From the Departments of Transforming growth factor Transforming growth factor TGF- Currently three different LTBPs have been cloned from mammals (LTBPs
1-3) and found to form the covalent association with TGF- LTBPs are mainly composed of EGF-like repeats and protein domains with
conserved patterns of eight cysteine residues, called 8-cysteine
repeats. The association between LTBP-1 and the LAP part of TGF- Reagents--
Pfu and AmpliTaq
thermostable DNA-polymerases were from Stratagene (La Jolla, CA) and
Perkin-Elmer, respectively. All other molecular biology enzymes were
from New England Biolabs (Beverly, MA).
Virology and
§ Medical Genetics,
ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References
s (TGF-
s) are
secreted by most cell types as latent high molecular weight complexes
consisting of TGF-
and its latency associated peptide (LAP)
propeptide dimers, covalently linked to latent TGF-
-binding proteins
(LTBPs). Currently, three different LTBPs are known (LTBPs 1, 2, and
3), all with highly similar protein domain structure consisting of
epidermal growth factor-like and 8-Cys repeats. The 3rd 8-Cys repeat of LTBP-1 mediates its association with TGF-
1·LAP. By using an
expressed sequence tag homologous to the 3rd 8-Cys repeat of human
LTBP-1 as a probe, a novel cDNA similar to known LTBPs was cloned
from human heart cDNA library. This cDNA was named LTBP-4 and
found to exist in at least four different forms, generated by
alternative splicing at the amino terminus and at the central epidermal
growth factor repeat domain. One of the alternative amino-terminal
forms contained an RGD sequence, indicating possible cell-surface
interactions with integrins. LTBP-4 gene was localized to
chromosomal position 19q13.1-19q13.2. The major LTBP-4 mRNA form
is about 5.1 kilobase pairs in size and is predominantly expressed in
the heart, aorta, uterus, and small intestine. Immunoblotting analysis
indicated that LTBP-4 was secreted from cultured human lung fibroblasts both in a free form and in a disulfide bound complex with a
TGF-
·LAP-like protein. Both LTBP-4 forms were also found to be
deposited in the extracellular matrix. The matrix-associated LTBP-4 was
susceptible to proteolytic release with plasmin. LTBP-4 is a new member
of the growing LTBP-fibrillin family of proteins and offers an
alternative means for the secretion and targeted matrix deposition of
TGF-
s or related proteins.
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
s
(TGF-
s,1 see Refs. 1-3)
belong to a large superfamily of growth-modulating polypeptides.
Members of the TGF-
superfamily regulate the growth and
differentiation of multiple cell types and the homeostasis of
extracellular proteolysis. They also have important roles in different
stages of development (reviewed in Refs. 1-4).
s remain biologically latent after secretion. The activity of
the mature TGF-
dimer is blocked by its amino-terminal propeptide
(LAP), which is cleaved apart from the mature TGF-
during secretion,
but remains associated with TGF-
by non-covalent interactions.
TGF-
is activated by its dissociation from LAP, which can be
accomplished for example by proteolysis (reviewed in Ref. 3). In most
cell types studied, including those of mesenchymal, epithelial, and
endothelial origin, TGF-
is secreted in a latent form associated
with latent TGF-
-binding protein (LTBP; Ref. 5). LTBPs are also
needed for the secretion and folding of TGF-
(6-8). The activity of
all the members of the TGF-
superfamily is under strict control
during developmental and pathological processes. Targeted deposition to
extracellular structures via association with binding proteins, like
LTBPs, would provide means to control the spatial activity of these
growth factors.
1·LAP
(9-13). The matrix association functions of LTBPs are important
characteristics of these proteins. LTBP-1 and -2 are assembled into ECM
rapidly after secretion (14, 11), the amino-terminal region being
responsible for the ECM association (15, 16). LTBPs have a high degree
of similarity to ECM microfibril proteins called fibrillins 1 and 2 (17, 18). In immunofluorescence and electron microscopic studies,
LTBP-1 and LTBP-2 have been found to associate with microfibrils (13,
19) similar to those previously shown to contain fibrillins. LTBP-1 and
-2 are released from the extracellular matrix by various proteinases
including plasmin, leukocyte elastase, and mast cell chymase (20, 21, 45). TGF-
, thus accumulates in the
microfibrillar ECM structures in a latent form, where it can be
released. The released large TGF-
complex is still latent, unless a
high concentration of plasmin is used (5, 22). The released complex is
then most likely activated at the cell surface (23, 24).
1 is
mediated by a disulfide bond between Cys-33 of LAP and a yet
unidentified cysteine in the 3rd 8-Cys repeat of LTBP-1 (15, 25),
providing a biological function for the 8-Cys protein domains. The
8-Cys repeats have been found only in five proteins, LTBPs 1-3, and
fibrillins 1 and 2. The current study was carried out to find novel
proteins containing the 8-Cys repeats that would possibly interact with
members of the TGF-
superfamily and extracellular microfibrils.
Through an EST data bank search we found a sequence for a new 8-Cys
repeat containing protein. By using this sequence as a probe, we cloned
a novel cDNA for a protein with high similarity to known LTBPs and
named it LTBP-4. We report here the cloning of human LTBP-4S as well as
three other alternative forms of it. LTBP-4 is deposited to the
extracellular matrix and has an ability to form complexes of high
molecular weight with heterologous TGF-
-like proteins.
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
Cell Culture-- SV40-transformed human kidney epithelial cells (293-T, American Type Culture Collection, Rockville, MD) and human embryonic lung fibroblasts (CCL-137, ATCC) were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 100 IU/ml penicillin, and 50 µg/ml streptomycin. All experiments were carried out under serum-free conditions. For the collection of conditioned medium, the cells were washed twice with serum-free medium, and the subsequently added serum-free medium was collected after specified periods.
CHO cell clones overexpressing human LTBP-1 (15) and LTBP-2 (45) have been described elsewhere.Antibodies--
Antibodies against human LTBP-1 and LTBP-2 were
kind gifts of Dr. C.-H. Heldin (Ludwig Institute for Cancer Research,
Uppsala, Sweden). Affinity purified rabbit anti-human TGF-1·LAP
antibodies (number 680) have been described previously (14).
cDNA Cloning and Sequence Analysis--
cDNA for human
LTBP-4 was cloned from two human heart cDNA libraries, obtained
from CLONTECH (Palo Alto, CA). Library HL3005q is
poly(T)-primed pCDM-8 plasmid library, and library HL3026a is both
poly(T)- and random-primed gt10 phage library. Libraries were first
screened using EST clone 302831 as a probe (see Fig. 1A).
Cloning was carried out essentially as described (27). Overlapping
cDNA clones were sequenced using ALF Express from Amersham
Pharmacia Biotech (Uppsala, Sweden) and ABI 373, ABI 377, and ABI 310 automatic DNA sequencers from Perkin-Elmer. The contig was assembled by
the aid of Gap4 program in the Staden software package (28).
cDNA Constructs--
The full-length cDNA of LTBP-4 was
constructed in pBluescript II (Stratagene) by ligating the 5' fragment
from clone 14.1.1 to clone D1-1 using an internal SphI site
(see Fig. 1A). The full-length LTBP-4 was subsequently
removed from pBluescript II as an EcoRI fragment and
inserted into pcDNA3 eukaryotic expression vector (Invitrogen,
Carlsbad, CA), generating construct pLTBP-4. Construct pTGF and
pLTBP-1 contained the full-length cDNA for human TGF-
1 and human
LTBP-1S, respectively, as described earlier (15).
Northern Hybridization Analysis--
A 3' NotI
fragment (nucleotides 3589-4808) or an
AgeI-NotI fragment (nucleotides 2826-3588) of
LTBP-4 cDNA was used as a probe in Northern and RNA master blot
hybridizations. The fragments were [32P]dCTP-labeled
using random priming kit (Amersham Pharmacia Biotech). In multitissue
Northern blots, -actin probe was used as a control. Hybridization of
both multitissue and RNA master blots were carried out according to the
manufacturer's instructions (CLONTECH). The amounts of RNA in RNA master blot have been equalized by the
manufacturer by comparing the expression levels of eight different
housekeeping mRNAs. Radioactivity levels in hybridized RNA master
blots were quantitated with a BAS-1500 bio-imaging analyzer (Fuji,
Tokyo, Japan).
Transfection of Cell Lines-- The 293T cells were seeded in 10-cm tissue culture dishes and transfected the following day as 70% confluent with 10 µg of the plasmids indicated. Transfections were carried out using a calcium phosphate transfection system (Life Technologies, Inc.) according to manufacturer's instructions. The day after transfection, the cells were washed twice and fed with serum-free medium, and the conditioned medium was collected after 48 h.
Isolation of the Extracellular Matrix, SDS-Polyacrylamide Gel
Electrophoresis, and Immunoblotting--
Extracellular matrices were
prepared according to Hedman et al. (29). Briefly, cell
cultures were washed once with phosphate-buffered saline (0.14 M NaCl, 10 mM sodium phosphate buffer, pH 7.4)
and then treated three times with 0.5% sodium deoxycholate in 10 mM Tris-HCl buffer, pH 8.0, at 0 °C for 10 min. The
plates were then washed again with phosphate-buffered saline and
allowed to dry overnight at room temperature. Cross-linked components
of the matrix were partially solubilized by digesting the sodium
deoxycholate-insoluble matrices by plasmin (0.3 caseinolytic units/ml)
in matrix digestion buffer (phosphate-buffered saline containing 1 mM Ca2+, 1 mM Mg2+, and
0.1% n-octyl-D--glycopyranoside) at 37 °C
for 1 h (14). Finally, released ECM proteins were dissolved in
nonreducing SDS-PAGE sample buffer.
Proteinase Digestion of Fibroblast-conditioned Medium-- Conditioned medium from confluent fibroblasts was collected for 3 days under serum-free conditions. Aliquots of the medium were treated with proteinases at 37 °C for 1 h. The following concentrations of proteinases were used: 50 nM plasmin; 10 nM chymase; 10 nM leukocyte elastase; 10 nM porcine pancreatic elastase; 100 nM cathepsin G; 500 nM cathepsin G; 500 nM cathepsin D (see Ref. 5).
Interspecies Genomic DNA Blot-- Zoo Southern blot (CLONTECH) contained 4 µg of EcoRI-digested genomic DNA from various species. The Zoo blot was hybridized using [32P]dCTP-labeled clone 1.1.1 A as a probe and washed first with 1× SSC, 0,1% SDS and subsequently with 0.1× SSC, 0,1% SDS at 42 °C.
Cloning of the Genomic Fragment Containing LTBP-4 and Fluorescence in Situ Hybridization-- A 3' NotI fragment (nucleotides 3589-4808) of LTBP-4 cDNA was used as a probe to clone the genomic DNA from a human genomic PAC library (Genome Systems Inc, St. Louis, MO). The PAC clone was further verified by amplifying an approximately 1.5-kilobase pair fragment with polymerase chain reaction using primers specific to LTBP-4 cDNA. The polymerase chain reaction product was sequenced and found to contain LTBP-4 exons and introns. The obtained PAC clone in vector pAd10SacBII was labeled with biotin-14-dATP by nick translation. The metaphase preparations made from human lymphocyte culture were pretreated with pepsin (0.2 mg/ml at 0.01 M HCl) for 10 min at 37 °C, and chromosomes were denatured in 70% formamide in 0.3 M NaCl, 0.3 M sodium citrate, pH 7.0 (2× SSC) at 64 °C for 2 min. Hybridization signals were detected by avidin-tetramethylrhodamine isothiocyanate and analyzed by Olympus fluorescence microscope equipped with a ISIS digital image analysis system (Metasystems, Altlussheim, Germany). The chromosome identity was verified by painting with a chromosome-specific probe according to manufacturer's instructions (Cambio, Cambridge, UK).
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RESULTS |
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Cloning of Human Latent TGF--binding Protein-4 (LTBP-4)--
To
determine whether there are yet unknown 8-Cys repeat containing
proteins, we searched an expressed sequence tag (EST) subsection of the
GenBankTM data bank (release 100.0, January 1997) against
the 3rd 8-Cys repeat of human LTBP-1 protein sequence using TBLAST
program (30). We obtained several overlapping clones with identical
sequences of various lengths, whose deduced amino acid sequence
contained an 8-Cys-like motif of an unknown protein. We used an EST
clone 302831 (GenBank accession number W19505) as an initial probe to
clone a homologous cDNA from human heart cDNA library. A large number of overlapping cDNA fragments were obtained, which were assembled to the full-length ORF sequence. Some of these cDNA fragments are depicted in Fig.
1A.
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Identification of Alternatively Spliced Forms of LTBP-4--
In
addition to LTBP-4S, we also obtained three different forms of LTBP-4
(see Fig. 1A), which we named as LTBP-4L, LTBP-42E, and
LTBP-4
E. LTBP-4S and LTBP-4L have different amino-terminal coding
sequences, whereas LTBP-4
2E and LTBP-4
E code for LTBP-4 forms
lacking one or two EGF-like repeats from the long stretch of EGF-like
repeats (Fig. 2A).
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Chromosomal Localization of the Human Gene for LTBP-4-- By using a NotI fragment of LTBP-4 cDNA as a probe, the corresponding genomic fragment was obtained from a human PAC library. The clone was verified to contain the LTBP-4 gene by partial sequencing. This PAC clone was used to analyze the chromosomal localization of the LTBP-4 gene. Human metaphase leukocyte chromosomes were hybridized with biotinylated LTBP-4 PAC probe and analyzed with a fluorescence microscope equipped with a digital imaging system. LTBP-4 gene was localized to chromosome 19, at the region of 19q13.1-19q13.2 (Fig. 4B). The localization to chromosome 19 was verified with painting using chromosome 19-specific probe.
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Conservation of the LTBP-4 Gene in Other Species-- To analyze whether the LTBP-4 gene is also present in other species, we probed the EcoRI-digested genomic DNA from different species using the LTBP-4 cDNA clone 1.1.1A (see Fig. 1A). The results of the Southern hybridization indicated that a gene homologous to human LTBP-4 exists in all mammalian species analyzed, including human, monkey, mouse, and rat (Fig. 4B). In addition, we have identified murine EST clones, representing mouse LTBP-4.3
Analysis of the Expression Levels of LTBP-4 in Different Tissues-- A NotI fragment of LTBP-4 was used as a probe to elucidate the expression levels of LTBP-4 in different tissues. Based on the hybridization results under high stringency conditions in Northern blots, the size of LTBP-4 mRNA was found to be approximately 5.1 kilobase pairs (Fig. 5A). This correlates well with the cloned LTBP-4S sequence of 4944 base pairs, implying that the cloned LTBP-4S represents the full-length ORF of this mRNA. The highest expression levels of LTBP-4 were found in the heart, uterus, and small intestine (Fig. 5, A and B). LTBP-4 mRNA from all tissues studied was of the same size, suggesting that the different forms of LTBP-4 are not detectable, because their small size differences are beyond the resolution of the agarose gels used for Northern hybridization analysis. Essentially identical results were obtained also from an another Northern analysis using an AgeI-NotI fragment of LTBP-4 (data not shown).
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Secretion of LTBP-4 by Human Lung Fibroblasts-- As fibroblastic cells are known to produce LTBP-1 and -2, we used the serum-free conditioned medium from human lung fibroblasts (CCL-137) to analyze the expression and secretion of the LTBP-4 protein. Polyclonal antibodies were raised in rabbits against two synthetic peptides of LTBP-4. These peptides were from the 3rd (epitope for the Ab 28-3) and 4th (epitope for the Ab 33-4) 8-Cys repeats of LTBP-4. Immunoblotting analysis of the conditioned medium from confluent fibroblasts under reducing conditions indicated that both Ab 28-3 and 33-4 detected LTBP-4 protein, which had a molecular mass of approximately 250 kDa (Fig. 6A).
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LTBP-4 Is Expressed Both in Free Form and in a Form Associated with
a Heterologous Protein That Binds Most Likely to the 3rd 8-Cys Repeat
of LTBP-4--
Since the TGF-1·LAP associates with LTBPs by a
disulfide bond, we analyzed next fibroblast-conditioned medium under
nonreducing conditions to detect possible complex formation between
LTBP-4 and a TGF-
1·LAP-related protein. Under nonreducing
conditions, both Ab 28-3 and 33-4 detected only the free form of
LTBP-4, which has molecular mass of approximately 215 kDa (Fig.
6B). Interestingly, Ab 33-4 detected also larger forms of
LTBP-4, approximately 240 and 300 kDa. These forms probably represent
LTBP-4 covalently complexed to a heterologous protein since the bands
were undetectable after reduction (Fig. 6B). These multiple
forms are characteristic to all LTBPs in SDS-PAGE under nonreducing
conditions (Fig. 6A, right lanes), previously reported to
represent complexes between LTBPs and TGF-
·LAP (14, 15, 32). When
the same samples were analyzed under reducing conditions, the LTBP-4
band detected with both Ab 28-3 and 33-4 had the same mobility, and the
larger forms of LTBP-4 (Fig. 6A) seen under nonreducing
conditions with Ab 28-4 were not detected. A likely explanation for the
fact that Ab 28-3 does not recognize the complexed higher molecular
weight forms of LTBP-4 is that the 3rd 8-Cys repeat of LTBP-4 might
associate with the heterologous protein(s) via a disulfide bond, thus
masking the epitope of this antibody.
LTBP-4 Associates Covalently with TGF-1--
The interaction
between LTBP-4 and TGF-
1 was studied using a mammalian cell
co-expression system. We have found earlier that the 3rd 8-Cys repeats
of LTBP-1 and LTBP-3 (15)4
bind TGF-
s 1-3 in a covalent manner. We constructed an expression vector pLTBP-4, containing the full-length cDNA of LTBP-4S. The construct was co-expressed with TGF-
1 cDNA in 293T cells, and the cell-conditioned medium was analyzed for complex formation between
TGF-
1 and LTBP-4S. Immunoblotting analysis under nonreducing conditions using Ab 680 against the LAP region of TGF-
1 indicated that co-expressed LTBP-4 formed a covalent complex with TGF-
1·LAP (Fig. 7, lane 1). As a
positive control, the cells were transfected with full-length human
LTBP-1S (Fig. 7, lane 2). When the cells were transfected
with only pTGF-
, the large complex was not seen (Fig. 7, lane
3).
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LTBP-4 Is a Protease-sensitive Extracellular Matrix Component-- LTBP-1 and -2 associate covalently with extracellular structures rapidly after secretion (11, 14). These complexes can be solubilized by plasmin digestion (14). The possible association of LTBP-4 with the extracellular matrices was studied using confluent human lung fibroblast cultures. The sodium deoxycholate-insoluble extracellular matrices of fibroblasts were partially solubilized with plasmin. Immunoblotting analysis of the plasmin-digested solubilized extracellular matrices using Ab 28-3 and Ab 33-4 revealed that LTBP-4 was present in these matrix fractions in a proteolytically processed form (Fig. 8). Under reducing conditions, LTBP-4 was detected as a single 200-kDa band (Fig. 8, two left lanes), whereas under nonreducing conditions, a major and 2-3 minor forms of LTBP-4 with apparent molecular masses of about 80-220 kDa were observed (Fig. 8, Non-reduced). The 220-kDa form detected with only Ab 33-4 most likely represent similar disulfide bonded LTBP-4 complexes, as seen from samples of conditioned medium (Fig. 7). With Ab 28-3, only the lower molecular weight forms were seen. As a control, the immunoblotting for plasmin-digested LTBP-1 detected a 120-140-kDa band (Fig. 8, right lane).
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Processing of LTBP-4 by Proteinases--
The sequence of LTBP-4
reveals plausible proteinase-sensitive regions, also called hinge
domains, having high local concentrations of both basic amino acids and
proline. These regions are found between the hybrid domain and the long
stretch of EGF-like repeats and near the carboxyl terminus after the
last 8-Cys repeat (see Fig. 2A). LTBP-1 has been found to be
susceptible to proteinase digestion at similar sites resulting in the
release of large latent TGF- complexes from the extracellular
matrix. An immunoblotting analysis from fibroblast-conditioned medium
was therefore carried out under reducing conditions with Ab 33-4 to
detect the susceptibility of LTBP-4 for digestion by various
proteinases. Of the proteinases used, plasmin, human mast cell chymase,
and leukocyte and pancreatic elastases processed LTBP-4 (Fig.
9). Digestion with plasmin, chymase, and
pancreatic elastase resulted in the formation of proteinase-resistant fragments with apparent molecular mass of about 230-220 kDa. Leukocyte elastase treatment resulted in the loss of the high molecular weight
form (Fig. 9), most likely due to a cleavage between the long stretch
of EGF-like repeats and the 3rd 8-Cys repeat (45) leading to the loss
of the peptide epitope of the antibodies used. Unlike the other LTBPs,
the amino acid sequence of LTBP-4 is rich with proline, and basic
residues in this region are indicative of an additional
protease-sensitive site. Digestion of the cell-conditioned medium with
cathepsins B, D, or G produced no detectable cleavage(s) in LTBP-4.
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DISCUSSION |
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The present report describes the cloning of a new member of the
LTBP family, LTBP-4, and several of its alternatively spliced forms.
Its structure is very similar to that of the other previously cloned
LTBPs, and, like the other ones, LTBP-4 also associates with the
extracellular matrix. LTBP-4 is mainly composed of EGF-like and 8-Cys
repeats, as the previously identified LTBPs and fibrillins. EGF like
repeats are found in multiple ECM and cell-surface proteins. The 8-Cys
repeats have been found thus far only in LTBPs and fibrillins, and the
only known function for these domains is the formation of a covalent
association between TGF-1 and the 3rd 8-Cys repeat of LTBP-1 (15,
25).
The amino termini of all LTBPs, including LTBP-4, contain two copies of
EGF-like repeats, one copy of 8-Cys repeat, and another 8-Cys repeat,
which is often also called a hybrid domain, since its sequence
resembles both EGF-like and 8-Cys repeats (Fig. 2A). The
amino termini of LTBPs are responsible for their association with the
extracellular matrix (Ref. 15 and also see Ref. 16). Following the
repetitive amino-terminal domain, there is a proteinase-sensitive region, rich in proline and basic amino acid residues. The large latent
TGF- complex is supposedly released from the extracellular matrix by
proteolytic digestion at this region (14). LTBP-1 deposited to the
extracellular matrix can be released by plasmin without its
amino-terminal matrix binding domain (15).
The central parts of all LTBPs and fibrillin family members are
composed of a long stretch of EGF-like repeats. In LTBP-4 this part
consists of 12-14 repeats and is over half of the total protein size.
The region of EGF-like repeats has been suggested to form a helical
rod-like structure, like similar regions in fibrillins (33). In LTBP-1
it has been found out that TGF-1-binding region is near its carboxyl
terminus, where a typical structure of 8-Cys---EGF---EGF---8-Cys
repeats is found. TGF-
1 is covalently associated with the first
8-Cys repeat of LTBP-1 in this domain (Fig. 2A, Refs. 15 and
25).
In LTBP-1 and -2 and in fibrillin-1, there exist at least two
alternative forms of the amino-terminal region (16, 34). We found two
alternative forms of the amino terminus of LTBP-4. The longer form
extends further to the 5' direction than was obtained from clone D1-1
(see Figs. 1A, 2A, and 3A). The
fragment of the longer form obtained in clone D1-1 does not encode for
any EGF-like or 8-Cys-like repeats. Instead, it encodes a nonrepetitive
amino acid sequence, like those found in the amino termini of other LTBPs. An RGD sequence, a known target for integrin-mediated cell adhesion (reviewed in Ref. 35), is present in the amino terminus of the
shorter LTBP-4 form (LTBP-4S) at amino acid position 30. In addition to
LTBP-4, the RGD sequence is also found in LTBP-2, human isoform of
LTBP-1, and in both fibrillin-1 and -2. Recombinant protein fragments
containing the RGD sequence of fibrillin-1 have been found to interact
with purified integrin V
3 (36, 37). The
existence of RGD sequences in LTBPs could thus be involved in the
targeting of the latent TGF-
complexes to cell-surface activation.
Another possibility is that the RGD motif is functional in the
microfibril assembly process. Whether the alternative amino-terminal forms of LTBP-4 are due to alternative splicing or the use of different
promoter regions is not known at present. Since the amino termini of
two other LTBPs, namely LTBP-1 and LTBP-2, have been found to be
responsible for the extracellular matrix interactions, the different
amino-terminal regions of LTBP-4 may confer to different affinities to
various extracellular fibrillar structures.
In addition to the varying amino-terminal region of LTBP-4, we found
alternatively spliced regions in the long stretch of EGF-like repeats
(Fig. 2A). This region is neither required for extracellular
matrix deposition nor for the observed binding to TGF--like
molecules. We identified two independent cDNA clones, named
LTBP-4-
E and LTBP-4-
2E, in which the EGF-like repeat 14 or both
14 and 15 were missing. There are two propositions for the function for
these EGF-like repeats and the long stretch of EGF-like repeats found
in some matrix proteins, including LTBPs and fibrillins. One
possibility is that the long stretch of EGF-like repeats in fibrillins
forms a helical rigid rod-like structure, which acts as a "fiber
forming unit" of the fibrillin-containing microfibrils (33). The
alternative splicing found in the central parts of LTBP-4 is likely to
be involved in the regulation of the extracellular structures, most
likely fibrillin-containing microfibrils, that associate with LTBP-4.
The regulation of the number of EGF-like repeats in the central,
rod-forming regions of LTBPs and fibrillins may affect the length of
the rod or change the angle between the amino and carboxyl termini of
the monomers. The deletion of a dimer of Ca2+-binding
EGF-like repeats would reduce the length of an LTBP-4 monomer by about
5.7 nm (33). The alternative splicing of the microfibrillar proteins,
fibrillins and LTBPs, could give an explanation to the observed
variation of the length of monomers in these fibrils (38). On the other
hand, the EGF-like repeats are known to mediate protein-protein
interactions (39, 40), including those between cell-surface receptors
and their ligands (41). As LTBP-1 released from ECM has been suggested
to interact with the cell surface in the supposed TGF-
activation
process (24), the observed variability in the number of EGF-like
repeats in LTBP-4 may have a role in its interactions with molecules at
the cell surface.
The structural similarity between LTBP-4 and other LTBPs suggests
similar functions for all of them. We found by immunoblotting analyses
from normal human skin fibroblasts that LTBP-4 was, in addition to the
free uncomplexed form, also in complex with an unidentified protein.
This complex has the additional apparent molecular mass about the same
as the 1·LAP·LTBP complexes reported earlier. The ability of
LTBP-4 to make a covalent association with TGF-
1 was confirmed with
transient co-expression in 293T cells. The conserved structural
similarities between the members of the TGF-
superfamily suggest
that, in addition to the documented interaction between TGF-
1 and
LTBPs, also other members of TGF-
superfamily might get associated
with LTBPs or other 8-Cys repeats containing proteins.
LTBP-4 was localized to human chromosome 19q13.1-19q13.2. Mutations in the fibrillin-1 and -2 are responsible for the Marfan syndrome and congenital contractual arachnodactyly, respectively (reviewed in Ref. 42). LTBP-4 gene was found to be conserved in different mammalian species by an interspecies Southern blot and further supported by the existence of mouse LTBP-4 cDNA ESTs in GenBankTM. From the eukaryotic species studied, only yeast was found not to have a related gene in its genome. This is well in accordance with the results of another microfibrillar protein, fibrillin, which is found to exist in a broad variety of species, even as distant to human as the jellyfish. The conservation of LTBP-4 gene speaks for the important role for LTBPs both in the microfibril composition as well as a vehicle for the deposition of latent growth factors to extracellular structures.
The presence of four members of LTBPs and the shared structural and
functional similarities raises questions of the differences between
these proteins. The expression patterns of different LTBPs in different
tissues are only partially overlapping. LTBP-1 is mainly expressed in
heart, placenta, and lung (10, 11) and LTBP-2 in lung and skeletal
muscle, liver, and placenta (11). LTBP-4 is predominantly expressed in
aorta, heart, small intestine, ovaries, and uterus. However, the
expression levels of LTBP-4 in most fetal tissues were significantly
lower than in adult tissues. This might indicate that LTBP-4 is not
required for the initial formation of microfibrillary structures but
provides a way to store latent TGF- or related molecules in
extracellular fibrils. "Switching" of isoform expression during
development may be typical of the LTBP fibrillin family, since
fibrillin-2 has been shown to appear earlier and in a more transient
manner in the mammalian development than fibrillin-1, which is
expressed at later stages of development (43). As there exist at least
four members in the LTBP family, it is of interest to compare the
expression levels of different LTBPs in different stages of development
and thus to provide possible explanations and plausible biological
functions for the existence of multiple LTBPs.
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ACKNOWLEDGEMENTS |
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We thank Drs. Olli Ritvos and Sakari Knuutila for critical comments and Sami Starast for fine technical assistance.
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Note Added in Proof |
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During the preparation of this manuscript a report was published describing the cloning of an identical cDNA that was also named LTBP-4. Its 5' end was different from those described here, further indicating that there are numerous alternatively spliced forms of the protein (Giltay, R., Kostka, G., and Timpl, R. (1997) FEBS Lett. 411, 164-168).
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FOOTNOTES |
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* This work was supported by The Academy of Finland, Sigrid Juselius Foundation, Finnish Cultural Foundation, Finnish Cancer Organizations, Maud Kuistila Foundation, Novo Nordisk Foundation, Paulo Foundation, Biocentrum Helsinki, Helsinki University Hospital, and the University of Helsinki.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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF051344 (LTBP-4S), AF051345 (LTBP-4L partial cDNA, AF054501 (LTBP-4EGF), and AF054502 (LTBP-4
2EGF).
To whom correspondence should be addressed: The Haartman
Institute, Dept. of Virology, University of Helsinki, P.O. Box 21 (Haartmaninkatu 3), FIN-00014 University of Helsinki, Helsinki, Finland. Tel.: 358-9-1912-6476; Fax: 358-9-1912-6475.
1
The abbreviations used are: TGF-,
transforming growth factor-
; LAP, latency associated peptide;
LTBP-1, LTBP-2, LTBP-3 and LTBP-4, latent TGF-
-binding proteins 1, 2, 3, and 4; EGF, epidermal growth factor; ECM, extracellular matrix;
8-Cys repeat, a conserved protein sequence motif containing eight
cysteines found in LTBPs and fibrillins, also called LT domain;
LTBP-4S, LTBP-4L, LTBP-4
2EGF and LTBP-4
EGF different
alternatively spliced forms of LTBP-4; CHO, Chinese hamster
ovary; PAGE, polyacrylamide gel electrophoresis; ORF, open reading
frame; EST, expressed sequence tag; Ab, antibody.
3 J. Saharinen and J. Keski-Oja, manuscript in preparation.
4 J. Saharinen, C. Koski, and J. Keski-Oja, manuscript in preparation.
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