402 in
protein C, respectively) further support the importance of the
carboxyl-terminal region.33 These findings are consistent with
our results.
In this study, we constructed two structural models of
deletion mutants of activated protein C, the secretion form
PC410 and the ER retention form PC408, using energy optimization and MD calculation with water molecules. Although the
other models of protein C were previously published,11, 24-26
none of them were constructed with the water molecules. The
overall tertiary structural models of both PC410 and PC408
were superimposed with that of wild-type PC412. In general,
the serine protease family has a common structural feature of an
alpha-helix with three turns at the carboxyl-terminal region. In
activated protein C, the helix is calculated to be from Y399 to
K410. Therefore, before model construction, we speculated that
the ER retention form PC408 has the structural abnormalities on
the carboxyl-terminal alpha-helix. However, even though the
model was constructed with the water molecules, the PC408
model showed that the hydrophobic distances in the hydrophobic core in relation to the alpha-helix with three rounds from
Y399 to K410 were the same as those in PC410 and in trypsin
(Table 3). Accordingly, once the native folding of PC408
occurred, the structure of PC408 was estimated to be thermally
stable in the same manner as that of PC410. Therefore, it was
not apparent from the model construction that the removal of
carboxyl-terminal amino acids should result in structural changes
that may lead to improper folding and consequently, defective
secretion. Although the overall conformation was not so
different, the ER resident molecular chaperones may recognize
very subtle changes of the PC408 molecules. Alternatively, the
KATSUMI ET AL
molecular chaperones may recognize the intermediate structure
of the folding process of PC408 that would not be predicted by
the modeling algorithm.
ACKNOWLEDGMENT
We thank Drs J. Evan Sadler, Maurice J. Keller, Tadashi Matsushita,
and Koji Yamamoto for their critical reading of the manuscript and Dr
Kotoku Kurachi for helpful discussions. We also thank Tomoko
Ichihara, Satoshi Suzuki, Mieko Suzuki, and Chika Wakamatsu for their
excellent technical help.
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1998 91: 3784-3791
The Carboxyl-Terminal Region of Protein C Is Essential for Its Secretion
Akira Katsumi, Tetsuhito Kojima, Takao Senda, Tomio Yamazaki, Hiroaki Tsukamoto, Isamu Sugiura,
Shigeru Kobayashi, Toshiyuki Miyata, Hideaki Umeyama and Hidehiko Saito
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