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INTRODUCTION |
Fibrillin-1 and -2 are large, secreted glycoproteins known to be
components of extracellular matrix microfibrils located in the
vasculature, basement membrane, and various connective tissues and are
often associated with a superstructure known as the elastic fiber (for
reviews see Refs. 1 and 2). It has been clearly established that
mutations in the FBN1 gene can result in the Marfan syndrome
(for review see Ref. 3) and have been found in individuals with
familial aortic aneurysm (4, 5), familial ectopia lentis (6), and
Shprintzen-Goldberg syndrome (7). Studies using cell strains from
individuals with the Marfan syndrome have suggested a dominant negative
pathogenesis for FBN1 mutations, i.e. the product
of the mutant allele disrupts the assembly of microfibrils (8,
9).
The two known fibrillin proteins are highly similar with modular
structures consisting of repeating epidermal growth factor (EGF)1-like domains
interspersed between 8 cysteine domains similar to those found in the
latent transforming growth factor-
-binding protein family. Of the 47 EGF-like domains, 43 contain the residues necessary for binding
calcium. Near the amino terminus, fibrillin-1 contains a proline-rich
domain that is unique to the molecule, whereas fibrillin-2 contains a
glycine-rich domain in the analogous location. Both proteins have
homologous carboxyl-terminal domains of about 185 residues. No
functions have yet been ascribed to any fibrillin domains, and nothing
is known about their role in the assembly of fibrillin-containing microfibrils.
The fibrillins are known to undergo several post-translational
modifications that include
-hydroxylation of Asn/Asp residues, N-linked glycosylation, and proteolytic cleavage of both the
leader sequence and the carboxyl-terminal domain (10-12). Proteolytic cleavage of the fibrillin-1 carboxyl-terminal domain has been proposed
to occur after the arginine at position 2731 and results in the removal
of the last 140 residues. It has been suggested that a member of the
Furin/PACE family, which cleaves multibasic sites, is responsible for
the processing (12).
Due to the large size and great degree of similarity of fibrillin-1 and
-2, it is difficult to distinguish between the two molecules much less
experimentally evaluate the proteolytic processing of their
carboxyl-terminal domains. To better understand the processing of the
fibrillin-1 carboxyl-terminal domain, we have developed three truncated
fibrillin expression constructs, one with the full-length
FBN1 carboxyl-terminal domain, one with the domain deleted,
and one with a mutation near the putative cleavage site. These protein
constructs have been expressed in two eukaryotic model systems,
baculoviral expression in SF9 insect cells and CHO-K1 mammalian cell
lines. Both systems are able to produce and secrete each of the three
constructs. Here we show that proteolytic processing of the fibrillin-1
carboxyl-terminal domain occurs in the constructs expressed in these
systems. The loss of the expected region can be seen by SDS-PAGE
migration differences and immunologically by Western blotting. Impaired
cleavage observed in the construct with a mutated multi-basic site
confirms the involvement of that region in normal processing. The use
of secretion blocking agents shows that processing occurs
intracellularly in the CHO-K1 cells and most likely by an early
secretory pathway resident furin-like enzyme.
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EXPERIMENTAL PROCEDURES |
Construction of LEE, LEEC, and LEEC-P1 FBN1 Sequences
The LEEC construct was amplified from human mRNA and was
initially cloned in four parts (Figs. 1 and 2). The first part is "L," the leader sequence of FBN1 (exon 1). The second
part is "E1," first half of the EGF-like and 8-cysteine domains
(FBN1 exons 23-33). The third part is "E2," the second
half of the EGF-like and 8-cysteine domains (FBN1 exons
30-44); and the fourth part is "C," the carboxyl-terminal domain
(FBN1 exon 64-65). The FBN1 leader sequence and
carboxyl-terminal domains were each amplified using the primers
described below by reverse transcription of 2.0 µg of total RNA
isolated from human foreskin fibroblasts with 20 units of Moloney
murine leukemia virus reverse transcriptase (Boehringer Mannheim)
followed by 30 cycles of polymerase chain reaction under standard
conditions. Each cDNA was isolated from a low melting point agarose
gel and ligated into the pCRII vector (Invitrogen, San Diego, CA). The
E1 and E2 inserts were similarly amplified using 40 cycles of
polymerase chain reaction and Vent polymerase and cloned into the
pGEM-t vector (Promega, Madison, WI).
Description of Primers and cDNA Clones
cDNA Clone L-Exon 1 of FBN1--
These primers are designed
to amplify the coding region of the first exon of the FBN1
gene. The sense primer begins with an EcoRI restriction site
and Kozak consensus sequence followed by the initiating ATG codon. The
antisense primer contains two other unique restriction sites,
BspEI and BsiWI, that allow splicing to the next
domain or the in frame replacement or removal of the leader sequence.
Together they amplify a 195-bp product that encodes the first 56 amino
acids of fibrilin-1 that contain the putative hydrophobic signal
peptide leader sequence: leader sense primer, 5'-GGAATTCGCCACCATGCGTCGAGGGCGTCTG-3'; and leader antisense primer 5'-CCCCTCCGGATCCCGTACGGACATTG GGTCCTTTAAGC-3'.
cDNA Clone E1-Exon 23-33 of FBN1--
This primer pair is
designed to amplify a 1,455-bp product that corresponds to the first
codon of FBN1 exon 23 and continues into exon 33. The sense
primer begins with a BsiWI restriction site to allow
ligation to the 3' end of the leader sequence: E1 sense primer,
5'-GACTCGTACGGATATAGATGAATGTGAAGTGTTCC-3'; and E1 antisense
primer, 5'-TCCTTCCTTGCACAGACAGCGG-3'.
cDNA Clone E2-Exon 30-44 of FBN1--
These primers are
designed to amplify a 1,771-bp product that begins in exon 30 and
continues through exon 44 of FBN1. This sequence overlaps
with clone E1 and can be joined to it by a common SmaI
restriction site. The antisense primer contains a BspEI site for ligation to the carboxyl-terminal domain sequence: E2 sense primer,
5'-TTGTGTTATGATGGATTCATGG-3'; and E2 antisense primer, 5'-TCAGTCCGGAATTGCACTGTCCTGTGGA-3'.
cDNA Clone C-the Carboxyl-terminal Domain of FBN1--
These
primers are designed to amplify 572-bp coding sequence that begins with
the first amino acid after the last EGF-like domain of FBN1
and continues to the termination codon. The sense primer contains
5'-BspEI-NheI-3' sites to facilitate assembly and
substitution or removal of the domain. The antisense primer contains
5'-XbaI-NheI-3' sites to facilitate vector
transfer and carboxyl-terminal domain substitution or removal. The
carboxyl-terminal sense primer is
5'-GTCATCCGGAGCTAGCTCTGGAATGGGCATGGG-3' and the carboxyl-terminal
antisense primer is 5'-GCTCTAGAGCTAGCTTAATGAAGCAAAACCTGGAT-3'.
Assembly of the cDNA Clones
The FBN1 leader sequence clone insert was excised,
gel-purified, and ligated to the 5' end of the EGF1 clone using
XbaI and BsiWI restriction sites. As blue/white
selection was already disrupted, positive clones were identified by
colony lifts screened with polymerase chain reaction-generated
radioactive probe. The LS/EGF1 cDNA sequence was excised,
gel-purified, and ligated into position 5' of the EGF2 clone with
5'-ApaI and 3'-SmaI restriction sites thus
producing the sequence referred to as "LEE." The LEE sequence was
then inserted into the pcDNA3 expression vector (Invitrogen) using
EcoRI and XbaI sites. The carboxyl-terminal
domain was inserted into the pcDNA/LEE with 5'-BspEI and
3'-XbaI sites to yield the sequence referred to as
"LEEC." The leader sequence and carboxyl-terminal domains were
sequenced in both directions upon initial cloning. The E1 and E2 clones
were sequenced at each end. After each assembly step, the junctions
were sequenced to ensure the maintenance of the reading frame.
The
P1 mutant was produced by site-directed mutagenesis using the
FBN1 carboxyl-terminal clone C. Primer 90 (5'-CGTTTGTGCTGCTCCGTTTC-3') and CT sense were used to amplify a 200-bp
5' fragment, and primer 89 (5'-GAAACGGAGCAGCACAAACG-3') and CT
antisense were used to amplify a 400-bp 3' fragment. Equimolar amounts
of the two fragments were then used as templates to amplify the
full-length mutant carboxyl-terminal fragment using the following
conditions: denaturation at 94 °C/2 min, annealing at 55 °C/3
min, and extension at 72 °C/1 min for six cycles. The
carboxyl-terminal sense and antisense primers were then added to the
reaction, and 30 additional cycles were carried out. Amplified
fragments were cloned into pGEM-T and sequenced.
Bacterial Expression Construct
The FBN1 carboxyl-terminal domain was excised from
the initial clone described above using the 5'-BspEI and
3'-HindIII sites and ligated into the pQE32 expression
vector (Qiagen, Valencia, CA) using the 5'-XbaI and
3'-HindIII restriction sites. This vector contains an
initiating ATG followed by a sequence that codes for six histidine
residues, all of which are upstream of the inserted sequence. The
resulting protein contains an amino-terminal His-tag that can bind a
Ni2+ ion containing solid support resin to facilitate
purification (Qiagen). The recombinant FBN1
carboxyl-terminal protein contains the following additional residues
amino-terminal to the first residue of the domain:
MRGSHHHHHHGIRMRARYPGAS + (FBN1-Ser-2688 to His-2871
terminus). The full-length recombinant peptide contains 206 amino acids
with a predicted molecular mass of 23,266 Da and an isoelectric point
of pH 8.4. The protein sequence was confirmed by amino-terminal
sequencing of 15 residues.
Log phase cells growing in 500 ml of Luria broth at 37 °C were
induced to produce protein with 1.5 mM isopropyl
-D-thiogalactopyranoside. Five hours post-induction, the
cultures were pelleted, lysed with 8 M urea, pH 8.0, and
centrifuged at 12,000 × g for 20 min. The recombinant
protein with the 6× His tag was purified from the other bacterial
proteins in the crude lysate by binding to Ni2+ resin
column (Qiagen) and washing out non-bound contaminating proteins,
followed by elution with a low pH, 8 M urea wash. The recombinant protein was then dialyzed in 12,000-14,000 molecular weight cut-off dialysis tubing (Spectrum Medical, Laguna Hills, CA)
against a 0.5 M acetic acid solution and lyophilized.
Baculoviral/SF9 Cell Expression Constructs
The LEE, LEEC, and LEEC-P1 coding sequences were each cloned
into the pFastbac shuttle vector using 5'-EcoRI and
3'-XbaI sites, and this was used to transform DH10' cells
(Life Technologies, Inc.) containing bacmid. Plasmid/bacmid
recombination occurred through bacterial transposon Tn7 regions
flanking the coding insert, and positives were identified with
blue/white selection. Bacmid DNA was prepared by alkaline lysis and
transfected into serum-free SF9 cells using Lipofectin (Life
Technologies, Inc.) for 4 h at 27 °C. Five days after
lipofection, conditioned medium was removed, centrifuged to remove
debris, and used to infect new cultures of SF9 cells. Cultures
expressing protein were determined by Western blot of conditioned
medium. Subsequent expression experiments were conducted in either
serum-free SF-900IISFM (Life Technologies, Inc.) or IPL41 medium
containing 10% fetal bovine serum.
Expression Constructs and Establishment of Three Stable
Expression Lines in CHO-K1 Background
The LEE, LEEC, and LEEC-P1 coding sequences were each inserted
into the pEE14 vector using the 5'-EcoRI and
3'-BclI sites. This vector employs the cytomegalovirus
promoter/enhancer to drive expression and glutamine synthase minigene
driven by the SV40 late promoter to provide selection with methionine
sulfoximine (13). The cell lines were established as described
previously (14). Briefly, three 60-mm dishes containing adherent CHO-K1 cells (ATCC, CCL-61) at approximately 50% confluency were transfected with 9 µg/dish of purified plasmid DNA that had been previously incubated with 30 µl of Lipofectin (Life Technologies, Inc.) in 200 µl of serum-free medium (Glasgow minimum essential medium from
Washington University TC Support Center, St. Louis) for 30 min at room
temperature. Total volume per dish was 2.0 ml, and the duration of
transfection was 5 h after which 3.0 ml of normal serum-containing
non-selective medium was added overnight. The following day, each 60-mm
dish was subcultured into three 100-mm dishes in selective medium
(Glasgow minimum essential medium without glutamine and containing 10%
10,000 molecular weight cut-off dialyzed FBS and 25 µM
methionine sulfoximine). After 14 days under selection, clonal colonies
were subcultured into 15-mm wells and screened for expression by
Western blot of conditioned medium. Approximately 20 colonies were
isolated for each clone and of those 10, 50, and 90% were positive by
Western blot of conditioned medium for LEEC, LEE, and LEEC-P1,
respectively. For each construct, one clone was chosen based on
expression levels and was used in all subsequent experiments.
Protein Expression and Purification
Baculoviral--
SF9 cells were infected in either serum-free
medium (SF900II from Life Technologies, Inc.) or in the presence of
10% FBS in IPL41 medium (Life Technologies, Inc.). Protein could be
detected as early as 24 h postinfection and continued to be
produced up to 90 h postinfection. To protect against degradation
under serum-free conditions, medium was collected after 48 h
postinfection and (4-amidinophenyl)methanesulfonyl fluoride (Life
Technologies, Inc.) to 2 mM was added followed by
centrifugation at 16,000 × g for 15 min at 4 °C
prior to purification. Harvest of protein from serum-containing medium
was performed at 90 h postinfection.
Various methods were used to purify and concentrate protein from
serum-free conditioned medium; these include dialysis in 12,000-14,000
molecular weight cut-off tubing (Spectrum) against PBS and then
lyophilization, 40% ammonium sulfate precipitation, followed by
dialysis and lyophilization, trichloroacetic acid precipitation
followed by EtOH:ether wash and resuspension, and concentration by
50,000 molecular weight cut-off centrifuge filters (Spectrum). Highest
purity and yield were achieved with pressurized ultrafiltration through
Diaflow YM100 molecular weight cut-off filters (Amicon, Beverly, MA)
followed by fast protein liquid chromatography size fractionation using
a Superose 6 column (Amersham Pharmacia Biotech) at 0.5 ml/min 100 mM ammonium bicarbonate, pH 7.5.
Stable CHO-K1 Lines--
To produce conditioned medium
containing recombinant protein, LEE-, LEEC-, or LEEC-P1-expressing
cells were grown in selective medium until confluency and switched to
Hy-Q CCM5 serum-free medium (Life Technologies, Inc.) where they were
grown for up to 4 days. After conditioning, the medium was withdrawn
and centrifuged at 15,000 × g for 30 min to pellet
debris. Next the medium underwent stirred ultrafiltration under 2.5 pounds/square inch at 4 °C through a YM100 molecular weight cut-off
membrane (Amicon) as a purification and concentration step.
Fetal Bovine Cells--
Cell cultures established from fetal
bovine nuchal ligament, fetal bovine vascular smooth muscle (BSMC), and
fetal bovine chondrocytes (FBC) were used as a source of fibrillin for
positive controls. Protein was harvested in a manner similar to that
described for the CHO-K1 lines with the exception that when grown in
serum-free medium, DMEM with ITS+3 (Sigma) was used instead. The
conditioned medium was used directly or concentrated approximately 25×
with ultrafiltration at 2.5 pounds/square inch through XM300K molecular weight cut-off filters (Amicon).
Antibody Production
The peptides Fib15D (YLDIRPRGDNGDTAC) and FBN1-386
(GNEDGFFKINOKEGC) were made using an Applied Biosystems 431A protein
synthesizer by standard FMOC
(N-(9-fluorenyl)methoxycarbonyl) chemistry (Fig. 1 and 2).
The peptides were purified and cross-linked to rabbit serum albumin
(Sigma) using the heterobifunctional cross-linker m-maleimidobenzoyl-N-hydroxysuccinimide ester
(Pierce) at a molar ratio of approximately 15:1 peptide to rabbit serum
albumin. One milligram of peptide-rabbit serum albumin conjugate was
emulsified in Freund's complete adjuvant for the initial injection
into rabbits and Freund's incomplete adjuvant for subsequent
injections at 2-week intervals. After 6 weeks, the resulting polyclonal
antisera were collected and subjected to a caprylic acid precipitation step, followed by dialysis against PBS, lyophilization, and affinity purification against the immunogen peptide linked to Sulfa-link support
(Pierce). After elution with 100 mM glycine, pH 2.3, the purified antibody was dialyzed, lyophilized, and resuspended in PBS at
approximately 1.0 mg/ml.
The recombinant FBN1 carboxyl-terminal domain was expressed in
bacteria, column purified as described above, and used to generate polyclonal antibodies. The immune serum was subjected to a caprylic acid precipitation followed by affinity purification against the protein bound to the Ni2+ resin support. After elution with
8 M urea, pH 8.0, the purified antibody was dialyzed,
lyophilized, and resuspended in PBS at approximately 1.0 mg/ml.
Protein Radiolabeling
Baculoviral--
Medium was removed 24 h postinfection from
a 35-mm well containing 1 × 106 SF9 cells, and the
infected cells were washed with and then incubated for a 2-h starvation
in Cys-free Grace's medium with 5% dialyzed FBS. This was then
replaced with Cys-free Grace's medium supplemented with 100 µCi/1.0
ml/well [35S]Cys and 5% dialyzed FBS. Labeling continued
for 24 h after which the conditioned medium was collected.
CHO Stables--
Confluent 15-mm wells were incubated in
Cys-free DMEM with 5% dialyzed FBS for a 2-h starvation period and
then incubated for various times in the presence of 50 µCi of
[35S]Cys/200 µl medium/well. Chase medium was
non-selective Glasgow minimum essential medium 175 µl/well with 5%
FBS. The chase medium was collected after various times points. To
collect cell lysate, cells were washed twice with serum-free medium and
lysed with 175 µl/15-mm well of 1% Nonidet P-40, 50 mM
Tris, pH 8.0, with 1.5 mM (4-amidinophenyl)methanesulfonyl
fluoride, 1.0 µM aprotinin, 100 µM
leupeptin, and 4 mM Pefabloc (Life Technologies. Inc.). Cellular debris was removed by centrifugation at 16,000 × g for 5 min at 4 °C. For SDS-PAGE analysis, 15 ml per
lane of labeled medium or supernatant was typically loaded.
PNGse F Digestions
Baculoviral Protein--
5.0 ml of conditioned medium was
collected for 24 h from SF9 cells infected with LEEC baculovirus
and immunoprecipitated with FBN1 carboxyl-terminal antibody at 1:250
overnight at 4 °C. The IgG-protein complex was bound to solid
support with 100 µl of protein A-Trisacryl (Pierce) by a 1-h
incubation at room temperature followed by centrifugation and two
washes with 0.05% Tween 20 in PBS. For the PNGase F digestion, 5 µl
of the Trisacryl beads were added to each of two parallel tubes
containing 9 µl of water and 1.5 µl of 10× denaturing buffer and
boiled for 10 min. The tubes were centrifuged, and the supernatant was
removed to other tubes with 1.0 µl of 10× buffer, 1.0 µl of 10×
Nonidet P-40 solution, and 1.0 µl of PNGase (500 New England Biolabs
units) and digested at 37 °C for 1.5 h. The reaction was
stopped by the addition of reducing loading buffer, and the contents
were electrophoresed through a 7.5% SDS-polyacrylamide gel and
detected by Western blot.
CHO-K1 Protein--
CHO-K1 cell line stably expressing the LEE
construct was washed with and incubated in Cys-free DMEM for 2 h
(1 × 106 cells/35-mm well) and then incubated with
125 µCi of [35S]Cys/1.0 ml of medium/well for 24 h
in Cys-free DMEM with 5% dialyzed FBS. Medium was collected and spun
through a 50,000 molecular weight cut-off column to remove
unincorporated label and resuspended in fresh medium at the original
volume. Two 20-µl aliquots of conditioned medium were processed in
parallel. Each tube received 2.0 µl of denaturing buffer, was boiled
for 10 min, centrifuged, and then received 2.0 µl each of Buffer G7
and Nonidet P-40 10× (New England Biolabs, Beverly, MA), and one tube
also received 2.5 µl of PNGase F (1,250 NEB New England Biolabs
units), and both were incubated at 37 °C for 2 h. The reaction
was stopped by the addition of reducing loading buffer, and the
contents were electrophoresed through a 5.0% SDS-polyacrylamide gel,
fixed, dried, and exposed to x-ray film for 6 h.
Amino-terminal Sequencing
Baculoviral- and CHO-K1-expressed protein was collected from
conditioned medium and concentrated by ultrafiltration as described above. Twenty µl of concentrated sample was loaded with reducing buffer onto SDS-PAGE and run at 20 mA for 1.5 h. Proteins were transferred onto ProBlott polyvinylidene difluoride support (Applied Biosystems, Foster City, CA) and detected with Coomassie stain. Appropriate bands were excised and sequenced on an Applied Biosystems model 473A automated sequencer using Edman degradation.
Secretion Blocks
CHO-K1 cells expressing LEEC were grown to confluency with
normal medium in 15-mm wells. Before the introduction of
[35S]Cys pulse, all wells underwent a 2-h Cys-free
starvation as described above. Thirty min prior to the pulse, A23187
was added to a final concentration of 2 µM to the
appropriate wells. Fifteen min before the pulse, brefeldin A, ammonium
chloride, and monensin were added to the appropriate wells in final
concentrations of 10 µg/ml, 50 mM, and 10 µM, respectively. All cells were then pulsed for 30 min
as described above. After the pulse, the wells were washed once with
normal medium containing blocking agents and then incubated with 175 µl of normal cold chase medium containing blocking agents for 2 h. Chase medium was retained and analyzed directly by SDS-PAGE and
autoradiography. Cells were washed 3× with PBS then lysed as described
above. Cell lysate was microcentrifuged 15,000 × g at
4 °C for 10 min, and the supernatant was analyzed by SDS-PAGE and autoradiography.
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RESULTS |
Cloning and Assembly of Expression Constructs--
Using the
primers described under "Experimental Procedures," each of the
target sequences was amplified and assembled as described in Figs.
1 and 2. All three constructs, LEE, LEEC
and LEEC-P1, begin with the first 56 amino acids (exon 1) of
fibrillin-1. This is spliced to exons 23-44 of fibrillin-1 which
contains 17 calcium binding EGF-like domains, three LTBP-like repeats,
one RGD sequence, and eight potential sites of N-linked
glycosylation. In the LEEC and LEEC-P1 proteins, exon 44 is spliced to
the entire carboxyl-terminal domain of FBN1 which contains
the putative cleavage site and three potential sites of
N-linked glycosylation. The carboxyl-terminal domain of
LEEC-P1 contains the Arg to Ser mutation at the tetrabasic cleavage
site. The entire LEEC construct contains 1189 amino acids, has a pI of
pH 4.6, and a predicted molecular mass of 129,907 daltons. As seen in
other proteins containing several EGF-like domains, all constructs run
at a higher than expected molecular weight on SDS-PAGE.

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Fig. 1.
Domain structure of fibrillin-1 and
expression constructs. Diagram illustrating the domain
organization of fibrillin-1 and the three expression constructs LEE,
LEEC, and LEEC-P1. The regions recognized by three polyclonal
antibodies are indicated. The P1 mutant is depicted in the
box at bottom left and domain types are
illustrated at bottom right.
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Baculoviral Expression System Produces, Secretes, and Correctly
Processes LEE, LEEC, and LEEC-P1 Constructs--
Each of the three
fibrillin-1 constructs was expressed in SF9 insect cell lines. All were
produced at high levels with the expected post-translational
modifications (see below). Protein was produced in SF9 cells under both
serum-containing and serum-free conditions. Time course experiments
revealed that peak protein production occurred between 24 and 48 h
postinfection, although a significant amount of production continued
out to 90 h (data not shown). Harvest of protein from
serum-containing medium was performed at 90 h postinfection and
was often used for Western blots without further purification. In
general, production in the absence of serum resulted in higher levels
of protein degradation but had greater ease of purification due to
lower amounts of background serum proteins. The extent of degradation
was related to the progression of infection and subsequent lysis of the
host SF9 cells. Protein produced under both serum-free and
serum-containing conditions was used for glycosylation analysis,
amino-terminal sequencing, Western blots, and the solubility
experiments. The FIB15D, FBN1-CT, and FBN1-386 antibodies recognized
the appropriate constructs on Western blot (Fig. 3).
CHO-K1 Cells Stably Transfected with pEE14 Expression Vector
Produce, Secrete, and Correctly Process LEE, LEEC, and LEEC-P1
Constructs--
Transfection of each of the three pEE14 expression
vector-based constructs into CHO-K1 cells yielded several positive
clonal cell lines, with the exception of LEEC for which only two
positive clones were identified. Positive clones were selected for
further experiments based upon protein expression levels as identified by Western blots of conditioned medium. The LEE and LEEC-P1 cell lines
produced sufficient protein to identify by Western blot of medium
conditioned for 24 h. The LEEC line often required a concentration
step before immunological recognition by Western blot.
The use of serum-free conditions greatly facilitated purification by
ultrafiltration as described. Protein stability problems analogous to
the baculoviral system were not experienced under these conditions.
These preparations were used for amino-terminal sequencing and Western blotting.
Characterization of Antibodies to Regions of the Carboxyl-terminal
Domain of FBN1--
Bacterial expression of the FBN1
carboxyl-terminal cDNA followed by 6His-tag/Ni2+ column
purification yielded mg/ml quantities of purified protein (data not
shown). These preparations were used to produce the rabbit polyclonal
antibody (FBN1-CT antibody) to the fibrillin-1 carboxyl-terminal domain.
The FBN1-CT rabbit polyclonal antibody recognizes the LEEC and LEEC-P1
proteins by Western blot and immunoprecipitation in both baculoviral
and CHO-K1 systems (Fig. 3). Importantly, it recognizes both the
uncleaved and cleaved carboxyl-terminal domain as 44 residues of the
domain remain after processing. As expected, it does not recognize LEE.
The antibody also immunoprecipitates native, full-length fibrillin from
FBC (Fig. 3A), fetal calf ligament, or BSMC (not
shown)-conditioned medium and detects fibrillin by Western blot.
Antibody Fib15D, generated to a peptide sequence in the fourth
8-cysteine domain of fibrillin-1, recognizes all three constructs as
well as both processed and unprocessed forms of LEEC (Fig. 3B). Antibody FBN1-386, in contrast, recognizes an epitope
in the region of the carboxyl-terminal domain that is lost after cleavage (see map in Fig. 2). This
antibody only recognizes LEEC-P1 and the larger, unprocessed form of
the LEEC constructs as expressed in both baculoviral and CHO-K1 cell
systems (Fig. 3C). The
FBN1-386 antibody does not detect fibrillin in conditioned medium from FBC or BSMC cultures (data not shown).

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Fig. 2.
Amino acid sequence of expression
constructs. The sequences of LEE, LEEC, and LEEC-P1 are identical
from residue 1 to 1002. The LEEC and LEEC-P1 proteins also contain the
FBN1 carboxyl-terminal domain. The Arg to Ser substitution present in
the LEEC-P1 mutant is shown in bold above the sequence.
Residues introduced by restriction sites are shown in bold
letters. The putative FBN1 carboxyl-terminal domain cleavage site
is boxed. The peptide sequences used for the Fib15D and 386 antibodies are underlined. The first 54 residues of FBN1
(exon 1) were used as the leader sequence in all of our constructs.
Protein secreted by CHO and SF9 cells contains two populations of
amino-terminally processed LEEC as indicated by
arrows.
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Fig. 3.
Antibody recognition of secreted proteins
from baculoviral and CHO-K1 expression systems and FBC cells.
A, left, Western blot of LEE, LEEC, and LEEC-P1 protein from
baculoviral conditioned medium using FBN1-CT antibody (Ab).
A, right, immunoprecipitation of radiolabeled fibrillin
(arrow) from FBC conditioned medium. CM, total
conditioned medium. IP, immunoprecipitation using the
carboxyl-terminal antibody. B, Western blot of LEE, LEEC,
and LEEC-P1 protein from baculoviral and CHO-K1 conditioned medium
using the Fib15D antibody. C, Western blot of LEE, LEEC, and
LEEC-P1 protein from baculoviral and CHO-K1 conditioned medium using
the 386 antibody. For all Western blots, each gel was loaded with
identical samples. Upper and lower arrows
indicate cleaved and uncleaved proteins, respectively.
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Carboxyl-terminal Processing and Other Post-translational
Modifications--
The coding sequence of each of the constructs, LEE,
LEEC, and LEEC-P1, begins with exon 1 of fibrillin-1. To determine the signal peptide cleavage site, amino-terminal sequencing of protein expressed in both baculoviral and CHO-K1 systems was performed. Interestingly, both systems process exon 1 of fibrillin 1 in the same
way using a preferred cleavage site between Gly-24/Ala-25 and a
secondary site of Ala-27/Asn-28 (Fig. 2). The primary site is used 4-5
times more frequently than the secondary site.
Cleavage of the carboxyl-terminal domain also occurred in both cell
systems, as demonstrated by an electrophoretic mobility shift and the
loss of immuno-recognition by the FBN1-386 antibody (Fig. 3).
[35S]Cys pulse/chase experiments with CHO-K1 cells
displayed no significant differences in the secretion rates of LEE,
LEEC, and LEEC-P1 proteins at the time points studied (Fig.
4). Approximately 50% of the labeled
protein was secreted by 1 h, and most was secreted by 5 h. In
both expression systems, neither failure to cleave (LEEC-P1) nor the
absence of the carboxyl-terminal domain (LEE) disrupted secretion.
Although there was no difference in the ability of the cell to secrete
the P1 mutant compared with LEEC, processing of the carboxyl-terminal
domain of LEEC-P1 was less efficient (Fig.
5) in both SF9 and CHO cells. It is
important to note that PACE/furin family members are known to be
present and active in SF9 cells and correctly process multi-basic sites
(15-17).

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Fig. 4.
Pulse/chase of untransfected CHO-K1 and
transfected cell lines stably expressing LEE, LEEC, and LEC-P1
constructs. Each cell line was grown and labeled, and samples were
prepared as described under "Experimental Procedures." Time = 0 corresponds to the end of the 30-min pulse and the beginning of the
cold chase. The length of the chase is indicated in hours
below each lane. Molecular weight markers are indicated at
the sides. Arrows indicate unprocessed and
processed protein. Each figure contains cell lysate and medium from
untransfected CHO-K1 cells (A). B, CHO-K1 cells
expressing LEE. C, CHO-K1 cells expressing LEEC. The lane
designated "trypsin" indicates lysate from cells treated with
trypsin at T = 0 h. D, CHO-K1 cells
expressing LEEC-P1. LEEC is included for size reference.
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Fig. 5.
Inefficient processing of LEEC-P1
mutant. Western blot with the FBN1 carboxyl-terminal antibody of
conditioned medium from the baculoviral system (A) and the
CHO-K1 system (B). Both systems demonstrated less efficient
processing of the LEEC-P1 mutant. Upper and
lower arrows indicate unprocessed and processed forms,
respectively.
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It is interesting to note that we were sometimes able to detect
unprocessed LEEC in conditioned medium from the baculoviral system. The
amount detected varied according to the amount of virus used for
infection, i.e. high viral titers resulted in large amounts
of unprocessed protein, whereas lower viral levels resulted in more
efficient processing. This is likely to be an artifact of the
expression system, given that a baculoviral infection causes the
shutdown of host cell gene transcription-translation by 24 h.
Thus, the normal complement of host SF9 cell furins may be overwhelmed
by 72-96 h of high levels of transgene expression and not able to
efficiently process all of the protein produced (15). Unprocessed LEEC
was never detected in CHO-K1 cell conditioned medium.
PNGase F cleaves the linkage between an asparagine side chain and the
first N-acetylglucosamine unit of an attached polysaccharide chain. Protein produced by both systems was digested with PNGase F, and
this resulted in faster migration on SDS-PAGE (Fig.
6). These data indicate that both
expression systems are glycosylating asparagine residues. After PNGase
F digestion, the CHO-K1 LEEC-P1 protein, which contains 11 of the 14 potential N-glycosylation sites of full-length fibrillin 1, changes SDS-PAGE migration so that it is approximately equal to the
untreated LEE protein which has a calculated molecular mass that is 19 kDa less.

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Fig. 6.
PNGase digestion of glycosylated protein from
baculoviral and CHO-K1 systems. A, Western blot of LEEC
protein immunoprecipitated from the baculoviral system with the FBN1-CT
antibody and digested with PNGase. Both the processed and unprocessed
forms are sometimes visible in conditioned medium from the baculoviral
system (see text). B, autoradiography of
[35S]Cys-labeled LEE and LEEC-P1 protein isolated from
the CHO-K1 system and digested with PNGase. The PNGase-treated proteins
from both expression systems migrate more rapidly than untreated
protein due to the removal of Asn-linked sugars.
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Mapping Intracellular Cleavage Compartment Using Secretion Blocking
Agents--
The presence of processed LEEC in cell lysates suggested
that cleavage of the carboxyl-terminal domain occurs intracellularly. To rule out the possibility that processing was occurring
extracellularly on the cell surface, CHO cells were treated with
trypsin prior to lysis to remove surface-bound proteins. Fig.
4C shows that both processed and unprocessed LEEC were still
evident in the cell lysate after trypsin treatment, confirming that
both forms of the protein were inaccessible to trypsin digestion.
To better characterize the location and timing of intracellular
processing of the human fibrillin-1 carboxyl-terminal domain present in
the LEEC protein, four well known secretion blocking agents were
employed. Ammonium chloride is a weak base that blocks the secretory
pathway by neutralizing the pH of acidifying compartments of the
trans-Golgi and secretory vesicles. Monensin mediates a one-for-one
exchange of a proton for a monovalent cation such as sodium or
potassium (18). The use of ammonium chloride and monensin in
pulse-chase experiments resulted in the build-up of processed LEEC
(Fig. 7). The pH neutralization that
results from the use of agents such as ammonium chloride and monensin
have been shown to disrupt processing of secreted proteins normally carried out by the enzymes that reside in late compartments (19, 20).
The presence of processed LEEC under these conditions is consistent
with the idea that proteolytic processing occurs before and not in
these late secretory compartments.

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Fig. 7.
Secretion blocking of LEEC in CHO-K1
cells. Cells were preincubated with the various blocking agents,
pulsed for 30 min with [35S]Cys, and chased in the
presence of blocking agents for 2 h as described under
"Experimental Procedures." Gel A contains cell lysate.
Gel B contains conditioned medium. For both gels: lane
1, control with no blocking agents; lane 2, brefeldin
(10 µg/ml); lane 3, A23187 (2 µM);
lane 4, brefeldin (10 µg/ml) + A23187 (2 µM); lane 5, monensin (10 µM);
lane 6, ammonium chloride (50 mM).
Upper and lower arrows indicate unprocessed and
processed forms, respectively.
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Brefeldin A (BFA) disrupts anterograde transport from the ER to the
cis-Golgi by blocking the ability of ADP-ribosylation factors to
exchange GTP for GDP and become activated (21). Under circumstances
when forward transport is blocked, retrograde transport becomes visibly
apparent, and this leads to the redistribution of many Golgi resident
proteins back to the ER (for review see Ref. 22). The use of BFA in
pulse-chase experiments resulted in the build up of processed LEEC
(Fig. 7) indicating that in CHO-K1 cells, the cleavage occurs in the ER
or results from a Golgi resident enzyme that acts after the fusion of
the two compartments.
To refine further the cleavage compartment of LEEC in CHO-K1 cells, the
calcium ionophore calcimycin (A23187), which causes the equilibration
of calcium levels between the ER, Golgi, and cytoplasm, was employed.
There are strict calcium requirements for trafficking from the ER to
the Golgi (see Ref. 23 and for review see Ref. 24). By disrupting this
balance, calcimycin blocks protein exit from the ER (25, 26) without
altering protein synthesis or inhibiting glycosyltransferases (27, 28). When calcimycin is used in combination with BFA, retrograde flow of
Golgi contents to the ER is efficiently blocked (28).
The use of calcimycin alone or calcimycin with BFA in the pulse/chase
protein labeling studies results in the intracellular retention of both
unprocessed and processed LEEC (Fig. 7). Importantly, treatment with
calcimycin + BFA results in a form of LEEC with a SDS-PAGE migration
suggestive of incomplete glycosylation confirming that the inhibition
of retrograde flow from the Golgi has been successfully inhibited.
Because LEEC is already proteolytically processed at the point of this
block, we can conclude that processing is carried out by an enzyme that
resides early in the secretory pathway.
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DISCUSSION |
To study the processing of the carboxyl-terminal domain of
fibrillin-1, protein from three truncated fibrillin-1 constructs was
produced by baculoviral expression in SF9 cells and stable expression
from the pEE14 vector in CHO-K1 cells. In these expression systems,
each of the three constructs (LEE, LEEC, and LEEC-P1) was produced at
high levels with the expected post-translational modifications of
leader sequence cleavage, glycosylation, and carboxyl-terminal domain processing.
Amino-terminal sequencing of protein from both expression systems
revealed leader sequence processing at two sites: a primary site
between Gly-24/Ala-25 and a secondary site of Ala-27/Asn-28 (Fig. 2).
The secondary site was predicted by Pereira et al. (29) to
be the signal peptide cleavage site from primary sequence analysis of
the cloned cDNA. Cleavage at Gly-24/Ala-25, however, is an acceptable site based upon signal sequence structural requirements and
signal peptidase specificity. It has been reported that a multibasic
site in the FBN1 leader sequence was intracellularly processed when expressed in HT1080 cells (11). These constructs, however, employed the BM40 leader sequence inserted before the FBN1 leader sequence. This may explain the differences
between our results and the previously reported observations.
Proteolytic processing of the FBN1 carboxyl-terminal domain
in the LEEC construct occurred in both the baculoviral and CHO-K1 cell
systems. The inability of the FBN1-386 antibody to recognize processed
LEEC confirms that the region is lost after processing (Fig. 3).
Although the exact cleavage site was not determined, it is likely to
occur at the multibasic sequence indicated in Figs. 1 and 2. Tetrabasic
sequences of this type have been shown to be sites of cleavage by
members of the Furin/PACE family of proteases, and it has been
suggested that these enzymes are responsible for the processing event
in native fibrillin (12).
There are seven known mammalian members of the furin/PACE family that
are related to the bacterial subtilisins (30). Takahashi et
al. (31) have assigned a site preference order for one furin family member (PACE) using in vivo expression data coupled
with information from in vitro incubations with purified
PACE. They found the following: RXRXKR > XXRXKR >/= RXRXXR > RXXXKR > XXRXXR with an arginine
in the
P1 position being required. Sites with XXXXKR and
RXXXXR sequences were not processed. These rules are specific only for PACE. The other family members that have been examined, e.g. PACE4, have slightly different preferences
(32, 33).
In agreement with these established site preferences, the LEEC-P1
mutant, which replaces the required arginine with a serine in the
P1
position, shows less efficient processing than LEEC. It should be noted
that an acceptable site still exists in the
P1 sequence if the
recognition frame is shifted one residue upstream. In this frame,
however, the
P4 basic residue is then lost which would be expected to
lower the suitability of the site.
It is interesting to compare the putative processing site of
fibrillin-1 (PKRGRKRRS) to that of fibrillin-2 (PKKDSRQKRS). Data from
Milewicz et al. (12) indicates that the enzyme that normally
processes fibrillin-1 has a
P6 basic residue requirement. However,
there is not a basic residue in the
P6 position of fibrillin-2. This
suggests that fibrillin-1 and fibrillin-2 may be processed by different
enzymes, or with different efficiencies, or in different compartments
within the same cell. Given the wide tissue- and cell-specific
differences of expression in the furin family, it may be that the
cleavage of the carboxyl-terminal domain of fibrillins is not confined
to only one subcellular compartment or carried out by only one protease
and may vary by cell and tissue type. Fibrillin maturation within
different compartments of the secretory pathway may affect ECM assembly
by preventing or allowing interactions with chaperones or other matrix
components. In this way, tissue-specific or temporal expression of the
furin responsible for fibrillin processing may afford a level of
post-translational control of the maturation and matrix
deposition of the molecule.
To date, all known members of the PACE family are
calcium-dependent (34, 35), and modeling studies have
predicted a calcium-binding site, conserved from subtilisin, within
their catalytic domains. Raghunuth et al. (36) reduced the
efficiency of fibrillin cleavage in human fibroblasts by the addition
of 5 mM EGTA to culture medium. This would be expected to
hinder the activity of intracellular PACE which has half-maximal
activity at a calcium concentration of 0.2 mM (37). Also,
calcimycin-induced calcium depletion has been shown to inhibit a
Golgi-localized furin (38). Indeed, the calcium sensitivity of the
processing enzyme in the CHO-K1 protein expression system described
here is evident as the disruption of normal calcium levels by
calcimycin reduced the efficiency of the processing enzyme and leads to
the retention of the unprocessed form of LEEC that is still visible in
the samples incubated with calcimycin. This is consistent with the idea
that a furin family member is responsible for processing in this cell type.
Raghunath et al.2
added in vitro transcribed and translated LEEC (referred to
as "mini-fibrillin") to medium conditioned by cells transfected
with PACE, PACE4, PC1/3, and PC2 mutated by the removal of their normal
transmembrane domains to result in secretion. Each of the enzymes
tested was able to process LEEC. This work demonstrates that the
tetra-basic site in the fibrillin-1 carboxyl-terminal domain can be
recognized by these proteases. Wild type PACE is thought to localize to
the trans-Golgi network and therefore would be expected to process
proteins of the constitutive secretory pathway (for review see
Refs. 35, 39, and 40). The intracellular localization of other widely
expressed PACE family members such as PACE4, PC5/6, and PC8 has not
been determined but is also thought to be somewhere in the
constitutive secretory pathway (30, 33, 41).
The compartment in which the processing of full-length fibrillin occurs
is still unknown. Immunoprecipitation of radiolabeled fibrillin from
conditioned FBC medium yields only one band by autoradiography (Fig.
3A). Additionally, immunoprecipitated fibrillin is not
detected by Western blot with the FBN1-386 antibody made against an
epitope in the carboxyl-terminal fragment that is cleaved from the
full-length protein, yet the FBNI-386 antibody readily detects the
unprocessed domain of the LEEC-P1 mutant. The lack of significant
amounts of unprocessed fibrillin in conditioned medium indicates that
processing either occurs just after secretion in a quick and efficient
manner or occurs intracellularly in a way similar to the processing
undergone by our constructs in the CHO and SF9 systems.
Milewicz et al. (12) have proposed that carboxyl-terminal
domain processing must occur before fibrillin can be incorporated into
a linear microfibril. Studying a cell line that secretes fibrillin with
the entire carboxyl-terminal domain intact because of an arginine to
tryptophan mutation at position 2726, the authors found that the
unprocessed fibrillin was not incorporated into the matrix. Yet three
Marfan causing mutations have been described that result in the
omission of 69, 83, and 90% of the region normally lost after the
processing step. Two mutations, R2756X and
R2776X, result in a premature translation stop and the
omission of the last 116 and 96 residues of the domain, respectively
(42, 43). A third mutation is a two nucleotide deletion (8237 delGA)
that results in a frameshift and a premature stop. This allele codes correctly through residue 2746 and then ends with an out of frame stop
at 2757 (44). Importantly, premature intracellular polymerization of
fibrillin has not been reported for cells expressing these mutations.
These mutations are different from all other fibrillin-1 mutations in
that they affect a part of the molecule that is not present in the
mature protein. They show that this region somehow plays an important
role in the assembly or matrix incorporation of the molecule.
Handford et al. (45) has proposed a model of fibrillin
assembly in which monomers assemble into an aligned bundle. It may be
that the carboxyl-terminal domain of fibrillin serves to aid the
intracellular association of monomers into multimeric fibrillin building blocks (e.g. dimers or larger multimers), and once
that function has been accomplished, the domain can be processed, and other assembly steps can proceed. Other matrix molecules are known to
assemble intracellularly into large multimeric structures and then
undergo secretion, most notably laminin (46), tenascin (47), and
several of the collagens (48).
With the constructs described here, we have been able to more closely
observe the proteolytic processing of the fibrillin-1 carboxyl-terminal
domain. The loss of the expected region is shown by SDS-PAGE migration
differences and immunologically by Western blotting. Impaired
processing observed in the mutant LEEC-P1 construct confirms the
involvement of the multi-basic site in normal processing. Use of
secretion blocking agents has shown that cleavage occurs intracellularly in the CHO-K1 cells and is likely carried out by an
early secretory pathway resident furin-like enzyme.