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Blood, Vol. 94 No. 4 (August 15), 1999:
pp. 1337-1347
Multimerin Processing by Cells With and Without Pathways for Regulated
Protein Secretion
By
Catherine P.M. Hayward,
Zhili Song,
Shilun Zheng,
Roxanna Fung,
Menaka Pai,
Jean-Marc Massé, and
Elisabeth M. Cramer
From the Departments of Pathology and Molecular Medicine, Laboratory
Medicine, and Medicine, McMaster University, Hamilton, Ontario,
Canada; the Hamilton Health Sciences Corp, Hamilton,
Ontario, Canada; and INSERM U474, Hôpital Henri Mondor,
Créteil, France.
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ABSTRACT |
Multimerin is a massive, soluble, homomultimeric, factor V-binding
protein found in platelet  -granules and in vascular endothelium. Unlike platelets, endothelial cells contain multimerin within granules
that lack the secretory granule membrane protein P-selectin, and in
culture, they constitutively secrete most of their synthesized multimerin. To further evaluate multimerin's posttranslational processing and storage, we expressed human endothelial cell
prepromultimerin in a variety of cell lines, with and without pathways
for regulated secretion. The recombinant multimerin produced by these
different cells showed variations in its glycosylation, proteolytic
processing, and multimer profile, and human embryonic kidney 293 cells
recapitulated multimerin's normal processing for constitutive
secretion by human endothelial cells. When multimerin was expressed in
a neuroendocrine cell line capable of regulated protein secretion, it
was efficiently targeted for regulated secretion. However, the
multimerin stored in these cells was proteolyzed more extensively than
normally occurs in platelets, suggesting that endoproteases similar to those expressed by megakaryocytes are required to produce platelet-type multimerin. The impact of the tissue-specific differences in
multimerin's posttranslational processing on its functions is not yet
known. Multimerin's sorting and targeting for regulated secretion may be important for its functions and its association with factor V in
secretion granules.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
MULTIMERIN IS A massive, soluble protein
found in platelets, megakaryocytes, vascular endothelium, and the
fibrillary extracellular matrix of cultured endothelium.1-4
Like von Willebrand factor (vWF), multimerin is one of the largest
platelet and endothelial cell proteins, and it is composed of variably
sized, disulfide-linked homomultimers.4-6 However, unlike
vWF, multimerin is not detectable in normal plasma.1,5
Studies of multimerin's functions indicate it is the major platelet
binding protein for coagulation factor V.3 Because the
biologically active stores of coagulation factor V in platelets are
stored within  -granules as a complex with multimerin,3
multimerin could be important for regulating the factor V stored in
platelets. Furthermore, multimerin's unusual structure and similarity
to adhesive and extracellular matrix proteins, its normal sequestration
in resting platelets and vascular endothelium, and its associations
with activated platelets, endothelial cells, and the extracellular
matrix suggest that multimerin has roles in supporting platelet and
vascular functions at sites of vessel injury.1-7 But
studies of multimerin's functions have been limited by the quantities
of multimerin in human platelets and endothelial cells, the procedures
required to isolate multimerin from these cells,4,5,8 and
the lack of established models for producing recombinant multimerin
that resembles the forms normally produced in platelets and endothelial cells.
Multimerin's large homomultimers are assembled from a novel precursor
protein, prepromultimerin, that is expressed in megakaryocytes and
vascular endothelium.1,2,4,7 Prepromultimerin contains a
signal sequence, multiple N-glycosylation sites, an RGDS site, a
central coiled-coil region, epidermal growth factor (EGF)-like and
partial EGF-like domains, and a C-terminal region that resembles the
globular domain in the extracellular matrix proteins collagens type
VIII and X.7 In both platelets and endothelial cells, prepromultimerin undergoes extensive N-glycosylation, proteolytic processing, and multimerization to generate mature
multimerin.1,2,4 Although the prepromultimerin transcripts
expressed in platelets, megakaryocyte cell lines, and endothelial cells
are identical in size, there are differences in multimerin's
posttranslational processing by these cells.1,2,4 When
multimerin is synthesized and secreted by phorbol myristate acetate
(PMA)-treated Dami cells (a megakaryocyte cell line) and
endothelial cells, it contains large amounts of uncleaved promultimerin
and proportionally less of the high molecular weight forms found in
platelets.2,4 Promultimerin is cleaved to smaller forms in
platelets; these forms have been designated by different
laboratories as p-155 and p-170, or glycoprotein Ia*, because of
similarities in multimerin's reduced mobility with an unrelated
platelet protein, glycoprotein Ia.1,2,4-8 The size of
N-deglycosylated platelet multimerin and its cleavage near the RGDS
domain suggest that prepromultimerin is cleaved only in its N-terminal
region in platelets, to generate a form that contains the RGDS,
coiled-coil, EGF-like, and globular domains of
prepromultimerin.2,7,8 However, PMA-treated Dami cells and
cultured endothelial cells appear to process and proteolyze multimerin
differently from platelets, because their multimerin subunits do not
comigrate with platelet multimerin, before or after removal of their
N-linked carbohydrate.2,4
In platelets and endothelial cells, multimerin is contained within
dense core secretion granules, suggesting that multimerin has the
property to be targeted for regulated secretion in a variety of cell
types.2,4 However, there are unusual features of multimerin's distribution in platelets and endothelial cells. Unlike
most soluble proteins stored in platelet -granules, multimerin is
stored with vWF and coagulation factor V in the eccentric, electron-lucent zone.2,3,9-11 Multimerin's
storage in -granules is not altered by severe vWF deficiency,
indicating that it is sorted to the electron-lucent zone independently
of vWF.9 In human vascular endothelial cells, multimerin
and vWF are primarily sorted to different dense core granules, although
trace amounts of multimerin are present in some
vWF/P-selectin-containing Weibel-Palade bodies.4 Because
the internal granule membrane protein P-selectin12-17 sorts
to several types of secretion granules when expressed in heterologous
cells,18 its undetectable levels in the multimerin granules
within endothelial cells suggest that P-selectin and multimerin are
sorted differently by secretory pathways.4
Multimerin's variant processing and storage by platelets and
endothelial cells, its undetectable levels in normal plasma, and its
putative role as an intragranular factor V carrier protein led us to
investigate the processing and sorting of human prepromultimerin using
heterologous cells. Our objectives were to determine if multimerin
contained information to signal its sorting into the regulated
secretory pathway, to evaluate the effects of constitutive and
regulated secretory pathways on its processing, and to determine if
heterologous cells could model multimerin's normal production by human
platelets and endothelial cells.
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MATERIALS AND METHODS |
Cell preparation and culture.
Human platelets and endothelial cells were isolated as
described.2-6 Human embryonic kidney (HEK) 293 cells (gift
from Dr F. Graham, McMaster University, Hamilton, Ontario,
Canada) and the Chinese hamster ovary cell line CHO
(American Type Culture Collection [ATCC], Manassas, VA) were cultured
in Minimum Essential Medium alpha Medium (-MEM; GIBCO/BRL,
Burlington, Ontario, Canada) supplemented with 10% fetal bovine serum
(FBS), 10 mmol/L HEPES, 100 U/mL penicillin G, and 100 µg/mL
streptomycin. The monkey kidney cell line COS-1 and the mouse pituitary
tumor cell line AtT 20/D16V-F2 (ATCC) were cultured in Dulbecco's
modified Eagle's medium (D-MEM; GIBCO/BRL) supplemented with 10 mmol/L
HEPES, 0.12% sodium bicarbonate, 10% FBS, 100 U/mL penicillin G, and
100 µg/mL streptomycin.
Generation of the multimerin construct and cell transfections.
To obtain a full-length cDNA sequence encoding human
prepromultimerin,7 a fragment spanning the internal
EcoRI site was reverse transcribed from endothelial cell RNA
using Superscript II (GIBCO/BRL) and the primer
5'-GAGCAGATGTGCAAGCAAAGAT-3' for bp 3815 to 3836 in the
3' untranslated prepromultimerin cDNA. The region between 1834 and 3805 of the prepromultimerin cDNA was then amplified by 30 cycles
of the polymerase chain reaction (PCR) using pfu DNA polymerase
(GIBCO/BRL), the forward primer (bp 1834-1856)
5'AUGGAGAUCUCUTCAGAGGTGAATGTGAAGACATG-3', and the reverse
primer (bp 3783-3805)
5'-ACGCGUACUAGUCACTGGCTGTTTCTCAATAAAGG-3'. The resulting
PCR product was subcloned into pAMP19 (GIBCO/BRL), and double-stranded
sequencing indicated that the insert's coding region corresponded to
the published 3' prepromultimerin sequence.7 This
3' region of prepromultimerin was excised as an Nco
I-Kpn I fragment and subcloned into Nco I-Kpn
I-cut pGEM7Zf that contained the complete 5' region from the
prepromultimerin cDNA clone 17.7 The resulting
prepromultimerin construct, containing the complete coding sequence,
was excised as an Xho I-Kpn I fragment and subcloned into the expression vector pCMV5 (gift from Dr M. Stinski, University of Iowa, Iowa City, IA), and the correct composition and
orientation of the construct pCMV5-multimerin was verified by partial
sequencing and restriction mapping.7 The empty pCMV5 vector
was used for control, mock transfections.
For transient and stable transfection studies, cells were transfected
using lipofectamine (GIBCO/BRL) according to the supplier's recommendations. Briefly, cells were grown to 50% to 80% confluence in 35-mm wells and transfected using 2 µg of DNA/well and conditions determined to be optimal for control transfections using lipofectamine and the vector pGreen Lantern-1 (GIBCO/BRL; percentage of cells transfected evaluated by immunostaining4). To generate
stably transfected cell lines expressing multimerin, AtT 20 and HEK 293 cells were cotransfected using lipofectamine, 2 µg of
pCMV5-multimerin, and 0.2 µg of the neomycin resistance vector
pSV2neo/35-mm well. Forty-eight hours posttransfection, cells were
subcultured using media containing 0.5 mg/mL active G418.
Neomycin-resistant clones were selected, cloned by limiting dilution,
and evaluated for multimerin production using an enzyme-linked
immunosorbent assay (ELISA).19
Metabolic labeling.
Metabolic labeling studies2,4 were performed using
methionine-free D-MEM media supplemented with
35S-methionine (Mandel Scientific Co Ltd, Guelph, Ontario,
Canada; 0.5 mCi/mL for 30-minute pulse labeling studies of HEK 293, COS-1, and AtT 20 cells; 0.1 mCi/mL for other studies), 10% dialyzed FBS (HEK 293 and COS-1 cells), and 10% to 20% complete D-MEM. For
transient expression studies, cells were incubated with labeling media
44 to 52 hours posttransfection. To study multimerin secretion from
stably transfected AtT 20 cells, cells were plated at 40% confluence
and cultured overnight before labeling in media with 20% D-MEM. After
28 hours of continuous labeling, cells were washed and chased
consecutively for 12 hours and 1 hour in complete and then serum-free
media to allow secretion of all of the radiolabeled multimerin destined
for constitutive release. Postchase, cells were incubated with serum
free media, with or without the secretagogue 10 mmol/L 8-Br-cAMP (Sigma
Chemical Co, St Louis, MO) for 60 minutes, followed by
radioimmunoprecipitation analyses of the multimerin in the media and
cell lysate fractions. Radiolabeled multimerin was prepared from
passage 1 human umbilical vein endothelial cells and 125I
labeled platelets as previously described.4,5
Preparation of cell lysates and culture media.
Cell lysates (1% Triton X-100) and culture media were collected into
protease inhibitors as described4-6 or into buffer
containing 4.0 mmol/L pefabloc, 0.3 µmol/L aprotinin, 100 µg/mL
soybean trypsin inhibitor, 28 µmol/L E64, 1 µmol/L leupeptin, 5 mmol/L N-ethyl-maleimide, 1 µmol/L pepstatin, 100 µmol/L
phenanthroline, and 10 to 20 mmol/L EDTA. All buffers contained EDTA,
which effectively prevented multimerin degradation by calcium-dependent
proteases. The multimerin prepared by these different procedures had an
identical mobility on nonreduced and reduced gels. For quantitative
analyses, samples were harvested on day 3 from T25 flasks that were
80% to 100% confluent. To evaluate regulated secretion, transfected
AtT 20 cells were grown in 6-well plates for 2 days until 95%
confluent and then incubated in 0.5 mL of serum-free media with or
without 10 mmol/L 8-Br-cAMP for 60 minutes.
Glycoprotein analyses.
The multimerin content of cell lysates and culture media were evaluated
(triplicate studies) using an ELISA and a pooled platelet lysate
standard (1 milliunit of multimerin was defined as the amount in
106 platelets).19 For studies of regulated
secretion, the quantities of multimerin secreted were expressed as a
percentage of the total cell lysate multimerin antigen content, and a
1-tail t-test was used to determine if significantly more
multimerin antigen was released when the cells were stimulated with secretagogue.
Immunoblotting and radioimmunoprecipitation analyses were performed as
described.2,4 Briefly, the multimerin in equivalent volumes
of culture media and cell lysates (10% to 30% of T25 flask contents)
was affinity-purified using monoclonal antimultimerin (JS-1) capture
beads, followed by analyses using reduced 4% to 8% sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) or nonreduced
1.25% agarose/1% acrylamide gels6 and autoradiography or
immunoblotting with polyclonal antimultimerin.4,5 To
characterize N-linked carbohydrate, radioimmunoprecipitates were
treated with N-glycanase F, endoglycosidase H, or control buffer, as
previously described.2 For some studies, affinity-purified
recombinant multimerin was treated with sample buffer containing 0, 0.01, 0.2, 0.5, 1.0, 2.0, 5.0, 10, 25, or 100 mmol/L dithiothreitol (DTT),6 followed by analyses using 3% to 8% SDS-PAGE and
immunoblotting with polyclonal antimultimerin.
Immunofluorescent and immunoelectron microscopy.
Cells prepared for immunofluorescent antibody labeling were cultured
onto poly-D-lysine-coated glass coverslips. Proteins were visualized
using fluorescein isothiocyanate-conjugated or Texas-Red-conjugated
secondary antibodies and standard immunofluorescent microscopy or
confocal optical sectioning as previously described.4 The
primary antibodies used to label the cells included monoclonal (JS-1;
10 µg/mL)5 and polyclonal (1/200 dilution)
antimultimerin5 and rabbit polyclonal anti-ACTH (1/50
dilution; Cedarlane Laboratories Ltd, Hornby, Ontario, Canada).
Negative controls included cells labeled with normal mouse IgG and
normal rabbit IgG or without primary antibodies. The intracellular
distribution of multimerin and ACTH in control and
multimerin-transfected AtT-20 cells was investigated using electron
microscopy (EM), previously described methods for single and double
immunolabeling, and secondary antibodies conjugated with 10- and 15-nm
immunogold.4,20,21 For double-labeled EM preparations, thin
sections were labeled with polyclonal rabbit antimultimerin (1/200
dilution)5 before labeling the opposite sides of the
sections with antibodies to ACTH (1/30 dilution).4 To
exclude false-positive immunolabeling, the negative controls included
mock-transfected AtT 20 cells and, for the double-labeled preparations,
sections labeled without one of the primary antibodies.4
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RESULTS |
Intracellular distribution of multimerin in transfected heterologous
cells.
Immunofluorescent microscopy studies indicated that there was no
detectable multimerin staining in control or mock-transfected HEK 293, COS-1, or AtT 20 cells (Fig 1). When
multimerin was expressed in cell lines lacking regulated secretory
pathways (HEK 293 and COS-1 cells [Fig 1]; CHO cells [not shown]),
it was found throughout the cytoplasm, in a fine reticular
distribution, but no granules were evident (Fig 1). In contrast, within
transiently and stably transfected AtT 20 cells, multimerin was
localized in small granules, abundant at the distal cell tips (Figs 1
and 2; data representative of 12 of 12 clones), that were similar in size and distribution to the secretion
granules containing ACTH (Figs 3 and
4), but smaller than the multimerin
granules in endothelial cells (Fig 2).
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| Fig 1.
The distribution of multimerin in transfected HEK 293, COS-1, and AtT 20 cells immunolabeled with polyclonal
antimultimerin. Images acquired by standard immunofluorescent
microscopy (left and middle panels) and confocal optical sectioning
(right panels) are shown. Multimerin was not detected in control cells
transfected with the empty vector (panels labeled control). In HEK 293 and COS-1 cells transiently transfected with the multimerin cDNA
(panels labeled multimerin), recombinant multimerin was distributed
throughout the cytoplasm, without evidence of granule formation. In AtT
20 cells transiently (standard immunofluorescent microscope image) and
stably (confocal optical section image) transfected with the multimerin
vector, multimerin was predominantly located in small granule-like
structures that were most abundant at the distal cell tips (arrowheads;
N indicate cell nuclei).
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| Fig 2.
Confocal microscopy images (250-nm optical sections) of
human umbilical vein endothelial cells (HUVEC) and
quiescent and secretagogue-treated (+) AtT 20 cells. Cells were
immunolabeled with monoclonal antimultimerin after transfection
with the empty vector (control) or the multimerin expression vector.
The upper panels show images taken at a higher magnification.
Multimerin was identified in small granule-like structures
(permeabilized cells) that were concentrated at the cell tips of stably
transfected AtT 20 cells (arrows). Twenty minutes after stimulation
with 10 mmol/L 8-Br-cAMP, transfected AtT 20 cells showed intense
multimerin labeling in release patches (arrowheads) around the cells
that were not evident in control cells, and this redistribution was
associated with reduced multimerin granule labeling at the cell tips
(permeabilized cells).
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| Fig 3.
Electron micrographs of multimerin-transfected
AtT 20 cells, immunogold labeled with antibodies to ACTH (a) or
multimerin (b). (a) Cells labeled with immunogold for ACTH contained
gold particles within their secretion granules (sg), and the plasma
membrane (pm) and mitochondria (m) were not labeled (original
magnification × 60,000). (b) Cells labeled with immunogold for
multimerin contained multimerin in secretion granules (sg). The
mitochondria (m), as a control structure, and the plasma membrane (pm)
displayed no labeling for multimerin (original magnification × 60,000).
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| Fig 4.
Electron micrographs of multimerin-transfected AtT 20 cells, double-labeled for multimerin (10-nm gold) and ACTH (15-nm
gold). Some granules contained only multimerin (sg2), whereas others
contained both ACTH and multimerin (sg1, and inset; original
magnifications: main panel, ×60,000; inset, ×120,000).
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To determine if the structures containing multimerin in AtT 20 cells
had morphologic features of secretion granules, immunoelectron microscopy was performed. No significant multimerin labeling was observed with control AtT 20 cells. In AtT 20 cells stably transfected with the multimerin cDNA, there was abundant multimerin immunolabel within dense core secretion granules that resembled the ACTH storage granules (Fig 3). In multimerin-transfected AtT 20 cells that were
double-labeled (Fig 4), there was colocalization of multimerin and ACTH
in  50% of the dense core secretion granules that contained label for
these proteins, suggesting that they were similarly, but independently,
sorted to the secretion granules of AtT 20 cells. By electron
microscopy, the multimerin secretion granules in AtT 20 cells were
approximately two thirds the size of the multimerin granules in
endothelial cells (not shown), but they were the same size as the
ACTH-containing granules in AtT 20 cells.
Stably, multimerin-transfected AtT 20 cells were studied to determine
if they released multimerin when treated with 8-Br-cAMP, a secretagogue
that induces regulated secretion from AtT 20 cells.22 The
cell lysates and culture media from control and mock-transfected AtT 20 cells had undetectable multimerin in the ELISA. By 60 minutes after
treatment with 10 mmol/L 8-Br-cAMP, multimerin-transfected AtT 20 cells
released 8.9% of their cell lysate multimerin antigen (mean of 3 experiments; range, 8.1% to 10.4%), which was significantly greater
(P = .015) than the 2.4% released by quiescent cells (mean of
3 experiments; range, 2.0% to 2.8%). Furthermore, nonpermeabilized AtT 20 cells evaluated at 5 minutes (not shown) or 20 minutes after
stimulation with 10 mmol/L 8-Br-cAMP (Fig 2) showed intense labeling
for multimerin in release patches (Fig 2, arrowheads) on the external
surfaces and cell perimeters that were not evident on quiescent cells
(Fig 2). Secretagogue treatment was also associated with reduced
multimerin labeling of granules at the distal cell tips of stimulated
cells (Fig 2, permeabilized cells) and similar reduced, but not absent,
granule staining for ACTH at the cell tips (not shown). The multimerin
release patches on secretagogue-treated cells were much less apparent
by 60 to 90 minutes (not shown) when there was measurable
stimulus-induced release of multimerin into the culture media.
Processing of recombinant multimerin in heterologous cells, with and
without pathways for regulated secretion.
The cell lysates and culture media of the control and mock-transfected
cell lines did not contain detectable multimerin when evaluated by
ELISA, immunoblot (Fig 5), or
radioimmunoprecipitation assays (Fig 6A;
control AtT 20 cells). Small amounts of multimerin antigen (2 to 7 mU/T25 flask) were detected in the cell lysates of transiently
transfected CHO, COS-1, and HEK 293 cells, but most was found in their
culture media, consistent with its constitutive secretion (mU/T25 flask
culture media/3 days: 19 mU for CHO cells, 75 to 95 mU for COS-1 cells,
and 165 to 250 mU for HEK 293 cells). Comparisons of multimerin's
production and processing by AtT 20 cells and HEK 293 cells were
performed using stably transfected cells, because less than 1% of AtT
20 cells were successfully transfected by the vectors encoding
multimerin or the green fluorescent protein in transient expression
studies. The amounts of multimerin constitutively secreted by the
stably transfected clones ranged from 650 to 1,800 mU/T25 flask/3 days
for the HEK 293 cells to 400 to 1,315 mU/T25 flask/3 days for the AtT
20 cells. Stably transfected AtT 20 cells contained much more
multimerin in their cell lysates (490 to 640 mU/T25 flask) than stably
transfected HEK 293 cells (22 mU/T25 flask), suggesting that regulated
secretory pathways allowed multimerin to be retained within AtT 20 cells.
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| Fig 5.
Western blot comparing platelet (Plt) and endothelial
cell (EC) multimerin with recombinant multimerin synthesized by COS-1,
HEK 293, and AtT 20 cells. Transient transfection experiments are shown
in (A), and studies using stably transfected cells are shown in (B).
Cell lysates and culture media from cells transfected with the empty
vector (v) or the multimerin expression vector (m) were analyzed using
4% to 8%, reduced SDS-PAGE. Recently synthesized promultimerin
(proM), fully glycosylated promultimerin (proM*), and the platelet
multimerin subunits p-155 and p-170 are indicated.
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| Fig 6.
Pulse-chase metabolic labeling studies of recombinant
multimerin biosynthesis by AtT 20 cells. Radioimmunoprecipitates from a
30-minute pulse-labeling study (A; chase times indicated) and a 28-hour
continuous labeling study (B) were analyzed using 4% to 8% reduced
SDS-PAGE. In (B), labeled cells were consecutively chased for 12 hours
and 1 hour in cold media before 60 minutes of incubation (postchase)
with (+) or without ( ) the secretagogue 10 mmol/L 8-Br-cAMP. (A)
Multimerin was first synthesized as promultimerin (proM, Mr
160 kD), which migrated with a higher apparent molecular mass (proM*;
A) after additional processing of its N-linked carbohydrate. Multimerin
was constitutively secreted as proM* and smaller proteolyzed subunits.
(B) Proteolyzed multimerin, with a Mr of 122 kD (reduced),
persisted in the cell lysate after there was no further constitutive
secretion of radiolabeled multimerin during the chase and postchase
periods and it was secreted into the postchase medium only when the
cells were incubated with secretagogue.
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The predominant form of multimerin in the cell lysates of transfected
COS-1 (Fig 5), HEK 293 (Fig 5; stable and transient transfection
studies), and CHO cells (not shown) was recently synthesized
promultimerin (proM) that had not yet undergone full N-glycosylation.
Prolonged exposures (not shown) of these immunoblots confirmed that
only trace quantities of mature, fully glycosylated multimerin (proM*)
were contained in the lysates of HEK 293 and COS-1 cells, consistent
with their lack of regulated secretory pathways. Pulse-chase labeling
experiments (not shown) indicated that multimerin biosynthesis by
transfected HEK 293 cells and COS-1 modeled multimerin's processing
for constitutive secretion by human endothelial cells,4
because these cells constitutively secreted fully glycosylated
promultimerin (proM*) and smaller proteolyzed subunits but
did not retain mature multimerin. Although the multimerin subunits
secreted by COS-1 cells were smaller than those secreted by HEK 293 cells and human endothelial cells (Fig 5A), their similar mobility
after treatment with N-glycanase F (not shown) indicated that these
differences were due to less extensive N-glycosylation of multimerin by
COS-1 cells.
The processing of recombinant multimerin by AtT 20 cells was different
from the other cell lines. Transfected AtT 20 cells synthesized and
constitutively secreted multimerin that had subunits ranging in size
from 185 to 122 kD (Figs 5 and 6; Mr based on reduced 4%
to 8% SDS-PAGE). AtT 20 cell lysates mainly contained a 122-kD
multimerin subunit (smaller than the multimerin subunits produced in
platelets, endothelial cells, and the other cell lines) that comigrated
with the smallest form constitutively secreted by AtT 20 cells (Figs 5
and 6). Pulse-chase metabolic labeling studies (Fig 6) indicated that
AtT 20 cells first synthesized multimerin as its precursor
promultimerin (Fig 6, proM; 160 kD on 4% to 8% SDS-PAGE), which
subsequently migrated with a larger apparent molecular mass (proM*, Fig
6) due to further processing of its N-linked carbohydrate. Multimerin
was subsequently proteolyzed and constitutively secreted from AtT 20 cells as radiolabeled subunits that ranged in size from 122 to 185 kD
(Fig 6A). The form of multimerin retained within AtT 20 cells after
prolonged chases in cold media was the more extensively proteolyzed
122-kD subunit, indicating that there was more complete proteolytic
processing of the retained, compared with constitutively secreted,
multimerin by these cells (Fig 6B). Postchase, AtT 20 cells released
radiolabeled multimerin (Mr 122 kD, reduced) into the
culture media when incubated for 60 minutes with media containing
8-Br-cAMP, but not when they were incubated in media without
secretagogue, confirming that multimerin was processed for regulated
secretion in AtT 20 cells (Fig 6B; data representative of 2 of 2 experiments).
Comparisons of multimerin's subunit size after treatment with buffer,
N-glycanase F, or endoglycosidase H indicated that the multimerin
stored and secreted from AtT 20 cells contained mainly complex forms of
N-linked carbohydrate (Fig 7). As
previously reported, mature platelet (Fig 7) and endothelial cell
multimerin (not shown) were sensitive to N-glycanase F and resistant to
endoglycosidase H, indicating that they contained only complex forms of
N-linked carbohydrate.2,4 The recombinant multimerin
secreted by COS-1 and 293 cells was similarly endoglycosidase H
resistant (not shown). However, the multimerin stored and secreted by
AtT 20 cells contained small amounts of endoglycosidase H-sensitive
N-linked carbohydrate (Fig 7; data representative of 2 of 2 experiments), indicating that there was incomplete conversion of
multimerin's N-linked carbohydrate to complex forms when it was
synthesized in AtT 20 cells. After N-linked carbohydrates were removed
using N-glycanase F, 2 of the multimerin subunits constitutively
secreted from AtT-20 cells comigrated with the platelet multimerin
subunits p-155 and p-170, but AtT 20 cells also constitutively secreted
multimerin subunits that were larger and smaller than platelet p-155
and p-170, before and after N-deglycosylation (Fig 7). Because AtT 20 cell lysates contained a 122-kD subunit smaller than platelet p-155 and
p-170 after N-deglycosylation and smaller amounts of a subunit that
comigrated with platelet p-155 after N-deglycosylation (Fig 7), these
data confirmed that there was more extensive proteolytic processing of
multimerin when it was stored in AtT 20 cells compared with platelets.
In vitro proteolysis was not observed when affinity-purified platelet
multimerin was incubated overnight with undiluted culture media from
control and secretagogue-treated nontransfected AtT 20 cells (not
shown). These findings, and the less complete proteolysis of
constitutively secreted multimerin, suggested that intracellular endoproteases were responsible for producing the smaller subunits stored in AtT 20 cells.
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| Fig 7.
Comparisons of N-linked carbohydrate on platelet (plt)
and AtT 20 cell-derived multimerin. Cells were labeled for 28 hours as
in Fig 6B; their multimerin radioimmunoprecipitates were treated with
buffer, N-glycosidase F (endo-F), or endoglycosidase H (endo-H) and
then separated using reduced 4% to 8% SDS-PAGE (lane L, multimerin
constitutively secreted during the labeling; lane C, multimerin
remaining in the cell lysate postchase; lane S, longer exposure showing
the multimerin secreted postchase in response to 8-Br-cAMP). The
125I-labeled platelet multimerin subunits p-155 (arrows)
and p-170 (arrowheads) are indicated. The coprecipitated, 83-kD
platelet protein, which was resistant to N-glycosidase F and
N-glycosidase H, was not observed in all studies. The multimerin stored
in AtT 20 cells was more extensively proteolyzed than constitutively
secreted multimerin. Two of the multimerin subunits constitutively
secreted by AtT 20 cells (lanes L) comigrated with p-155 and p-170
after their N-linked carbohydrates were removed with N-glycanase F. The
major form of multimerin stored in AtT 20 cells (lanes C) was a
proteolytic product smaller than platelet multimerin, although AtT 20 cells also stored a subunit that comigrated with p-155 after
N-glycosidase F treatment. Endoglycosidase H-sensitive carbohydrate was
detected on the mature multimerin stored and secreted from AtT 20 cells, but not on platelet multimerin.
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Multimer analyses indicated that transfected AtT 20 cells
constitutively secreted variably sized multimerin multimers
(Fig 8, left panel) that were similar to
the multimers constitutively released by the other cell lines (not
shown) and by cultured endothelial cells.4 Like platelet
multimerin, the multimerin stored in transfected AtT 20 cells was
resolved into discrete bands on multimer gels (Fig 8, left panel),
likely due to its extensive and fairly uniform proteolytic processing.
However, the multimerin radioimmunoprecipitates prepared from
transfected AtT 20 cells and their culture media contained
proportionally less high molecular weight multimerin than platelets
(Fig 8, left panel), and identical findings were observed when similar
amounts of multimerin antigen from AtT 20 cells and platelets were
analyzed by immunoblotting (not shown). AtT 20 cells contained
disulfide-linked multimerin multimers that were larger than
intermediary forms generated by partial reduction (Fig 8, right panel),
indicating that, like platelets,6 AtT 20 cells produced
multimerin that was organized into trimers and larger multimers. AtT 20 cells contained small amounts of recently synthesized, promultimerin
(Fig 8; proM, nonreduced lane) that had not yet multimerized in
addition to larger quantities of recently synthesized promultimerin
that had already been incorporated into multimers. Fully glycosylated,
promultimerin (proM*) subunits and the smaller, mature proteolyzed
multimerin subunits were only detected within multimers (Fig 8, right
panel), indicating that multimerization occurred in these cells early,
before promultimerin's N-linked carbohydrates were processed to
complex forms and before promultimerin was proteolyzed to smaller
forms.
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| Fig 8.
The multimeric composition of multimerin synthesized by
AtT 20 cells. The left panel shows multimerin radioimmunoprecipitates
from platelets and AtT 20 cells (fractions as in Figs 6B and 7)
analyzed using nonreduced agarose/acrylamide gels. The right panel
shows a Western blot of cell lysate from AtT 20 cells, analyzed on 3%
to 8% SDS-PAGE after treatment with different concentrations of DTT
(the multimerin subunits proM and proM*, native multimers, and
intermediary forms generated by partial reduction are indicated).
Similar to platelet multimerin,6 the multimerin stored and
secreted from AtT 20 cells was composed of different sized multimers
(left panel). Only small amounts of proM that had not yet multimerized
were detected in the cell lysates (right panel).
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DISCUSSION |
The proteins sequestered in platelets and endothelial cells are
important for changing the functions of these cells at sites of vessel
injury. Individuals with -granule protein deficiency (eg, gray
platelet syndrome) and humans and animals with deficient stores of vWF
or P-selectin suffer from bleeding and impaired vascular
function.11,12,23-28 The mechanisms that regulate protein trafficking, storage, and secretion in platelets and endothelial cells,
although important, are only partially understood. The proteins that are known to be stored and secreted by human endothelial cells are endogenously synthesized.4,15-17,29-31 In
contrast, platelets direct many different types of proteins into their
-granules, including proteins that are not normally targeted for
regulated secretion and proteins that they incorporate from the
plasma.10,11 Of the proteins stored by endothelial cells,
vWF,32 P-selectin,18,33 and tissue-type
plasminogen activator34 have properties that result in
their targeting for regulated secretion when they are expressed in
cells capable of regulated protein secretion. Our current study
indicates that this is also true for multimerin and suggests that
multimerin's presence in platelet -granules and endothelial cell
granules reflects its ability to be sorted by their regulated secretory pathways.
Although our previous studies of multimerin in cultured endothelial
cells suggested that multimerin might not be efficiently targeted for
regulated secretion,4 multimerin was targeted for regulated
secretion in AtT 20 cells with an efficiency similar to that reported
for other secretory proteins.35 The much larger quantities
of multimerin produced by our transfected AtT 20 cell lines may have
facilitated multimerin's storage, due to a concentration-dependent enhancement of its condensation and sorting for regulated secretion. Because helper proteins, including the granins expressed by
neuroendocrine cells, regulate the sorting and proteolytic processing
of some stored secretory proteins,36,37 similar factors
could exist to regulate multimerin's processing and storage in
different cell types. In endothelial cells4 and AtT 20 cells, multimerin is sorted with a variable degree of overlap to
granules that store other endogenously synthesized secretory proteins,
and in platelets, multimerin sorts away from the majority of proteins
that are stored in the central matrix of -granules.2,3,9
These findings suggest that exclusionary factors such as homoaggregate
formation influence multimerin's sorting for regulated secretion in a
variety of cell types.
Variations in posttranslational processing have been implicating in
producing the different forms of multimerin made by platelets, Dami
cells, and endothelial cells, because these cells express identically
sized prepromultimerin transcripts.1,7 Our current study
confirms that there are cell-type-dependent variations in multimerin's glycosylation and proteolytic processing. Given
multimerin's high content of N-linked carbohydrate, it is not
surprising that its glycosylation by different cells produces forms
that vary in their molecular mass.1,2,4 The human embryonic
kidney cell line HEK 293 proved the most useful to recapitulate
multimerin's normal processing by the constitutive secretory pathways
of human endothelial cells, and these cells produced recombinant
multimerin that was functional in its ability to bind human factor V
(unpublished observations). However, none of the cell
lines investigated, including AtT 20 cells, produced platelet-like multimerin.
Many secretory proteins, including multimerin, are synthesized as
proproteins that are cleaved by proprotein converting
endoproteases.35,36 The similar proteolytic processing of
multimerin by the constitutive secretory pathways of diverse cells such
as COS-1, HEK 293, Dami, and endothelial cells indicates that
ubiquitously expressed endoproteases, perhaps enzymes such as furin,
cleave multimerin to smaller forms. Because these cells proteolyze
multimerin differently from platelets, our findings suggest that a
proprotein converting enzyme with a restricted tissue distribution
cleaves promultimerin in platelets; however, the proprotein-converting
enzymes expressed by megakaryocytes are not yet known. In platelets,
promultimerin is cleaved N-terminal to its RGDS domain to generate
platelet multimerin.8 The smaller, N-deglycosylated size of
the major form of multimerin stored in AtT 20 cells indicates that
these cells contain different multimerin-cleaving endoproteases than
platelets. We have observed that similar, more extensive proteolysis
also occurs when multimerin is synthesized and stored in rat insulinoma
RIN-5F cells (unpublished observations). The quantities
of multimerin produced by AtT 20 cells did not permit analyses of
N-terminal sequences. Because the multimerin stored in AtT 20 cells
contained the monoclonal antibody JS-1 epitope, we suspect that
endoproteases in AtT 20 cells remove an N-terminal region from
promultimerin that includes the RGDS domain, or the C-terminal,
globular domain distal to the JS-1 epitope.7 AtT 20 cell
are known to express highly restricted endoproteases (such as
prohormone convertases 1 and 2) that cleave substrates with dibasic
amino acid sequences, although the substrate specificities of these
enzymes are only partially understood.36 Prepromultimerin
contains a number of potential cleavage sites for these proteases,
including a Lys-Lys site at amino acids 340-341, located downstream
from the N-terminal site where promultimerin is cleaved in
platelets.7,8 The functional significance of multimerin's
aberrant proteolysis by AtT 20 and RIN-5F cells is not yet known, but,
because it could alter or remove domains that support multimerin's
functions in platelets, AtT 20 and RIN-5F cells may not be appropriate
for evaluating some of multimerin's functions.
Multimerin's large size and multivalent structure may facilitate its
attachment to activated platelets and endothelial cells and its
assembly into fibrillary scaffolds in the extracellular matrix.1,2,4-6 Our current study indicates that
multimerin's interchain disulfide bonds form early during
biosynthesis, likely in a pre-Golgi compartment, before its N-linked
carbohydrates are converted to complex forms. Because the multimerin
produced by the heterologous cells contained proportionally less high
molecular weight multimers than platelet multimerin, these cells may
lack components that optimize multimer formation in platelets. It is also possible that multimerin's more extensive proteolysis during storage in AtT 20 cells interferes with the formation or stability of
its multimers, despite preserving its organization into trimers and
larger multimers.
Platelets contain large amounts of factor V that they store complexed
with multimerin,3 but the mechanisms that facilitate factor
V storage in platelets are uncertain. The factor V released by
activated platelets may enhance factor V's delivery to sites of tissue
injury, because it is sufficient to support hemostasis in individuals
with plasma factor V deficiency.38-40 Controversies exist
regarding the origins of the factor V stored in platelets and suggest
that the high concentrations of factor V in platelets are the result of
megakaryocyte synthesis of factor V or factor V's uptake from the
plasma.41-44 Our current and previous studies indicate that
multimerin can be stored independently of factor V in
platelets,19 endothelial cells,4 and
heterologous cells. Multimerin's ability to bind exogenous factor V
indicates that multimerin-factor V complexes could form during or after
biosynthesis.3 At least several mechanisms could facilitate
factor V's storage with multimerin in secretion granules. One
possibility is that multimerin functions as a carrier protein, destined
for regulated secretion, that diverts coexpressed factor V from
constitutive into regulated secretory pathways.36,45,46
Examples of this type of sorting include the diversion of factor VIII
into storage granules when it is coexpressed with its plasma carrier
protein vWF.46 Factor V's similarity to factor
VIII47 suggests that it may not be independently targeted
for storage in secretion granules. Recent studies indicate that
complexes of plasma-derived and megakaryocyte-synthesized proteins can
also be incorporated into -granules.48 Further studies
are needed to determine if factor V is stored complexed with multimerin
in -granules due to multimerin's interactions with
megakaryocyte-synthesized and/or plasma-derived factor V. Because
multimerin does not circulate in detectable amounts in normal plasma,
it likely forms complexes with factor V in megakaryocytes.
Multimerin's ability to be targeted for regulated secretion in a
variety of cells may facilitate its normal storage and sequestration within platelets and vascular endothelium, and this may be important for regulating its functions. Although HEK 293 cells process multimerin similarly to human endothelial cells, the production of platelet-like multimerin appears to require cells that possess pathways for regulated
secretion, in addition to megakaryocyte-like proprotein converting
endoproteases. Defects in protein storage (such as gray platelet
syndrome) may alter multimerin's delivery to sites of blood vessel
injury and change how and where multimerin associates with platelets,
endothelial cells, factor V, and the extracellular matrix, with
hemostatic consequences.
| |
FOOTNOTES |
Submitted February 3, 1999; accepted April 15, 1999.
Supported by grants from the Medical Research Council of Canada
(C.P.M.H.), from the Heart and Stroke Foundation of Ontario (C.P.M.H.),
and from l'Association pour la Recherche sur le Cancer (ARC) and
Fondation pour la Recherche Medicale (FRM; to E.M.C.). C.P.M.H. is a
Research Scholar of the Heart and Stroke Foundation of Canada.
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.
Address reprint requests to Catherine P.M. Hayward, MD, PhD, Room 2N32,
McMaster University Medical Centre, Hamilton Health Sciences Corp, 1200 Main St W, Hamilton, Ontario, Canada L8N 3Z5.
| |
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ABSTRACT
INTRODUCTION