From the Department of Biochemistry and Molecular Biology, Shriners Hospital for Children, Oregon Health and Science University, Portland, Oregon 97239, the § Department of Cell Biology and Medicine, New York University, New York, New York 10016, and the ¶ Department of Medical Molecular Biology, University of Lübeck, Ratzeburger Allee 160, D-23538 Lübeck, Germany
Received for publication, September 10, 2002, and in revised form, November 4, 2002
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ABSTRACT |
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Latent transforming growth factor The fibrillins and latent transforming growth factor Extensive immunolocalization data combined with structural analyses of
the fibrillin-1 monomer and fibrillin-containing microfibrils (1,
13-15) have established that fibrillin-1 is a major structural component of connective tissue microfibrils. In addition, genetic evidence in humans (16, 17) and mice (18, 19) has confirmed that
fibrillin-1 performs a significant role in the maintenance of
microfibrils and elastic fibers.
Fibrillin-2, whose structure is predicted to be highly similar to
fibrillin-1, has also been immunolocalized to microfibrils (20).
However, in contrast to fibrillin-1, the contribution of fibrillin-2 to
microfibril structure is temporally and spatially restricted. In
situ hybridization studies in mice indicated that expression of
the fbn2 gene is most prominent in the early
developing fetus (20). Genetic evidence in humans (5, 21) suggests that
fibrillin-2 plays a more restricted role in the maintenance of
microfibrils and elastic fibers in postnatal connective tissues. Recent
immunolocalization studies demonstrate a ubiquitous early distribution
of fibrillin-2 in fetal tissues followed by a restricted distribution
in postnatal tissues (22). Mice produced by gene targeting experiments
recapitulate the features (contractures of large and small joints) of
the human disease congenital contractural arachnodactyly caused by
mutations in fibrillin-2 (23). In addition, fibrillin-2 null mice
revealed an unexpected role for fibrillin-2 in limb patterning, because
the mutant animals display syndactyly (23).
LTBP-1 forms a complex with latent TGF- LTBP-1 becomes immobilized into the extracellular matrix of tissue
culture cells in a covalent manner involving tissue
transglutaminase-mediated cross-linking of a region in the N-terminal
sequence of LTBP to an undefined matrix protein (28). In tissue
culture, LTBP-1 colocalizes with both fibronectin and fibrillin-1 (29,
30). LTBP-1 has been immunolocalized to fibrillin-containing
microfibrils in the skin (31) and bone (30, 32) and to microfibrillar structures in the heart (33). LTBP-2 has also been immunolocalized to
fibrillin-containing microfibrils (34) and exogenous LTBP-2 can be
incorporated into the extracellular matrix by fibroblasts (35). Studies
of mRNA have indicated that LTBPs are differentially expressed in
various tissues. However, surveys of the distribution of LTBP-1 protein
in tissues have not been conducted. Moreover, the relationship between
LTBPs and fibrillins within microfibril structures is not understood.
The investigations presented here were undertaken to determine whether
LTBP-1 is present in extracted fibrillin microfibrils, whether LTBP-1
and fibrillin bind to each other, and if so, which regions of the two
proteins interact.
Production of Recombinant Polypeptides--
Recombinant human
fibrillin polypeptides used in these investigations have been
previously described and characterized (15, 22, 36-38). These are
depicted schematically in Fig.
1B. All recombinant fibrillin
polypeptides were expressed using 293 human embryonic kidney cells.
Full-length recombinant human LTBP-1 was expressed in insect cells as
previously described (25). To make the expression construct rL1N,
coding for Asn21 to Ile629 of LTBP-1,
clone pACUW-51 was amplified with sense primer LTBP-1-1S (5'-CTGCTAGCAAACCACACTGGCCGCATCAAG-3') introducing a
NheI restriction site at the 5' end, and antisense primer
LTBP-1-1AS
(5'-CTCGAGTCAATGATGATGATGATGATGTATGCAGTTAGTACCCTCCTC-3'), introducing the sequence for 6 histidine residues, a stop codon, and a
XhoI restriction site at the 3' end. The resulting
NheI-XhoI fragment was subcloned into the
expression vector pCEP/
The cDNA that was used to obtain expression construct rL4K
(Ser1301-Ala1587 of LTBP-4) was derived from
normal human dermal fibroblast RNA by reverse transcriptase-PCR. PCR
amplification of the cDNA used sense primer rL4K-S
(5'-GATCGCTAGCATCCAACGAGAGCCAGAGCC-3') and antisense primer
rL4K-AS
(5'-GAGTCTCGAGCTCAGTGATGGTGATGGTGATGGGCCCGGGGCCGTGCGG-3'), which introduced, respectively, a 5' NheI restriction site
and a 3' sequence for 6 histidine residues, a stop codon, and an
XhoI site. An 894-bp
NheI-XhoI-restricted insert was subcloned into the expression vector pCEP4/
Mouse LTBP-3 specific sense primer L3-4F
(5'-CCAGAAGGAGAGTCTGTGGC-3') and antisense primer L3-4R
(5'-TGTGGGCACTTGTGACACTT-3') were designed based on the published
sequence (10) and used in reverse transcriptase-PCR to amplify a
fragment of mouse LTBP-3 cDNA (nucleotide 460-902, with the A in
the translation start codon ATG designated as +1) from RNA extracted
from 2T3 mouse osteoblast cells. This fragment was used as a probe for
screening of a mouse heart cDNA library
(Clontech). Several full-length LTBP-3 cDNA
clones were isolated and cloned as EcoRI-EcoRI
fragments into pBluescript SK (Stratagene) vector. Several errors were
found in the original sequence by analyzing these cDNA
clones.2 To make the
expression vector rL3K, encoding the region from the beginning of CR4
to the COOH terminus, a fragment of the cDNA was amplified by PCR
using sense primer L3CR4C-S (5'-CGGCTAGCCCCAAAGAGACGTGAAGTG-3') and antisense primer L3CR4CAS
(5'-CCGCTCGAGTCAGTGGTGGTGGTGGTGG-CGGCGGCGCTGAGGCAC-3'), introducing an NheI site at the 5' end and the sequence for
6 histidine residues, a stop codon, and an XhoI site at the
3' end. The NheI-XhoI fragment was subcloned into
the expression vector pCEP4/
Schematic representations of the LTBP constructs used for these studies
are shown in Fig. 1A. For stable episomal expression, 293 EBNA cells (Invitrogen) were transfected with the expression plasmids
by a calcium phosphate precipitation method as described previously
(40). Purification of the recombinant peptides was accomplished using
chelating chromatography (HiTrap chelating, Amersham
Biosciences) (15) followed by molecular sieve chromatography, using Superose 6 (Amersham Biosciences) in 50 mM Tris-HCl,
pH 7.5, 0.15 M NaCl (TBS) for rL1M and rL1C or 50 mM Tris-HCl, pH 7.5, 1 M NaCl for rL1N.
Each of the expressed polypeptides was secreted into the medium
resulting in yields of more than 0.5 µg/ml. N-terminal sequence analysis of the purified peptides using Edman degradation and amino
acid analysis confirmed the expected polypeptide sequence and also
demonstrated the purity of the peptides. SDS-PAGE analysis under
nonreducing and reducing conditions revealed that the LTBP-1, LTBP-3,
and LTBP-4 recombinant polypeptides were secreted as monomers from 293 cells (Fig. 2).
Antibodies and Immunoassays--
Mouse monoclonal antibodies 201 and 69 to fibrillin-1 have been characterized previously (1, 13, 15,
38). Polyclonal anti-fibrillin-1 9543 was also characterized (18, 22).
Monoclonal antibodies, 75G, and 42E, were generated using full-length
LTBP-1 expressed by Sf9 insect cells (25). Mouse
monoclonal antibody 246 against the TGF-
The specificity of monoclonal antibodies was determined by ELISA, as
described (41). Recombinant fibrillin-1 subdomains, rF11 and rF6, and
LTBP-1 subdomains, rL1N, rL1M, and rL1C, were used to coat microtiter
plates at 10 µg/ml. The antibodies were diluted in TBS. For Western
blot analysis, serum-free conditioned medium from normal skin
fibroblasts was collected for 48 h (13). Proteins in the medium
were precipitated, subjected to 7.5% SDS-PAGE, and analyzed by
immunoblotting as previously described (42).
Extraction and Size Fractionation of LTBP-1 from
Tissues--
Extracts of normal adult human skin, bovine calf tendon,
and human fetal membranes were prepared as follows. Nonexposed human skin (~1 g) was obtained as excess tissue from a skin grafting procedure. Bovine tendon (~1.5 g) was dissected from an 80-cm crown
to rump fetal calf (~245 days gestation, almost full-term) obtained
from a local slaughterhouse. A 40-ml suspension of fetal membranes,
washed and homogenized as described (42), was also extracted as follows.
Tissue samples were minced, and the pellets were washed briefly with 50 mM Tris-HCl, pH 7.5, containing 10 mM
CaCl2, and 1 mM phenylmethylsulfonyl fluoride.
The buffer was removed after centrifugation, and the pellet was
extracted in 6 M guanidine hydrochloride, 50 mM
Tris-HCl, pH 7.5, containing 1 mM phenylmethylsulfonyl fluoride for 72 h at 4 °C with vigorous shaking. The
supernatant was collected after centrifugation, and the pellet was
extracted in the same buffer for 48 h followed by centrifugation
and another 24 h of extraction. The supernatants were pooled and
concentrated to 5 ml using an Amicon concentrator (cut-off
Mr = 30,000). Sieve chromatography under
dissociative conditions was performed using a Sepharose CL-2B (Amersham
Biosciences) molecular sizing column (90-ml total volume), equilibrated
in 4 M guanidine HCl, 50 mM Tris-HCl, pH 7.5, at a flow rate of 0.1 ml/min. The fractions were collected every 2 ml.
Protein concentrations were determined using a BCA protein assay kit
(Pierce) with bovine serum albumin as the standard. Dot blot analysis
was performed using 2.5 µl of spotted fractions and either polyclonal
antibody 39 or monoclonal antibodies 69, 75G, and 42E, as described
(42). Western blot analysis was performed using two combined
consecutive fractions. Guanidine hydrochloride was eliminated by
ethanol precipitation as described (43).
Microfibrils were also isolated from tissues using crude collagenase
(Sigma) digestion. Procedures used were the same as those we have
previously detailed (42).
Immunolocalization Studies--
Light and electron microscopic
immunohistochemical procedures were the same as those we have
previously described (44). Tissues were frozen in hexanes for light and
confocal microscopy. Fluorescein isothiocyanate-conjugated rabbit
anti-mouse IgG (Sigma) was used for immunofluorescence microscopy,
using 8-µm sections. For confocal microscopy, 25-µm sections were
incubated with primary antibodies, followed by Alexa Fluor 488 goat
anti-mouse IgG or Alexa Fluor 594 goat anti-rabbit IgG (Molecular
Probes, Eugene, OR). Stained sections were viewed with a Leica TCS SP2
confocal microscope and merged images were generated using Leica
software. For electron microscopic immunolocalization, fresh tissue
blocks were first incubated with dilutions of primary antibody,
followed by a gold-conjugated second antibody, and then embedded and
prepared for electron microscopy.
Binding Studies--
Interactions between LTBP and fibrillin
were investigated by solid phase ELISA binding or blot overlay assays.
For ELISA binding assays, multiwell plates were coated with purified
LTBP-1 peptides (50 nM, 100 µl/well) in 15 mM
Na2CO3 and 35 mM
NaHCO3, pH 9.2, at 4 °C overnight. Coated wells were
blocked with 5% nonfat dry milk in TBS at room temperature for 1 h. Recombinant fibrillin-1 polypeptides were serially diluted 1:2 in
2% milk, TBS, containing 2 mM CaCl2 or
5 mM EDTA, and incubated in the wells for 3 h.
Monoclonal antibodies against soluble ligands were diluted in 2% milk,
TBS and used to detect the bound ligands, after a final incubation with
enzyme-conjugated secondary antibodies. Color reaction of the enzyme
immunoassay was achieved using p-nitrophenyl phosphate (Sigma tablets) or 1 mg/ml 5-aminosalicylic acid. Absorbance was determined at 405 nm using a Titertek Multiskan.
For blot overlay assays, serum-free conditioned media was collected
from High FiveTM cells that were transfected with
recombinant LTBP-1. 1 ml of media was precipitated using
trichloroacetic acid, resolved by SDS-PAGE, and transferred to a
nitrocellulose membrane. After blocking with 5% nonfat milk in TBS at
room temperature for 1 h, the membrane was incubated with
recombinant fibrillin peptides (50 µg/ml or 1 µM) in
2% nonfat milk in TBS at 4 °C overnight or room temperature for
3 h. Monoclonal antibodies diluted in 2% milk, TBS were used to
detect the bound ligands, after a final incubation with
enzyme-conjugated secondary antibodies. The blots were developed by
color reaction using 4-chloro-1-naphthol (Bio-Rad).
LTBP-1 and Fibrillin-1 Are Co-distributed in Some
Tissues--
Although immunolocalization studies of LTBP-1 have been
performed using cultured cells and tissues, surveys of tissue
distribution have not been published. In addition, most published
results have relied upon polyclonal antiserum 39. To immunolocalize
LTBP-1 with greater confidence and to establish tissue distribution
patterns, monoclonal antibodies were generated using purified
full-length recombinant human LTBP-1 expressed in insect cells. Two
monoclonal antibodies (mAb 75G and mAb 42E), which immunoblotted and
immunoprecipitated LTBP-1 produced by insect cells (data not shown),
were selected for further characterization. Epitopes for these
antibodies were mapped by immunoblotting using four LTBP-1 recombinant
polypeptides (Fig. 1A) expressed in 293 human embryonic
kidney cells. MAb 75G recognized an epitope in rL1M, but did not bind
to rL1N or rL1C (data not shown). mAb 42E bound to a site close to the
C-terminal region in rL1C, not in rL1K (data not shown). In addition,
mAb 75G and mAb 42E recognized authentic LTBP-1 present in the medium of cultured human fibroblasts (NSF lane, in Fig. 7).
75G and 42E displayed no reactivity with authentic fibrillin in
fibroblast-conditioned medium, nor with rF11 and rF6, the two
recombinant halves of fibrillin-1 (data not shown).
When tested using a panel of human tissues, mAb 75G and mAb 42E yielded
similar immunohistochemical results. In tissues such as tendon,
perichondrium, and blood vessels, the staining patterns for LTBP-1 and
fibrillin-1 were apparently identical. In tendon and perichondrium,
long fluorescent fibrils were evident after staining with anti-LTBP-1
(Fig. 3, A and L)
or with anti-fibrillin-1 (Fig. 3B). Matrix around blood
vessels was densely stained with antibodies to LTBP-1
(arrowheads in Fig. 3, C, K, and
L) and fibrillin-1 (Fig. 3D). However, in other
tissues such as skeletal muscle (Fig. 3, I compared with
J) and lung (Fig. 3, C compared with
D) where fibrillin-1 was abundant, LTBP-1 appeared to be
absent or more limited in spatial distribution. In lung, bright
staining for LTBP-1 was found around blood vessels but faint staining
outlined the airways (Fig. 3C, arrow). In
peripheral nerves, LTBP-1 appeared to be present primarily in the outer
nerve sheath (Fig. 3, E and L,
asterisks), whereas fibrillin-1 was found in all three connective tissue sheaths of the nerve (Fig. 3F). In skin, fibrillin-1
was present throughout the dermis (Fig. 3H), whereas LTBP-1
was much less abundant and was concentrated primarily in hair follicles (Fig. 3G) and scattered fibers (Fig. 3K).
These studies demonstrated that LTBP-1 is not present in all tissues
containing fibrillin microfibrils. Co-distribution of LTBP-1 and
fibrillin was prominent in tendon, perichondrium, cartilage, and all
blood vessels. Partial co-distribution was found in tissues like skin,
lung, and peripheral nerve.
Confocal and Electron Microscopic Immunolocalization Demonstrate
Colocalization of Fibrillin-1 and LTBP-1--
Tissues demonstrating
strong LTBP-1 immunofluorescence were chosen for additional analyses to
determine whether LTBP-1 and fibrillin-1 are colocalized. In the
developing fetal foot, tendons were well labeled with LTBP-1 antibodies
(Fig. 4A) and fibrillin-1 antibodies (Fig. 4B). The merged image (Fig. 4C)
indicated that in the tendon most of the fibrillar staining directed by
LTBP-1 antibodies was labeling the same fibrillar structures that were stained by fibrillin-1 antibodies.
In addition, neonatal foreskin, fetal bovine tendon, and fetal bovine
aorta were immunolabeled with anti-LTBP-1 mAb 75G and examined by
electron microscopy. As has been reported, immunogold labeling was
observed on microfibrils (Fig. 5,
A and C). However, in contrast to the periodic
labeling obtained with antibodies to fibrillin-1 (1, 15) (Fig.
5B) and with polyclonal antibodies to LTBP-1 (30), LTBP-1
antibody-directed gold labeling was sparse and irregular (Fig. 5,
A and C).
Microfibril Extracts from Tissues Do Not Contain LTBP-1--
To
determine whether LTBP-1 is contained within microfibrils or is a
microfibril-associated molecule, extraction studies were performed.
Bovine calf tendon, human fetal membranes (data not shown), and human
skin, tissues in which LTBP-1 co-localizes with fibrillin, were used.
Microfibrils were extracted either by crude collagenase digestion or
denaturation in guanidine HCl. After fractionation using Sepharose
CL-2B chromatography, fibrillin microfibrils were eluted in the void
volume fraction both from the collagenase digests (human fetal
membranes and human skin) and the guanidine HCl extracts (human fetal
membranes and fetal bovine tendon), as shown by dot blot analysis with
several anti-fibrillin-1 antibodies and rotary shadowing (Fig.
6 and data not shown). Interestingly, no
immunoreactivity with LTBP-1 antibodies (mAb 75G, mAb 42E, or pAb 39)
was detected in the microfibril fractions from any of the tissues
examined, including collagenase and guanidine extracts of human skin
and bovine tendon (Figs. 6 and 7) and
collagenase and guanidine extracts of human fetal membranes (data not
shown). These results suggested that LTBP-1 is not an integral
structural component of the beaded string microfibril.
When LTBP-1 was extracted from tendon and skin using guanidine HCl,
Western and dot blots using mAb 75G (data not shown), mAb 42E and pAb
39 (data not shown) detected LTBP-1 in the included fractions of the
Sepharose CL-2B column with a molecular mass close to 200 kDa (Fig. 7).
This molecular mass, similar to that of authentic LTBP-1 found in
normal skin fibroblasts (NSF lane, in Fig. 7), corresponds
to the complex of LTBP and LAP (25). Indeed, in human skin extracts,
LTBP-1 was detected as a complex with LAP (Fig. 7B). The
small apparent differences in molecular masses of the LTBP-1 complexes
from these two tissues may reflect either the presence of
differentially spliced isoforms or proteolytic degradation in the case
of skin.
LTBP-1 Binds to Fibrillins--
Blot overlay assays were utilized
to screen for regions of fibrillins that might interact with LTBP-1.
Insect cell-conditioned medium containing full-length recombinant human
LTBP-1 (and multiple other proteins) (Fig. 2A) was subjected
to SDS-PAGE, transferred to nitrocellulose, and used as substrate for
the binding assays. Recombinant human fibrillin-1 and fibrillin-2
polypeptides (Fig. 1B) were purified and used as ligands for
these assays. Detection of bound ligands was accomplished by using
specific mAbs to fibrillins or by using a monoclonal antibody that
reacts with the histidine tag present at the C-terminal end
of some fibrillin recombinant polypeptides. When the large N- and
C-terminal halves of fibrillin-1 (rF11 and rF6, respectively) were
tested, rF11 bound to LTBP-1 but rF6 did not (Fig.
8A). When subregions of rF11
were tested, rF23 and rF38 were positive, whereas other regions of rF11
(rF20 and rF31) were negative (Fig. 8A). These data
localized the binding site in fibrillin-1 to the four domains contained
in rF38 (see Fig. 1B). When fibrillin-2 recombinant
polypeptides were used as ligands, rF37 and rF46 interacted with LTBP-1
(Fig. 8B), suggesting that the homologous region of
fibrillin-2 was also capable of binding to LTBP-1.
To define the binding site in LTBP-1, recombinant LTBP-1
polypeptides, rL1N, rL1M, rL1C, and rL1K were tested in both blot overlay (data not shown) and ELISA. Results indicated that the major
binding site for fibrillin-1 is contained in the C-terminal region
of LTBP-1, rL1C (Fig. 9A). A
small subdomain of rL1C, rL1K, contained the fibrillin-1 binding site
(Fig. 9B). When ELISAs were performed to compare the binding
of fibrillin-1 and fibrillin-2 with LTBP-1, fibrillin-1 polypeptides
displayed much higher affinities for LTBP-1 than fibrillin-2
polypeptides (Fig. 9A). Incubation of ligands in the
presence of EDTA did not affect the LTBP-1/fibrillin interaction (data
not shown).
To test whether homologous regions of other LTBPs might
perform similar functions, recombinant LTBP-3 and LTBP-4 polypeptides were expressed and purified. In ELISA binding assays, fibrillin ligands
interacted equally well with rL1K and rL4K, but not with rL3K (Fig.
9B). As observed for the binding to rL1K, the same region of
fibrillin-1 (rF23 but not rF31) contained the binding site for rL4K
(Fig. 9B). Additional studies that precisely define the
binding site in LTBP-1 and LTBP-4 are required to understand why these
bind to fibrillin and LTBP-3 does not.
Beaded string structures have been extracted from various tissues
and shown by immunolabeling to contain periodically spaced fibrillin
molecules (14, 15). Together with the known extended structure of
fibrillin (13), these studies have suggested that the strings
connecting the globular beads in the beaded microfibrils are fibrillin
molecules. However, molecules similar in shape to fibrillins may also
constitute the string-like connecting filaments in microfibrils. LTBPs
are candidate molecules to be considered, because they have been
immunolocalized to microfibrils and because they are structurally
homologous to fibrillins.
Procedures for extraction of microfibrils are now routinely utilized
(45, 46). These procedures rely upon crude collagenase digestion of
tissues to release microfibrils from the insoluble extracellular matrix
environment. We have recently found that highly purified bacterial
collagenase does not release fibrillin microfibrils from tissues,
suggesting that microfibrils are not simply trapped in the matrix by
collagen fibers (42). Further digestion with crude collagenase, which
contains other proteases, is required to release microfibrils. These
results indicate that molecules linking microfibrils to the insoluble
extracellular matrix must be degraded to release microfibrils. We have
demonstrated that versican is one of these molecules (42).
In the current investigations, we have shown that LTBP-1 is not a
component of crude collagenase-digested microfibrils. Therefore, LTBP-1
is likely not to be a connecting string in these microfibrils. Because
LTBP-1, unlike fibrillins, contains several regions of protein
sequences that may provide sensitive sites for proteolysis (47), LTBP-1
may have been degraded in the digestion protocol. Hence, it is possible
that LTBP-1 was present in the collagenase-digested beaded microfibrils
as degraded stubs but undetected with the available antibodies. To
address this possibility, microfibrils were extracted from tissues
using nondegradative procedures and fractionated by sieve
chromatography. Denaturing guanidine HCl extractions released small
amounts of microfibrils found in fractions that failed to react with
antibodies specific for LTBP-1. However, LTBP-1 immunoreactive
materials close to the size of complexes containing full-length LTBP-1
and LAP were detected in the guanidine extracts. Taken together, these
data suggest that LTBP-1 is not substantially cross-linked into the
beaded string microfibril structure and may not be an integral
component of microfibrils.
Results from immunoelectron microscopic analyses of tissues are
consistent with the conclusion that LTBP-1 may not be an integral component of microfibrils. In the present studies, labeling of tissues
with anti-LTBP-1 was always sparse and never periodic. In contrast,
immunolabeling of calvarial cell cultures revealed some periodic
labeling of microfibrils using an anti-LTBP-1 serum (30). It may be
that cells in culture can secrete and deposit larger amounts of LTBP-1
into the extracellular matrix than was observed in the tissues we sampled.
Because immunolocalization experiments indicated that LTBP-1 is
associated with microfibrils, binding studies were conducted to
determine whether LTBP-1 interacts with fibrillin. Binding was detected
using well characterized recombinant fibrillin polypeptides in blot
overlay assays with either full-length recombinant LTBP-1 or authentic
LTBP-1 present in fibroblast cell culture medium. ELISA binding studies
demonstrated that the interaction was not calcium-dependent
and that the interaction between fibrillin-1 and LTBP-1 appeared to be
much stronger than the interaction between fibrillin-2 and LTBP-1.
These studies mapped the binding site for fibrillin to the C-terminal
region of LTBP-1, a region corresponding to the domains contained in
peptide rL1K. This result is consistent with a study showing that the
C-terminal region of LTBP-1 can associate with extracellular matrix in
cell culture experiments (48). The binding site for LTBP-1 in
fibrillin-1 is contained within four domains (rF38) close to the
N-terminal end of fibrillin-1. The homologous region of fibrillin-2
(rF46) may also mediate binding to LTBP-1, but with lower affinity than
fibrillin-1.
Because LTBP-1 is similar in structure and function to other LTBP
family members, it seemed plausible that other LTBPs might interact
with fibrillins, using binding sites homologous to rL1K. To test this
hypothesis, recombinant polypeptides were constructed for mouse LTBP-3
(rL3K) and human LTBP-4 (rL4K). Binding by fibrillin to the LTBP-4
polypeptide was strong, equivalent to the interaction with LTBP-1, and
specifically mediated by the same region in fibrillin. However, the
comparable C-terminal region of LTBP-3 did not appear to bind to the
N-terminal region of fibrillin-1 (rF23), suggesting that LTBP-3 may
specifically bind to other sites in fibrillins or to another fibrillin,
that it uses other domains to mediate interactions with fibrillins, or
that it does not interact with fibrillins. Immunolocalization of LTBP-3
and LTBP-4 to microfibrils in tissues has not yet been performed.
Based on these findings, we propose a model of LTBP-1 in association
with the extracellular matrix and with fibrillin networks (Fig.
10). The N-terminal region of LTBP-1 is
cross-linked to the extracellular matrix (28). In our investigations,
the N-terminal third of LTBP-1 (rL1N) did not display any binding to
fibrillin-1 or fibrillin-2. Therefore, it is likely that the N-terminal
region of LTBP-1 is cross-linked to components other than fibrillins. We cannot, however, exclude that the N-terminal region of LTBP-1 interacts with components of microfibrils other than the fibrillins. Because our studies of extracted microfibrils did not demonstrate the
presence of LTBP-1 in the beaded strings, we suggest that the
N-terminal region of LTBP-1 is associated with matrix molecules close
to microfibrils, but not present within the beaded microfibril.
-binding
protein 1 (LTBP-1) targets latent complexes of transforming growth
factor
to the extracellular matrix, where the latent
cytokine is subsequently activated by several different mechanisms.
Fibrillins are extracellular matrix macromolecules whose primary
function is architectural: fibrillins assemble into ultrastructurally
distinct microfibrils that are ubiquitous in the connective tissue
space. LTBPs and fibrillins are highly homologous molecules, and
colocalization in the matrix of cultured cells has been reported. To
address whether LTBP-1 functions architecturally like fibrillins,
microfibrils were extracted from tissues and analyzed immunochemically.
In addition, binding studies were conducted to determine whether LTBP-1
interacts with fibrillins. LTBP-1 was not detected in extracted beaded-string microfibrils, suggesting that LTBP-1 is not an integral structural component of microfibrils. However, binding studies demonstrated interactions between LTBP-1 and fibrillins. The binding site was within three domains of the LTBP-1 C terminus, and in fibrillin-1 the site was defined within four domains near the N
terminus. Immunolocalization data were consistent with the hypothesis that LTBP-1 is a fibrillin-associated protein present in certain tissues but not in others. In tissues where LTBP-1 is not expressed, LTBP-4 may substitute for LTBP-1, because the C-terminal end of LTBP-4
binds equally well to fibrillin. A model depicting the relationship
between LTBP-1 and fibrillin microfibrils is proposed.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-binding
proteins (LTBPs)1 are members
of a family of homologous molecules. The fibrillins and LTBPs contain
multiple calcium-binding epidermal growth factor-like modules
interspersed by a domain module (the 8-Cys or TB module), so far found
only in these two proteins. Fibrillin-1 (1-4) and fibrillin-2 (5, 6)
share a highly similar overall structure. Both molecules are of
equivalent size (~350 kDa) and domain organization. In contrast,
LTBP-1 (7, 8), LTBP-2 (9), LTBP-3 (10), and LTBP-4 (11, 12) are each
smaller than the fibrillins and variable in size.
and targets it to the
extracellular matrix (7). Latent TGF-
consists of the mature growth
factor plus the TGF-
propeptide, also known as the latency associated peptide (LAP). LAP binds to TGF-
by noncovalent
interactions, and the association of LAP with TGF-
prevents the
growth factor from binding to its receptor. During the secretory
process, CR3, the second 8-Cys module, in LTBP-1 becomes
disulfide-linked to the latency associated propeptide (LAP) of TGF-
(24, 25). The processed TGF-
remains noncovalently bound within this
complex of LAP and LTBP. Studies of 8-Cys modules from fibrillin-1
indicate that the 8 conserved cysteine residues in this module form 4 intrachain disulfide bonds (15). The solution structure of one 8-Cys
module from fibrillin-1 has been determined (26), indicating the
position of disulfide bonds. The structure of the 8-Cys module in
LTBP-1, which is disulfide-linked and complexed with latent TGF-
, is not yet known. However, based upon the conformation of the fibrillin 8-Cys motif, binding of LTBP-1 to LAP has been proposed to occur through Cys4 and Cys7 or Cys2 and
Cys6 (26). LTBP-3 and LTBP-4, but not LTBP-2, also interact
with LAP (27).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Schematic representations of the recombinant
polypeptides used in this study. A, LTBP-1,
LTBP-3, and LTBP-4 polypeptides. B, fibrillin-1 and
fibrillin-2 polypeptides.
2III4, which contains the sequence for the
BM40/SPARC signal peptide (39). To make the expression construct rL1M,
coding for Asp588 to Phe1139 of LTBP-1,
template was amplified with LTBP-1-2S
(5'-CTGCTAGCAGATATTGATGAGTGTACTCAGCAGGTC-3') and LTBP-1-2AS
(5'-CTCTCGAGTCAATGATGATGATGATGATGAAAGCACTGCAGTTTCACAGG-3'). For
rL1C, coding for Asp1097 to
Glu1394, the primer set LTBP-1-3S,
(5'-CTGCTAGCAGATGCAGATGAATGCCTACTTTTTG-3') and LTBP-1-3AS
(5'-CTCTCGAGTCAATGATGATGATGATGATGCTCCAGGTCACTACTGTCTTTCTC-3') was used. rL1K, coding for Arg1181 to Glu1394,
was amplified with LTBP-1-29S
(5'-AGCTGCTAGCACGACCGGCTGAGTCAAACGAAC-3') and LTBP-1-3AS.
The correct in-frame insertion of all constructs and the sequence of
PCR amplified products were confirmed by sequence analysis using a DNA
sequencer (Applied Biosystems 373A).
2III4. The entire insert of the
resulting construct, designated pCEPSP-rL4K, was then verified by DNA sequencing.
2III4. The correct orientation of the
insert and the sequence of the PCR amplified fragment were verified by
DNA sequencing.
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Fig. 2.
Coomassie Blue-stained gels of recombinant
LTBP-1 polypeptides used in these investigations. A,
full-length LTBP-1 protein present in conditioned insect cell medium.
LTBP-1 was identified by immunoblotting (data not shown). This sample,
run without disulfide bond reducing agent, was used for blot overlay
experiments. B, purified recombinant LTBP-1, LTBP-3, and
LTBP-4 polypeptides. Samples were run on SDS-PAGE without reducing
agent.
1 propeptide, known as the
LAP, was purchased from R & D Systems (Minneapolis, MN), and rabbit
polyclonal antibody 39 against human LTBP-1 was purchased from BD Pharmingen.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 3.
Immunofluorescence staining of tissues using
antibodies specific for LTBP-1 (A, C,
E, G, I, K, and
L) and fibrillin-1 (B, D,
F, H, and J). Sections in
C and D were 20-week human fetal lung, and
sections in K and L were from an 8-year-old human
toe. A (tendon), B (perichondrium), E
and F (peripheral nerve), G and H
(skin), and I and J (skeletal muscle) were
16-week human fetal tissues. The arrow in C
indicates LTBP-1 staining around an airway. Arrowheads in
C, K, and L point to blood vessels,
and asterisks in L designate peripheral nerves.
Bar = 50 µm.
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Fig. 4.
Colocalization of LTBP-1 and fibrillin-1 in
tendon. Confocal microscopy using LTBP-1 mAb 75G (A)
and fibrillin-1 pAb 9543 (B) demonstrated colocalization of
LTBP-1 and fibrillin-1 (C) in tendon of a 15-week fetal
human foot specimen. Bar = 80 µm.
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Fig. 5.
Immunolocalization of LTBP-1. MAb 75G,
followed by immunogold-labeled microfibril bundles in neonate skin
(A) as well as elastic fiber microfibrils in fetal bovine
aorta (C). The middle panel (B)
demonstrates typical periodic labeling of microfibrils in neonate skin
using anti-fibrillin-1 mAb 69. Bars = 100 nm.
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Fig. 6.
Analyses of collagenase-digested microfibrils
from human skin. A, Sepharose CL-2B chromatograph,
indicating the elution position of beaded strings of microfibrils,
identified by rotary shadowing electron microscopy. B, dot
blot analysis demonstrated the presence of fibrillin-1 (mAb 69) in the
microfibril fractions but not in subsequent fractions; dot blot
analyses of the same fractions with mAb 75G and pAb 39 failed to detect
LTBP-1 in the microfibril fractions, but revealed LTBP-1 in subsequent
fractions. Each row contains replicate dotted samples from the
microfibril fractions through the later eluting peak.
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Fig. 7.
Analyses of guanidine HCl extracted
tissues. A, Sepharose CL-2B chromatography of extract
from fetal bovine tendon followed by Western blotting of fractions
revealed LTBP-1 in fractions 28-44 but not in the earlier microfibril
containing fractions (16-20). B, Sepharose CL-2B
chromatography of human skin followed by Western blotting of fractions
revealed both LAP and LTBP-1 in fractions 32-40. Each fraction
contains 2 ml (fractions 32-40 are equivalent to 64-80 ml of elution
volume). Gels were run without reducing agent. Partially purified
authentic LTBP-1 from fibroblast cell culture medium (NSF)
was applied as a control and to demonstrate molecular mobility in these
gels. V0 and Vt are the same
for A and B.
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Fig. 8.
Blot overlay assays of immobilized
recombinant full-length LTBP-1 in insect cell medium (Fig.
2A) incubated with fibrillin ligands and
antibody probes. Control lanes, where soluble ligand was omitted
but antibody probe was incubated with the blot, are identified with the
antibody used for detection. A, recombinant fibrillin-1
ligands that bound to LTBP-1 included rF11 and subdomains rF23 and
rF38. rF11 was detected with mAb 201. Detection of rF6 was with mAb 69. Antihistidine was used to detect rF23, rF38, rF31, and rF20.
B, recombinant fibrillin-2 polypeptides rF37 and rF46,
detected by mAb 161, also bound to LTBP-1.
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Fig. 9.
ELISA binding assays using recombinant LTBP-1
polypeptides and recombinant fibrillin ligands. A,
fibrillin ligands bound preferentially to rL1C and demonstrated higher
affinity of fibrillin-1 (rF23) compared with fibrillin-2 (rF37).
B, fibrillin-1 ligands (rF11 and rF23, but not rF6) bound
equally well to rL1K and rL4K, but did not bind to rL3K.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 10.
Proposed model of LTBP-1 in relation to
fibrillin microfibrils and other extracellular matrix components.
This model is adapted from Ref. 24. The N-terminal end of LTBP-1 is
transglutaminase cross-linked to the extracellular matrix. The small
latent TGF- complex is bound covalently to CR3 of LTBP-1. The
numbers, 1 and 2, mark protease-sensitive sites
in LTBP-1. Interaction between LTBP-1 and fibrillin takes place between
the C-terminal region of LTBP-1 and the N-terminal region of fibrillin.
Latent TGF-
may be targeted and sequestered by the interactions
occurring at both the N-terminal and C-terminal ends of LTBP-1.
Networks such as the one depicted in Fig. 10 can be modified in vivo in a tissue-specific manner. LTBPs may be present in different tissues, as shown for LTBP-1 in this investigation. Fibrillins are also present in tissues in differential temporal and spatial patterns. Additional information regarding tissue distributions of the fibrillins and the LTBPs is required. However, we can speculate that in skeletal muscle, for example, fibrillin-1 may interact with LTBP-4, because LTBP-1 is not present in this tissue and LTBP-4 is highly expressed in skeletal muscle (11). In the developing and postnatal lung, fibrillin-1 may interact with LTBP-1 in blood vessels and cartilage and with LTBP-1 and LTBP-4 in the airways.
Based upon our findings, we propose that interactions between LTBP-1
and fibrillin-1 may stabilize latent TGF- complexes in the
extracellular matrix. It is possible that loss of fibrillin-1 would
abolish this stabilization and lead to activation of TGF-
. Activation of TGF-
was recently demonstrated in the lungs of fibrillin-1-deficient mice.3
In Marfan syndrome, caused by mutations in fibrillin-1, loss of
fibrillin-1 microfibrils leads to multiple phenotypic features that may
result in part from activation of TGF-
. This hypothesis will be
tested in future investigations.
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ACKNOWLEDGEMENT |
---|
We thank Sara Tufa for excellent technical assistance.
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FOOTNOTES |
---|
* This work was supported by grants from the Shriners Hospitals for Children (to L. Y. S. and D. R. K.), the Scleroderma Foundation (to L. Y. S.), the Deutsche Forschungsgemeinschaft (to D. P. R.), and National Institutes of Health Grants CA 23753 and CA 34282 and DE13742 (to D. B. R.).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.
Current address: Dept. of Dermatology, Nagoya City University
Medical School, Kawasumi 1, Mizuho-cho, Mizuho-ku, Nagoya, Aichi, Japan
467-8602.
To whom correspondence should be addressed: Shriners Hospital
for Children, 3101 S.W. Sam Jackson Park Rd., Portland, OR 97239. Tel.:
503-221-3436; Fax: 503-221-3451; E-mail: LYS@SHCC.org.
Published, JBC Papers in Press, November 11, 2002, DOI 10.1074/jbc.M209256200
2 Y. Chen, unpublished result.
3 E. R. Neptune, P. A. Frischmeyer, D. A. Arking, L. Myers, T. E. Bunton, B. Gayraud, F., Ramirez, D. Rifkin, R. Ono, L. Sakai, and H. C. Dietz, submitted for publication.
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ABBREVIATIONS |
---|
The abbreviations used are:
LTBP, latent
transforming growth factor -binding protein;
LAP, latency associated
peptide;
TGF-
, transforming growth factor
;
ELISA, enzyme-linked
immunosorbent assay;
TBS, Tris-buffered saline;
mAb, monoclonal
antibody.
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