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J. Biol. Chem., Vol. 277, Issue 52, 50795-50804, December 27, 2002
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From the Departments of
Received for publication, October 16, 2002
Fibrillin-1 and fibrillin-2 constitute the
backbone of extracellular filaments, called microfibrils. Fibrillin
assembly involves complex multistep mechanisms to result in a
periodical head-to-tail alignment in microfibrils. Impaired assembly
potentially plays a role in the molecular pathogenesis of genetic
disorders caused by mutations in fibrillin-1 (Marfan syndrome) and
fibrillin-2 (congenital contractural arachnodactyly). Presently, the
basic molecular interactions involved in fibrillin assembly are
obscure. Here, we have generated recombinant full-length human
fibrillin-1, and two overlapping recombinant polypeptides spanning the
entire human fibrillin-2 in a mammalian expression system.
Characterization by gel electrophoresis, electron microscopy after
rotary shadowing, and reactivity with antibodies demonstrated correct
folding of these recombinant polypeptides. Analyses of homotypic and
heterotypic interaction repertoires showed N- to C-terminal binding of
fibrillin-1, and of fibrillin-1 with fibrillin-2. The interactions were
of high affinity with dissociation constants in the low nanomolar range. However, the N- and C-terminal fibrillin-2 polypeptides did not
interact with each other. These results demonstrate that fibrillins can
directly interact in an N- to C-terminal fashion to form homotypic
fibrillin-1 or heterotypic fibrillin-1/fibrillin-2 microfibrils. This
conclusion was further strengthened by double immunofluorescence
labeling of microfibrils. In addition, the binding epitopes as well as
the entire fibrillin molecules displayed very stable properties.
Microfibrils are extracellular supramolecular aggregates
found in many elastic and non-elastic tissues (1). Ultrastructurally, they appear as beaded filaments with a periodicity of 50-55 nm (2).
The backbone of microfibrils are constituted by fibrillins, a family of
large extended proteins (3, 4). Other proteins such as
microfibril-associated glycoprotein
(MAGP)1-1 and -2 (5, 6),
fibulin-2 (7), versican (8), and latent transforming growth factor
Fibrillins consist of characteristic extracellular repetitive domains
such as the calcium-binding epidermal growth factor-like domains
(cbEGF) also found in many other extracellular proteins, and the
8-cysteine-containing domains (8-CYS) found exclusively in fibrillins
and LTBPs (4, 11-13). Fibrillin-1 and fibrillin-2 share 100% homology
on the domain level and ~68% homology on the overall amino acid
residue level. Based on this homology, one would predict a similar
mechanistic basis and architecture for the supramolecular assembly of
both fibrillins. Whether fibrillin-1 and fibrillin-2 have intrinsic
properties to self-assemble into homotypic or heterotypic microfibrils
or both is not clear at present. Based on developmental studies, it is
intelligible that adult tissues chiefly contain homotypic fibrillin-1
microfibrils, because fibrillin-2 generally is expressed early during
mammalian embryogenesis and tends to disappear later (14). However,
during development fibrillin-1 and fibrillin-2 often coincide in many tissues such as skin, lung, heart, aorta, central nervous system anlage, but are individually expressed in certain regions of kidney, liver, rib anlagen, and elastic cartilage (4, 15). In situations where
both fibrillins are expressed simultaneously, it is theoretically possible that each fibrillin isoform forms separate homotypic fibrils
or both isoforms together form heterotypic microfibrils. Different
organization of fibrillins in tissues have different functional
consequences that may be relevant in pathological situations.
Mutations in the genes for fibrillin-1 (FBN1) on chromosome 15 and
fibrillin-2 (FBN2) on chromosome 5 are responsible for the genetic
disorders Marfan syndrome (MFS, MIM no. 154700) and congenital
contractural arachnodactyly (MIM no. 121050), respectively (for recent
reviews, see Refs. 16 and 17). Although these autosomal dominant
disorders share some skeletal complications such as arachnodactyly,
scoliosis, and chest deformities, the cardiovascular and ocular
features characteristic for MFS are typically absent in congenital
contractural arachnodactyly. How mutant fibrillin-1 and -2 molecules
exert dominant negative effects in these disorders is presently
unknown. Certain lines of evidence point to the possibility that
fibrillin assembly mechanisms are compromised. For instance,
pulse-chase and immunofluorescence experiments have revealed reduced
amounts of mutant fibrillin-1 deposited into the extracellular matrix
of many fibroblast strains obtained from individuals with MFS (18-21).
These data suggest that many mutations in fibrillin-1 impair the
ability to assemble into microfibrils. Similar analyses of fibrillin-2
mutations in cell culture are presently lacking.
Aggregation of small N-terminal regions of fibrillins in recombinant
systems has suggested that homotypic dimerization is involved in the
multistep assembly mechanism of fibrillins (22-24). The static
organization of fibrillin-1 in microfibrils has been examined by
several groups and various techniques. Labeling of microfibrils with
specific antibodies, high resolution structure of cbEGF modules, and
analysis of transglutaminase cross-links have led to various models of
fibrillin alignment in microfibrils (25-29). Despite the unresolved
controversy whether fibrillin molecules are arranged in a nonstaggered
or in a staggered fashion, common to all models is a head-to-tail
arrangement of fibrillin molecules originally proposed by Sakai and
co-workers in 1991 (25). Mapping of monoclonal antibody epitopes in
fibrillin-1 molecules and correlation with the epitopes in microfibrils
revealed that the N- and the C-terminal ends of the fibrillin molecules
are located in or close to the bead structures (25, 26). However, it is
not clear whether microfibrillar backbone formation requires
fibrillin-1 and fibrillin-2 to directly interact with themselves and
with each other, or requires adapter molecules to connect fibrillin molecules in a head-to-tail fashion.
Here, we have analyzed in detail the spectrum of homotypic and
heterotypic molecular interactions of fibrillin-1 and fibrillin-2. The
results demonstrate direct head-to-tail interactions of fibrillin-1 alone, and of fibrillin-1 with fibrillin-2. However, fibrillin-2 alone
was not able to self-interact in a N- to C-terminal fashion, indicating
a different assembly mechanism for fibrillin-2. The results presented
suggest that fibrillins are able to form microfibrils in a direct
head-to-tail fashion without the aid of adapter molecules.
Cloning of Human Fibrillin-2 cDNA
Human fibrillin-2 cDNA was synthesized by reverse
transcription of total RNA isolated from MG-63 cells (American Type
Culture Collection) using the antisense (as) primers specified below. The obtained cDNAs were used as templates for polymerase chain reactions (PCR) using the same antisense primers in combination with
appropriate sense (s) primers (see below) and the high fidelity polymerase Pfx (Invitrogen). The 5' fragment rFBN2-0s (nt
1-1782) was synthesized by PCR amplification using primers 0s
(5'-ATTACCGCTAGCAACGGCTCGGCATCATGGGGAGAAGACGGAGGC-3') and 0as
(5'-ATCTGAGTTCACGCATCGACC-3'), which introduced a Kozak sequence
and a NheI recognition site for cloning at the 5' end. Primer pairs 5s (5'-TAGACACTAATGAGTGTGTCGC-3') and 8as
(5'-GAGGTTGAACTTCATGTTGACG-3'); 1s (5'-GGAAGCTTCAAATGCCGTTGC-3') and
4as (5'-GACAGATGCATCTGAAGGATCC-3'); and 9s
(5'-ACCTGTCACTGGATACAGAGG-3') and 9as
(5'-TTCAAAGTCTAAGACAGGGAGG-3') were used to amplify fragment
rFBN2-1g (nt 6041-8424), rFBN2-4a (nt 3277-6127), and rFBN2-9s (nt
8288-8872), respectively. All PCR products were cloned into the
pCR-BluntII plasmid (Invitrogen). The remaining portions (nt 432-3317)
of the fibrillin-2 cDNA originated from a construct
(pBS-UP Construction of Expression Plasmids
Fibrillin-2 Constructs--
To construct an expression plasmid
for the C-terminal half of human fibrillin-2 (rFBN2-C, positions
1531-2771), the NheI-NotI 9028-bp fragment from
pBS-rFBN2full was subcloned into the
NheI-NotI-restricted expression vector
pcDNA3.1 (Invitrogen), resulting in plasmid pcDNA-rFBN2full. To
add a sequence for a 6-histidine tag and a stop codon at the 3' end,
template rFBN2-1g was amplified by PCR using oligonucleotides 7s
(5'-TTTTGGGTCCTATGAATGCACG-3') and 14as (5'-ATCCGAATCAGCGGCCGCTCACTAGTGATGGTGATGGTGATGCTTAGAATAGCCGTTGATTTTGC-3'). The 1483-bp Bsu36I-NotI fragment of the resulting
PCR product was ligated into the
Bsu36I-NotI-restricted pcDNA-rFBN2full,
resulting in plasmid pcDNA-rFBN2full-his. The 6-histidine tag and
the stop codon replaced the sequence for part of the C-terminal unique domain from position 2772 to 2911. To modify the 5' end of the construct, template rFBN2-4a was amplified by PCR using
oligonucleotides 15s (5'-CGTAGCTAGCAGATATTGATGAGTGTGCAGATCC-3')
and 15as
(5'-CTGCTACTCGAGAGCGGCCGCTCACTAGTGATGGTGATGGTGATGCATGCAGTTGTGGCCTCCATTGACC-3'). A 460-bp BbvCI-NheI fragment of the PCR
product was ligated into the
BbvCI-NheI-restricted pcDNA-rFBN2full-his,
resulting in plasmid pcDNA-rFBN2-C-his. The
XbaI-NheI-restricted sequence from plasmid pBS-BM40 coding for the signal peptide of the human BM40 protein, which
confers efficient expression and secretion of the expressed protein,
was fused via the NheI site at the 5' site of the construct. The resulting plasmid was designated pDNSP-rFBN2-C. Expression and
processing resulted in the secretion of a protein (rFBN2-C) with
additional 4 N-terminal and 6 C-terminal amino acid residues preceding
and following the authentic domains
(Ala-Pro-Leu-Ala-Asp1531-Lys2771-(His)6).
To prepare the plasmid coding for the N-terminal half of human
fibrillin-2 (rFBN2-N, position 1-1732), the PCR product described above for the C-terminal construct was restricted by XhoI
(479 bp) and inserted into the XhoI-restricted plasmid
pcDNA-rFBN2full-his, resulting in pcDNA-rFBN2-N. Expression of
this plasmid resulted in a protein (rFBN2-N) that includes the sequence
for the authentic fibrillin-2 signal peptide and a 6-histidine tag at
the C-terminal end
(Met1-Met1732-(His)6). The most
likely prediction for the cleavage site of the signal peptide in
fibrillin-2 is between position 28 and 29 (Thr-Ala-Gly28 Fibrillin-1 Constructs--
The expression plasmids to express
the N-terminal half (pDNSP-rF16) and the C-terminal half
(pcDNA-rF6H) of human fibrillin-1 have been described in detail
previously (33). Both plasmids have been utilized to construct an
expression vector for full-length human fibrillin-1 as follows. A
3869-bp NotI-ApaI fragment was excised from
plasmid pcDNA-rF6H and subcloned into
NotI-ApaI-restricted pDNSP-rF16. A 320-bp
duplicated fragment was removed by restriction with PmlI,
followed by religation of the plasmid. The resulting plasmid was
designated pcDNA-rFBN1 and produces a secreted polypeptide (rFBN1)
with the sequence
Ala-Pro-Leu-Ala-Ser19-Lys2725-(His)6.
The correct DNA sequences at the restriction sites utilized have been
verified by DNA sequencing.
Transfection of Cells and Identification of Recombinant
Clones
For stable expression, the expression plasmids were linearized
by PvuI restriction and transfected into human embryonic
kidney cell 293 (American Type Culture Collection) using established methods (34). 10 h after transfection, selection was started with
500 µg/ml G418 (Calbiochem) and continued for 1 week. Thereafter, the
concentration of G418 was reduced to 250 µg/ml. After ~4 weeks, individual clones were transferred into 24-well plates and propagated to confluence. After washing with phosphate-buffered saline (PBS), the
wells were incubated with serum-free culture medium (1 ml) for 2 days.
Identification of clones expressing the recombinant proteins was
achieved by conventional gel electrophoresis, followed by Coomassie
Blue staining and Western blotting with specific monoclonal antibodies
against the expressed proteins. Typically, 50-80% of the analyzed
clones stably expressed the recombinant proteins.
Production of Recombinant Proteins
Stably transfected cell clones were routinely cultured in
Dulbecco's modified Eagle's medium (Invitrogen) supplemented
with 2 mM glutamine, 100 units/ml penicillin, 100 µg/ml
streptomycin, and 10% (v/v) fetal calf serum at 37 °C in a 5%
CO2 atmosphere. For large scale production, the clones were
propagated in triple layered flasks (Nalge Nunc International) to
confluence; washed twice with 20 mM HEPES, pH 7.4, 150 mM NaCl, 2.5 mM CaCl2; and incubated with serum-free culture medium for 48 h. The conditioned medium (2-2.5 liters) was harvested, supplemented with 1 mM phenylmethylsulfonyl fluoride (Fluka), and concentrated
to ~50 ml by ultrafiltration (YM30 membranes, Millipore). After
dialysis against 20 mM phosphate, pH 7.2, 1 M
NaCl (equilibration buffer), the medium was passed over a High-Trap
chelating column loaded with Co2+ (1-ml column size,
Amersham Biosciences) equilibrated in the same buffer. After washing
the column with equilibration buffer, bound proteins were eluted with a
linear imidazole gradient in equilibration buffer (0-250
mM imidazole in 30 ml) and fractioned in 1-ml aliquots. The
fractions were analyzed by gel electrophoresis followed by Coomassie
Blue staining and by Western blot analyses. Fractions containing the
recombinant proteins were pooled and dialyzed against 50 mM
Tris-HCl, pH 7.4, 150 mM NaCl (TBS).
Quantification of Protein Concentrations
Aliquots (50 µl) were supplemented with 450 µl of 6.67 M guanidine HCl in TBS and incubated at room temperature
for 30 min. The absorbance at 280 nm was determined on a Ultrospec 3000 spectrophotometer (Amersham Biosciences). Calculation of the
molar extinction coefficient followed an established method ( Alternatively, protein concentrations were determined by amino acid
analyses. The proteins were hydrolyzed in 6 N HCl under N2 for 24 h at 110 °C, and the amino acid
composition was determined on a Biochrom 20 analyzer (Biochrom).
Circular Dichroism Measurements
The purified recombinant proteins in TBS were diluted to a
concentration of 0.5 mg/ml. Spectra from 190 to 260 nm were recorded at
20 and 100 °C in a 1-mm quartz cuvette on a Jasco J-715 instrument.
Electron Microscopy
Purified recombinant proteins were dialyzed against 100 mM NH4HCO3 and adjusted to
concentrations of 0.25 mg/ml. The samples were diluted with 0.05%
(v/v) acetic acid to a final concentration of 60 µg/ml and mixed with
glycerol to a final concentration of 50% (v/v) glycerol. The samples
were sprayed onto freshly cleaved mica from a distance of 25 cm and
dried under vacuum for 2-3 h in an Edwards Auto 306 vacuum coater
(Edwards). Rotary shadowing was performed by platinum evaporation for
15 s, 50 mA, 2.5 kV at an angle of 5° and a distance of 12 cm,
followed by carbon evaporation for 2 s, 100 mA, 2.5 kV at an angle
of 90°. The replicas were floated onto a very clean surface of
distilled water and then supported using 400-mesh copper grids.
Replicas were examined at 100 kV in a transmission electron microscope
(Zeiss TEM 109).
Antibodies
Polyclonal antisera were produced in rabbits commercially
(Biotrend, Cologne, Germany) against the recombinant polypeptides rFBN2-N, rFBN2-C, and an ~110-kDa N-terminal fragment of human fibrillin-2, rF37, described previously (30). The antisera were characterized by a standard ELISA technique. Generation and properties of anti-human fibrillin-1 polyclonal antisera B9543 and Protein Interaction Assays
Solid Phase Binding Assay--
Multiwell plates (96 wells,
MaxiSorp, Nalge Nunc International) were coated overnight with purified
recombinant proteins (10 µg/ml, 100 µl/well) in TBS at 4 °C.
Nonspecific binding sites were blocked for 1 h with 5% (w/v)
nonfat milk in TBS. Each of the following incubations was performed in
TBS/5% nonfat milk including either 5 mM CaCl2
or 10 mM EDTA at room temperature (~ 20 °C) and was
followed by three washes with TBS including 0.05% (v/v) Tween 20. Coated proteins were incubated with serial dilutions (1:3) of the
soluble ligands (0.14-100 µg/ml) for 2 h. Incubation (1.5 h)
with the primary polyclonal antibodies (1:200-1:1250 diluted) against
the soluble ligands was followed by incubation with horseradish
peroxidase-conjugated secondary goat anti-rabbit antibody (1:800
diluted; Bio-Rad) for 1.5 h. Color development was performed with
1 mg/ml 5-aminosalicylic acid (Sigma) in 20 mM
phosphate buffer, pH 6.8, including 0.1%
H2O2 for 3-5 min and stopped by adding 2 M NaOH. Color yields were determined at 492 nm using a
Microplate EL310 (Bio-Tek Instruments).
To determine the stability of fibrillin interaction epitopes, the
immobilized or the soluble binding ligands were heat-treated in
discrete steps between 20 and 95 °C for 10 min and then used in the
above described solid phase binding assay.
Kinetic Binding Studies--
For kinetic binding studies of
recombinant fibrillin-1 and fibrillin-2 fragments by surface plasmon
resonance, a Biacore biosensor was used (Biacore 3000; Biacore AB).
Purified recombinant fibrillin-1 and fibrillin-2 fragments were
biotinylated using activated NHS-LC-biotin as instructed by the
supplier (Pierce). Biotinylated fragments were coupled in TBS to a
streptavidin sensor chip SA (Pioneer), which resulted in 1000-2500
response units. Binding studies were performed with soluble recombinant
fibrillin-1 or fibrillin-2 fragments in concentrations of 12.5-1000
nM in TBS at flow rates of 10 µl/min. The binding sites
of the immobilized ligands were regenerated by injection of a mixture
of detergents (0.075% (v/v) each of CHAPS, Zwittergent 3-12, Tween
80, Tween 20, Triton X-100) after each cycle. After subtraction of the
blank curves, representing binding to bovine serum albumin, the
association and dissociation rate constants were determined by separate
ka/kd fitting all curves at once with the 1:1 Langmuir association/dissociation model
(BIAevaluation software version 3.0, Biacore AB). Although this model
produced the best fits, the observed interactions probably diverge
somewhat from the 1:1 binding model because ka
values slightly decreased with increasing concentrations of the soluble
ligands. Mass transfer limitations were not apparent.
Immunofluorescence
Primary dermal fibroblasts from a 1-year-old individual and
primary osteoblasts from a 42-year-old individual were cultivated in
the first (fibroblasts) or fourth (osteoblasts) passage using culture
conditions as described above for the recombinant cell clones.
Confluent cells were trypsinized and seeded at densities of 7.5 × 104 cells/well of a 8-well chamber slide (Permanox; Nalge
Nunc International). After 4 days, the cells were washed in PBS, and
fixed in 70% (v/v) methanol, 30% (v/v) acetone for 5 min, and
rehydrated in PBS. The nonspecific binding sites were blocked with 1:10
diluted normal goat serum for 30 min. The cells were incubated with
monoclonal anti-fibrillin-2 antibody mAb 48 (1:200) and the polyclonal
anti-fibrillin-1 To generate recombinant human fibrillin-2 fragments, the human
fibrillin-2 cDNA was cloned from MG-63 cells. The cloned sequence was compared with the published sequence for human fibrillin-2 cDNA, and 19 differences have been identified (Table
I, GenBankTM accession no.
NM_001999; Refs. 4 and 31). Based on sequence homology of the cloned
and published sequence for human fibrillin-2 cDNA with the
sequences for human fibrillin-1 (GenBankTM accession no.
NM_000138; Refs. 11-13) and human fibrillin-3
(GenBankTM accession no. AB053450; Ref. 39), we
conclude that all of the observed differences are correct in the
sequence presented here. In some instances they likely represent
polymorphisms (Table I). Some of the observed variations have been
reported previously (30, 40).
To analyze the mechanisms of how fibrillins assemble into higher
ordered structures, we have generated new recombinant fragments of
fibrillin-2 as well as a recombinant full-length fibrillin-1 polypeptide. For fibrillin-2, we produced the N-terminal half rFBN2-N
(position 1-1732) and the C-terminal half rFBN2-C (position 1531-2771) in human 293 cells (Fig. 1).
rFBN2-N and rFBN2-C span the entire fibrillin-2 amino acid sequence
except part of the C-terminal unique domain (position 2772-2911),
which in analogy to fibrillin-1 processing presumably is cleaved by
furin-type proteases (41, 42). Similarly, the recombinant fibrillin-1 full-length construct rFBN1 comprises the entire fibrillin-1 sequence except the processed C-terminal domain (position 2726-2871; Fig. 1).
Additionally, two recombinant halves of fibrillin-1, rF16 and rF6H,
described previously were used in this study (33). For clarity, these
fragments have been renamed for this study to rFBN1-N and rFBN1-C (Fig.
1).
Homo- and Heterotypic Fibrillin-1 and -2 Interactions Constitute
the Basis for the Assembly of Microfibrils*
,,,
, and**
Medical Molecular Biology,
§ Chemistry, and Dermatology, University of
Lübeck, D-23538 Lübeck and ¶ Klinikum Neustadt,
D-23730 Neustadt, Germany
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-binding protein (LTBP)-1 and -2 (9, 10) were found associated with
microfibrils. Although it is clear that one of the basic functions of
fibrillins is the formation of the microfibrillar backbone through a
complex multistep assembly mechanism, the functional importance for the
associated proteins is presently obscure.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
22-3) described previously (30). The cDNAs from these
constructs were combined to a full-length fibrillin-2 construct in
several steps within the pBluescript II SK(+) plasmid using appropriate
restriction enzymes (New England Biolabs Inc.). The resulting plasmid,
pBS-rFBN2full, contained fibrillin-2 cDNA positions 1-8872. The
entire sequence of the fibrillin-2 cDNA was obtained by commercial
sequencing (Agowa, Berlin, Germany). Differences to the published
fibrillin-2 sequence (NM_001999; Refs. 4 and 31) are summarized and
interpreted in Table I.
Gln29-Pro) (32). Therefore,
the secreted protein likely starts at position 29. Correct ligation of
all constructs have been verified by DNA sequencing.
= nTrp × 5500 + nTyr
× 1490 + nCys-S-S-Cys × 125 [M
1 × cm1]) (35).
-rF6H, as
well as the specificity of monoclonal anti-human fibrillin-2 antibody
mAb 48 have been described elsewhere (36-38). B9543 and mAb 48 were
generous gifts from Dr. Lynn Y. Sakai (Shriners Hospital for
Children, Portland, OR).
-rF6H (1:400) in PBS for 1 h, followed by
three washes with PBS. After incubation with 1:200 diluted goat
anti-rabbit fluorescein conjugate (Jackson ImmunoResearch) and
Cy3-conjugated Affinipure goat anti-mouse IgG (Jackson ImmunoResearch),
the fibrillin-1 and fibrillin-2 networks were visualized by
fluorescence microscopy with a Axioplan microscope (Zeiss). Digital
images were recorded using a 3CCD color video camera (Sony) and
AxioVision software version 3.0 (Zeiss).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Comparison of the cloned and published human fibrillin-2 cDNA
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Fig. 1.
Schematic representation of recombinant human
fibrillin-1 and fibrillin-2 polypeptides used in this study. The
overview shows the domain model for both fibrillins. Full-length rFBN1,
rFBN2-N, and rFBN2-C have been generated for this study. rFBN1-N and
rFBN1-C have been generated previously and described as rF16 and rF6H,
respectively (33).
The recombinant fragments rFBN2-N and rFBN2-C were synthesized and
secreted into the culture medium of recombinant cell clones at
concentrations of ~5 µg/ml/day. Full-length rFBN1 was produced in
significantly lower amounts of ~0.5 µg/ml/day. All recombinant polypeptides were purified to homogeneity by chelating chromatography (Fig. 2). The molecular masses for
rFBN2-N (~210 kDa nonreduced and ~225 kDa reduced), rFBN2-C (~175
kDa nonreduced and ~185 kDa reduced), and rFBN1 (~322 kDa
nonreduced and ~345 kDa reduced) corresponded well with the expected
masses for these polypeptides. Freshly prepared and purified rFBN1
resulted in single bands in Coomassie-stained gels (Fig. 2). However,
rFBN1 tended to precipitate in solution after a few days, or upon
repeated freezing/thawing cycles (data not shown). The recombinant
halves of fibrillin-1 and fibrillin-2 were soluble and did not
precipitate in solution. All recombinant polypeptides reacted in
Western blot analyses with specific mono- and polyclonal antibodies,
which are dependent on correct disulfide bonds, indicating correct
three-dimensional structures of the polypeptides (data not shown).
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The new recombinant polypeptides were visualized by electron microscopy
after rotary shadowing. rFBN2-N (Fig.
3A) and rFBN2-C (Fig.
3B) showed extended shapes similar to what was observed for
the corresponding recombinant fibrillin-1 polypeptides (26). Occasionally, kinks and bends have been observed in both molecules. The
length of the molecules were 74.9 ± 4.1 nm (rFBN2-N,
n = 62) and 68.2 ± 4.8 nm (rFBN2-C,
n = 30). The shape of the rFBN1 molecules was also
threadlike and extended (Fig. 4). Again,
kinks and bends could be observed within the molecules (Fig. 4). Length
measurements of these molecules showed that 43.1% were in the range of
monomers between 100 and 180 nm (139 ± 24 nm), 13.8% in the
range of dimers between 240 and 320 nm (274 ± 21 nm), and 3.4%
in the range of trimers between 380 and 460 nm (416 ± 27 nm). The
remaining particles likely represented proteolytically truncated
products of monomers, dimers, and trimers. These data suggest that
monomeric fibrillin-1 can associate to multimers in TBS buffer.
Interestingly, the molecules appeared to connect at their ends with
each other, because no significant overlap between two molecules have
been observed in dimers and trimers (Fig. 4). Occasionally, kinks or
globules were detected in the region where two molecules are in contact
with each other. These results suggested that fibrillin-1 molecules can
interact with each other without the support of other molecules.
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To further analyze the self-interaction properties of fibrillin-1 and
fibrillin-2, as well as the ability of fibrillin-1 to interact with
fibrillin-2, binding activities of various combinations of the
recombinant halves of fibrillin-1 and fibrillin-2 have been analyzed by
solid phase binding assays (Fig. 5).
Strong self-interactions were observed between the N- and the
C-terminal halves of fibrillin-1 (Fig. 5A). Surprisingly,
the corresponding constructs of fibrillin-2 did not show significant
self-interaction properties (Fig. 5B). Fibrillin-1 also
interacted strongly with fibrillin-2. The N-terminal half of
fibrillin-1 clearly showed dose-dependent binding to the C-terminal half of fibrillin-2 (Fig. 5C), and the N-terminal
half of fibrillin-2 interacted with the C-terminal half of fibrillin-1 (Fig. 5D). All of the observed interactions were dependent
on calcium (Fig. 5).
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The association (ka) and dissociation rate
(kd) constants of the homotypic and heterotypic
fibrillin binding interactions, and the dissociation constants
(KD), have been determined by surface plasmon
resonance (Fig. 6). The kinetic data
obtained from such experiments are summarized in Table
II. In these experiments, high affinity
self-interaction of fibrillin-1 has been observed between the rFBN1-N
and -C (Fig. 6A; KD = 3.8-25.6
nM), whereas the corresponding fragments of fibrillin-2 did
not significantly interact (Fig. 6B). Heterotypic
interaction between fibrillin-1 and fibrillin-2 was observed for
combinations of rFBN1-N with rFBN2-C (Fig. 6C;
KD = 4.3-20.5 nM), and rFBN2-N with rFBN1-C (Fig. 6D; KD = 23.1-82.0
nM). These results correlated well with the data obtained
by solid phase binding assays and demonstrated that the homotypic
interaction of fibrillin-1 and the heterotypic interaction of
fibrillin-1 with fibrillin-2 are of high affinity.
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The stabilities of the interacting epitopes have been studied by heat
inactivation experiments (Fig. 7). The
recombinant fibrillin polypeptides were incubated at increasing
temperatures followed by analyses of the homo- and heterotypic
interactions in solid phase binding assays. Most binding epitopes were
inactivated only when the fragments were treated with high temperatures
above 80 °C. In these cases, the inactivation temperatures resulting
in 50% inhibition of binding were in the range between 83 and
87 °C. Only when the C-terminal fragments of fibrillin-1 or
fibrillin-2 were heat-treated and used as soluble ligands with
immobilized rFBN1-N did the inactivation temperatures resulting in 50%
binding inhibition decrease somewhat to 68 °C (rFBN1-N/heat-treated
rFBN2-C) and 77 °C (rFBN1-N/heat-treated rFBN1-C). These data
demonstrate that the binding epitopes for homo- and heterotypic
fibrillin N-to-C interactions represent very stable regions.
|
These results prompted us to analyze the stability of the entire
recombinant fragments by circular dichroism spectra. The spectra of the
recombinant polypeptides were recorded at low temperatures (20 °C)
and compared with spectra recorded at high temperatures (100 °C)
(Fig. 8). Spectra of all fragments at low
temperature showed molar ellipticities similar to what has been
observed for shorter fragments of fibrillin-1 (43). These spectra are
characteristic of large amounts of -structures. The spectra of the
polypeptides recorded at 100 °C only marginally differed from the
spectra recorded at 20 °C. These results demonstrate that the
polypeptide chains maintained much of their secondary structures even
at 100 °C. In addition, denaturation experiments using guanidinium
hydrochloride demonstrated similar stabilities of the recombinant
proteins (data not shown). When the binding inactivation experiments
and the analyses by circular dichroism spectra at different
temperatures are taken together, we conclude that fibrillins are very
stable proteins.
|
The ability of fibrillin-1 and fibrillin-2 to interact in microfibrils
has been examined by double-immunofluorescence experiments (Fig.
9). First, the polyclonal
anti-fibrillin-1 antiserum (-rF6H) was tested for potential
cross-reactivity with fibrillin-2 by ELISA (Fig. 9A). Only
minor cross-reactivity of -rF6H raised against the C-terminal half
of human fibrillin-1 was observed with the C-terminal half of human
fibrillin-2 at high concentrations (1:50-1:200 dilutions). This
cross-reactivity was negligible at dilutions of 1:400 and higher. Even
less cross-reactivity of the -rF6H antiserum was observed with the
N-terminal halves of human fibrillin-2 and -1 (Fig. 9A). The
specificity of the monoclonal mAb 48 antibody exclusively for
fibrillin-2 has been reported elsewhere (38). In dermal fibroblasts
from a 1-year-old donor, fibrillin-1 (Fig. 9B,
green signal) and fibrillin-2 (Fig.
9C, red signal) were detectable in a
microfibrillar network. Superimposition of both fluorescence signals
clearly demonstrated that both fibrillins were localized to the same
microfibrils (Fig. 9D). On the other hand, osteoblasts from
a 42-year-old donor showed a fibrillin-1 microfibrillar network (Fig.
9E), but fibrillin-2 could not be detected (Fig.
9F). Consequently, superimposition of both signals only
resulted in a green signal exclusively representing the presence of
fibrillin-1 (Fig. 9G). These results demonstrate that
fibrillin-1 alone can form microfibrillar structures, and that
fibrillin-1 and fibrillin-2 can occur in the same microfibrils. These
findings clearly correspond with the in vitro binding
interaction of fibrillins described above.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
|
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Microfibrils 10-12 nm in diameter are supramolecular aggregates in the extracellular matrix consisting of fibrillins and other matrix proteins. Fibrillins are repetitively aligned within microfibrils and constitute their structural backbone. At present, the mechanistic basis for formation of the fibrillin backbone is unknown, as is whether fibrillins exclusively form homotypic microfibrils consisting of only one isoform or heterotypic microfibrils containing both isoforms. Because impaired assembly mechanisms may precipitate the pathogenetic pathways of genetic disorders caused by mutations in fibrillin-1 and fibrillin-2, it is important to answer these questions. In this study, we have analyzed in detail homotypic and heterotypic interaction repertoires and stabilities of fibrillin-1 and fibrillin-2.
Fibrillins cannot be extracted from tissues in their native form because they are heavily cross-linked by reducible and nonreducible cross-links (28, 44). A feasible alternative to obtain fibrillin for mechanistic studies is its recombinant expression in mammalian cells. Previously, we have recombinantly produced two halves of fibrillin-1 and demonstrated correct folding and functional properties (7, 26). Here, we have produced two corresponding halves of fibrillin-2 spanning the entire processed fibrillin-2 molecule. These constructs resembled the corresponding counterparts of fibrillin-1, as judged by gel electrophoretic analysis and electron microscopy after rotary shadowing. The extended shapes together with the reactivity with antibodies requiring native epitopes, stabilized by disulfide bonds, suggested correct folding of these recombinant polypeptides. A similar rationale applies to rFBN1, a new recombinantly expressed full-length fibrillin-1. We anticipate that this full-length construct will be also, beyond the scope of this study, a useful tool for the analysis of fibrillin assembly mechanisms. rFBN1 as well as the recombinant halves of fibrillin-2 showed several bends and sharp kinks within the molecules, indicating the potential to fold back on themselves. In rFBN1, for instance, kinks were obvious close to one end and in the center of the recombinant molecules. Potentially, these sites correspond to regions containing the proline-rich domain and the third or fourth 8-CYS domain. A folding mechanism in these regions has been hypothesized to facilitate condensation of the fibrillin molecules within a microfibril (26, 29).
It has previously been established that fibrillins are oriented in microfibrils in a head-to-tail fashion and that the N- and C-terminal ends of the molecules are located either in or very close to the bead structures (25, 26). Theoretically, the connections between fibrillin molecules could be mediated by adapter molecules or, alternatively, by direct fibrillin interactions in a head-to-tail manner. It has been observed by immunogold localization that MAGP-1 is located at or close to the bead structures, and thus it potentially could function as a fibrillin bridging molecule in microfibrils (5). However, it has been demonstrated that MAGP-1 does only interact with fibrillin-1 via an epitope located at the N-terminal region of fibrillin-1, whereas the C-terminal region of fibrillin-1 does not harbor a binding site for MAGP-1 (33). Therefore, MAGP-1 alone cannot function as an adapter to connect fibrillin molecules in the bead regions of microfibrils. Other fibrillin-binding ligands such as fibulin-2 (7), LTBP-1 (45), or versican (8) have been described, but they are present only on some but not all microfibrils and they are typically not periodically aligned along microfibrils.
In this study, we have found for fibrillin-1 very strong interactions between the N- terminal and the C-terminal halves with dissociation constants in the low nanomolar range. Surprisingly, for fibrillin-2, this type of interaction could not be observed, although various protein interaction assays have been employed. Self-interaction properties in a head-to-tail fashion were also observed for the recombinant full-length fibrillin-1 polypeptide, which represented the C-terminally processed form of fibrillin-1, because it lacks the small C-terminal portion usually processed by furin-type proteases (41, 42). This polypeptide clearly showed the tendency to precipitate in physiological buffer solutions, indicating self-interaction properties. When rFBN1 molecules were analyzed ultrastructurally by electron microscopy after rotary shadowing, monomers, dimers, trimers, and, very occasionally, tetramers were observed. Because the analyzed samples originated from the cleared supernatant of a rFBN1 preparation, we suspect that the precipitate of this preparation is formed by even higher molecular aggregates. The multimers of rFBN1 found among the population of molecules appeared often as continuous strings without obvious overlapping regions of the molecules (Fig. 4). In some instances globules or kinks indicated the end of one and the beginning of the next molecule. These results suggest that the interaction epitopes are located relatively close to the processed ends of the molecules. We have found that recombinantly expressed fibrillin proteins representing N-terminal parts of the molecules are not straight as suggested from the domain structure, but often display a curved or globular shape (data not shown). Thus, the globules occasionally found between two rFBN1 molecules potentially could represent the N-terminal region. In this light, it is possible that the N-terminal interaction epitope is not located strictly at the N terminus of the fibrillin-1 molecule but somewhat further C-terminal, for instance, in the region of the proline-rich domain.
To further understand the mechanism of fibrillin-2 assembly, which appears not to involve homotypic self-interaction, we determined the ability of fibrillin-2 and fibrillin-1 to interact heterotypically with each other. Interestingly, we have found strong interaction properties between fibrillin-1 and fibrillin-2 with dissociation constants again in the low nanomolar range. The N-terminal half of fibrillin-2 interacted with the C-terminal half of fibrillin-1, and, vice versa, the C-terminal half of fibrillin-2 interacted with the N-terminal half of fibrillin-1. Because the recombinant halves of the fibrillins used in these assays are relatively large (185-225 kDa), we cannot draw a conclusion as to which domains are responsible for mediating the fibrillin homo- and heterotypic interactions. Interestingly, when smaller and well established recombinant fragments of fibrillin-1 were utilized to narrow down the binding epitopes for fibrillin-2, no binding activity could be observed (data not shown). One interpretation of such results is that the binding epitopes are stabilized by regions of the molecules that are not in the immediate vicinity of the binding epitope, e.g. by stabilizing long range structural effects. Previously, long range structural effects have been reported in fibrillin-1 (43). Another interpretation is that binding epitopes are located in regions at the ends of the recombinant subfragments used and thus are potentially truncated.
In addition, we have used a functional assay to assess the stability of the fibrillin interaction epitopes. Heat denaturation of recombinant proteins prior to the established interaction assay showed that the interaction epitopes are very stable regions of the molecules. Only treatment with high temperatures resulted in loss of interaction properties, e.g. in denaturation of the binding sites, an observation that corresponds well with the overall structural stability determined. The basis for this high temperature stability of fibrillin proteins very likely resides in the many stabilizing disulfide bonds. The cbEGF-like domain as well as the 8-CYS domains are stabilized by three and four disulfide bonds, respectively (26, 27). This stability is probably a prerequisite to serve as components of systems underlying mechanical forces such as for instance elastic tissues.
In conclusion, the presented data suggest mechanisms for homo- and
heterotypic fibrillin assembly, which are discussed in the following
and schematically visualized in Fig.
10. The schematic representation in
Fig. 10 is shown for the parallel nonstaggered model previously
proposed (26), but the mechanisms are applicable to all other fibrillin
alignment models (27-29). First, fibrillin-1 alone can homotypically
form the backbone of a microfibril in the absence of adapter molecules
and in the absence of fibrillin-2 (Fig. 10A). This type of
mechanism could be further confirmed by the observation that
osteoblasts from a 42-year old donor deposited a microfibrillar network
consisting of fibrillin-1 in the absence fibrillin-2 (Fig. 9).
Generally in mammalian development, fibrillin-1 expression persists for
longer periods of time, whereas fibrillin-2 tends to disappear earlier
(14). Based on this observation, homotypic fibrillin-1 microfibrils are
likely the predominant form in adult organisms. The data presented here
provide an explanation of how fibrillin-1 can form microfibrils in the
absence of fibrillin-2. Second, the data strongly suggest that
fibrillin-1 and fibrillin-2 can co-polymerize in a head-to-tail fashion
to form heterotypic microfibrils (Fig. 10B). Based on the
high homology of the domain structures of fibrillin-1 and -2, it is
conceivable that both fibrillins fit into the same geometric
constraints of a single microfibril. This mechanism is supported by our
observation that dermal fibroblasts from a 1-year-old donor deposited a
microfibrillar network containing both fibrillin-1 and fibrillin-2
within the same microfibrils (Fig. 9). Ultrastructural immunogold
co-localization of fibrillin-1 and -2 within the same microfibril is
presented elsewhere by Charbonneau et al. (38). Early in
mammalian development, fibrillin-1 and fibrillin-2 are co-expressed in
most tissues (4, 15). Based on the ability of fibrillin-1 and
fibrillin-2 to interact with each other with very high affinity, we
predict in situations where both fibrillins are expressed
simultaneously, they will form heterotypic microfibrils. Third, the
data presented also predict that fibrillin-2 alone, in the absence of
fibrillin-1, cannot form homotypic microfibrils by corresponding N- to
C-interacting mechanisms. It is clear that fibrillin-2 is expressed in
some tissues during development where fibrillin-1 is not expressed (4,
15). In these situations, fibrillin-2 may assemble into homotypic
microfibrils by alternative mechanisms. Such mechanisms may require
molecular adapters, or a new member of the fibrillin family,
fibrillin-3, may play a critical role (39). Alternatively, fibrillin-2
assembles into another, not yet recognized form of supramolecular
aggregates.
|
| |
ACKNOWLEDGEMENTS |
|---|
We are grateful for the excellent technical assistance of Karin Wießmann, Thies Köhli, and Heiko Steenbock, and for helpful discussions with Dr. Holger Notbohm. We thank Dr. Lynn Y. Sakai for providing antibody mAb 48 and antiserum B9543.
| |
FOOTNOTES |
|---|
* This work was supported by Deutsche Forschungsgemeinschaft Grants SFB367-A1, RE1021/3-2, and RE1021/4-2.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
** To whom correspondence should be addressed: Dept. of Medical Molecular Biology, University of Lübeck, Ratzeburger Allee 160, D-23538 Lübeck, Germany. Tel.: 49-451-500-4086; Fax: 49-451-500-3637; E-mail: reinhardt@molbio.mu-luebeck.de.
Published, JBC Papers in Press, October 23, 2002, DOI 10.1074/jbc.M210611200
| |
ABBREVIATIONS |
|---|
The abbreviations used are:
MAGP, microfibril-associated glycoprotein;
8-CYS, 8-cysteine-containing
domain;
as, antisense;
cbEGF, calcium-binding epidermal growth
factor-like domain;
CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid;
ELISA, enzyme-linked immunosorbent assay;
LTBP, latent transforming
growth factor -binding protein;
mAb, monoclonal antibody;
MFS, Marfan syndrome;
nt, nucleotide(s);
PBS, phosphate-buffered saline;
s, sense;
TBS, Tris-buffered saline.
| |
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TOP
ABSTRACT
INTRODUCTION
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RESULTS
DISCUSSION
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S. A. Cain, C. Baldock, J. Gallagher, A. Morgan, D. V. Bax, A. S. Weiss, C. A. Shuttleworth, and C. M. Kielty Fibrillin-1 Interactions with Heparin: IMPLICATIONS FOR MICROFIBRIL AND ELASTIC FIBER ASSEMBLY J. Biol. Chem., August 26, 2005; 280(34): 30526 - 30537. [Abstract] [Full Text] [PDF]
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K. Tiedemann, T. Sasaki, E. Gustafsson, W. Gohring, B. Batge, H. Notbohm, R. Timpl, T. Wedel, U. Schlotzer-Schrehardt, and D. P. Reinhardt Microfibrils at Basement Membrane Zones Interact with Perlecan via Fibrillin-1 J. Biol. Chem., March 25, 2005; 280(12): 11404 - 11412. [Abstract] [Full Text] [PDF]
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A. Marson, M. J. Rock, S. A. Cain, L. J. Freeman, A. Morgan, K. Mellody, C. A. Shuttleworth, C. Baldock, and C. M. Kielty Homotypic Fibrillin-1 Interactions in Microfibril Assembly J. Biol. Chem., February 11, 2005; 280(6): 5013 - 5021. [Abstract] [Full Text] [PDF]
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 L. Schaefer, D. Mihalik, A. Babelova, M. Krzyzankova, H.-J. Grone, R. V. Iozzo, M. F. Young, D. G. Seidler, G. Lin, D. P. Reinhardt, et al. Regulation of Fibrillin-1 by Biglycan and Decorin Is Important for Tissue Preservation in the Kidney During Pressure-Induced Injury Am. J. Pathol., August 1, 2004; 165(2): 383 - 396. [Abstract] [Full Text] [PDF]
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T. Vollbrandt, K. Tiedemann, E. El-Hallous, G. Lin, J. Brinckmann, H. John, B. Batge, H. Notbohm, and D. P. Reinhardt Consequences of Cysteine Mutations in Calcium-binding Epidermal Growth Factor Modules of Fibrillin-1 J. Biol. Chem., July 30, 2004; 279(31): 32924 - 32931. [Abstract] [Full Text] [PDF]
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C. C. Werneck, B. C. Trask, T. J. Broekelmann, T. M. Trask, T. M. Ritty, F. Segade, and R. P. Mecham Identification of a Major Microfibril-associated Glycoprotein-1-binding Domain in Fibrillin-2 J. Biol. Chem., May 28, 2004; 279(22): 23045 - 23051. [Abstract] [Full Text] [PDF]
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M. J. Rock, S. A. Cain, L. J. Freeman, A. Morgan, K. Mellody, A. Marson, C. A. Shuttleworth, A. S. Weiss, and C. M. Kielty Molecular Basis of Elastic Fiber Formation: CRITICAL INTERACTIONS AND A TROPOELASTIN-FIBRILLIN-1 CROSS-LINK J. Biol. Chem., May 28, 2004; 279(22): 23748 - 23758. [Abstract] [Full Text] [PDF]
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 A. M. Kapoun, F. Liang, G. O'Young, D. L. Damm, D. Quon, R. T. White, K. Munson, A. Lam, G. F. Schreiner, and A. A. Protter B-Type Natriuretic Peptide Exerts Broad Functional Opposition to Transforming Growth Factor-{beta} in Primary Human Cardiac Fibroblasts: Fibrosis, Myofibroblast Conversion, Proliferation, and Inflammation Circ. Res., March 5, 2004; 94(4): 453 - 461. [Abstract] [Full Text] [PDF]
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Daniel. V. Bax, S. E. Bernard, A. Lomas, A. Morgan, J. Humphries, C. A. Shuttleworth, M. J. Humphries, and C. M. Kielty Cell Adhesion to Fibrillin-1 Molecules and Microfibrils Is Mediated by {alpha}5{beta}1 and {alpha}v{beta}3 Integrins J. Biol. Chem., September 5, 2003; 278(36): 34605 - 34616. [Abstract] [Full Text] [PDF]
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