From the Institut de Biologie et Chimie des Protéines, CNRS UPR 412, Université Claude Bernard, 7, Passage du Vercors, 69367 Lyon Cedex 07, France
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ABSTRACT |
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A heparin binding region is known to be present
within the triple helical part of the 1(V) chain. Here we show that
a recombinant
1(V) fragment (Ile824 to
Pro950), referred to as HepV, is sufficient for heparin
binding at physiological ionic strength. Both native individual
1(V)
chains and HepV are eluted at identical NaCl concentrations (0.35 M) from a heparin-Sepharose column, and this binding can be
inhibited specifically by the addition of free heparin or heparan
sulfate. In contrast, a shorter 23-residue synthetic peptide,
containing the putative heparin binding site in HepV, fails to bind
heparin. Interestingly, HepV promotes cell attachment, and
HepV-mediated adhesion is inhibited specifically by heparin or heparan
sulfate, indicating that this region might behave as an adhesive
binding site. The same site is equally functional on triple helical
molecules as shown by heparin-gold labeling. However, the affinities
for heparin of each of the collagen V molecular forms tested are
different and increase with the number of
1(V) chains incorporated
in the molecules. Molecular modeling of a sequence encompassing the
putative HepV binding sequence region shows that all of the basic
residues cluster on one side of the helical face. A highly positively
charged ring around the molecule is thus particularly evident for the
1(V) homotrimer. This could strengthen its interaction with the
anionic heparin molecules. We propose that a single heparin binding
site is involved in heparin-related glycosaminoglycans-collagen V
interactions, but the different affinities observed likely modulate
cell and matrix interactions between collagen V and heparan sulfate
proteoglycans in tissues.
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INTRODUCTION |
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Collagen V is a fibrillar collagen that plays an important role in fibrillogenesis, and it also acts as an adhesive substrate for a large variety of cells and binds to a number of extracellular components through its major triple helical domain (1). Collagen V interacts with matrix proteoglycans such as the two small proteoglycans decorin and biglycan (2), the proteoglycan form of macrophage colony-stimulating factor (3), the cell surface proteoglycan syndecan-1 (4, 5), and as shown recently, the membrane spanning proteoglycan NG2 (6). Some of these interactions are mediated by the core proteins, but others depend on the glycosaminoglycan chains such as the heparan sulfate chains.
Apart from in vitro binding of collagen V to membrane-spanning proteoglycans, the suggestion that heparan sulfate interacts with collagen V was supported by inhibition experiments showing a reduction of cell attachment to collagen V in the presence of heparin (7). It has been shown already that cell focal adhesion on fibronectin requires the cooperation of both cell transmembrane proteoglycans and integrin receptors (8). Because we have demonstrated already that cell-collagen V interactions involved integrins (9, 10), the binding of membrane-spanning proteoglycans could reinforce cell attachment to collagen V and, in that sense, would be of physiological importance. Therefore, to understand the role of collagen V-heparan sulfate proteoglycan interactions, it appears essential to characterize the specific domain(s) of the molecule responsible for binding to heparin, a glycosaminoglycan related to heparan sulfate.
Collagen V is a typical fibrillar collagen containing a 300-nm-long
triple helical domain that presents different molecular forms in
tissue. The predominant molecular form found in most tissues is the
heterotrimer [1(V)]2
2(V), whereas the
1(V)
2(V)
3(V) molecule is only extracted from human placenta
(1). The homotrimer [
1(V)]3 occurs in cultures of
hamster lung cells and was suggested to be present in embryonic tissue
(11-13). It was produced recently as a recombinant molecule (14). The
heterotrimeric [
1(V)]2
2(V) molecular form was shown
previously to bind to heparin at physiological salt concentrations.
This activity was attributed to a proteolytic NH2-terminal
30-kDa fragment of the
1(V) chain (15).
Aside from a requirement for the 1(V) chain, the features of the
different stoichiometries of the collagen V triple helix necessary for
heparin binding remain unknown. Such studies have been hampered by the
fact that determination of minimal sites on the collagenous triple
helix is difficult. Thus in this manuscript, to locate more precisely
the heparin binding site, we have combined several approaches: (i)
recombinant technology, in which collagenous domains and the molecule
isotypes can be engineered and generated in quantities sufficient for
biochemical and functional analysis; (ii) a synthetic peptide to narrow
the sequence involved in heparin-collagen V recognition; (iii) electron
microscopy to visualize the site on triple helical molecules. Using
these approaches it has been possible to establish that a recombinant
fragment of the
1(V) chain (Ile824I to
Pro950) but not a synthetic peptide encompassing the
putative heparin binding site binds to heparin and heparan sulfate.
Moreover, we provide evidence for a cell adhesive function of this
fragment through a cell surface heparan sulfate proteoglycan. This
region corresponds to a heparin binding site common to the three known collagen V molecular forms, although increasing heparin affinities were
correlated positively with the number of
1(V) chains incorporated in
the molecule. Based on molecular modeling of the portion of the
chain which includes the putative binding sequence, we propose a model
to explain the different affinities observed for the distinct collagen
V molecular forms.
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EXPERIMENTAL PROCEDURES |
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Construction, Expression of HepV--
The HepV module cDNA
(nucleotides 2595-2976) was generated by polymerase chain reaction
using as template the clone 302 kindly provided by Dr. Takahara (16).
Two oligonucleotides flanking the desired sequence were
designed. One corresponding to the 5'-end of the module carried
an EcoRI site
(5'-TATGAATTCCCATCAAGGGTGATCGGGGGGAGA-3'), and the second,
corresponding to the 3'-end of the module, introduced a PstI
site and a stop codon
(5'-TATCTGCAGATTAGGGTCCCCGTTCACCAGGAGGGCCAGCTGG-3'). The resulting polymerase chain reaction product of 399 base pairs was subcloned in the EcoRI and PstI sites of a
pT7-7 expression vector (17). The plasmid obtained, named pHepV, thus
encoded the heparin binding site of 1(V) under the control of the
Escherichia coli phage T7 promoter. The sequence of the
recombinant DNA was checked thoroughly.
Protein Purification-- For purification of the recombinant fragment HepV, the bacterial supernatant was dialyzed against 35 mM Tris/HCl, pH 7.4, filtered, and applied to a HPLC cation exchange chromatography on a 1-ml Resource-S column (Amersham Pharmacia Biotech) (Waters 625 LC system) and eluted by a linear NaCl gradient (0-300 mM) in the same buffer. The fraction eluted at 200 mM NaCl and containing HepV was purified further by anion exchange chromatography through a HiTrapQ (Amersham Pharmacia Biotech) column. The column was equilibrated in 200 mM NaCl, 35 mM Tris/HCl, pH 7.4. The unbound fractions contained purified HepV as analyzed by 15% SDS-PAGE.
Collagen V [Synthetic Peptide and Other Reagents-- The peptide GTPGKPGPRGQRGPTGPRGERGP referred to as HepP was synthesized according to standard protocols using Fmoc (N-(9-fluorenyl)methoxycarbonyl) chemistry in a Milligen 9050 synthesizer. The peptide was purified by reverse phase HPLC and was shown by amino acid analysis to have the correct primary structure. Glycosaminoglycans were obtained from commercial sources: heparin and chondroitin sulfate C from Sigma and heparan sulfate from Jacques Boy, Reims, France.
Analysis of Heparin Binding by Heparin Affinity Chromatography-- Heparin-Sepharose affinity columns (HiTrap Heparin, Amersham Pharmacia Biotech) were equilibrated in phosphate-buffered saline (PBS) (buffer A) or in 35 mM Tris/HCl, pH 7.4 containing 200 mM NaCl (buffer B). Protein samples were loaded onto a column, and a programmed linear gradient of 150-500 mM NaCl in buffer A and 200-500 mM NaCl in buffer B was applied at a flow rate of 0.5 ml/min. To determine heparin affinities to individual chains, collagen V samples were heat-denatured (60 °C for 20 min) before loading. Fractions (1 ml) were collected, and the elution profile of protein samples was determined by monitoring the absorbance at 206 nm. The different fractions were then analyzed by SDS-PAGE as described above except fractions obtained with the peptide, which were analyzed by amino acid analysis.
Analysis of the capacity of glycosaminoglycans to inhibit HepV binding to heparin and the synthetic peptide to release HepV bound to heparin were both carried out in batch with heparin-Sepharose CL-6B resin (Amersham Pharmacia Biotech). The resin was equilibrated with 50 mM Tris/HCl, pH 7.5 containing 150 mM NaCl for glycosaminoglycans inhibition experiments. For glycosaminoglycan inhibition experiments, 5 µg of HepV was incubated in the absence or presence of free heparin, heparan sulfate, or chondroitin sulfate for 2 h at room temperature before being applied to the resin for an additional 30 min. Unbound material was recovered by gentle centrifugation after the addition of 1 ml of starting buffer to the samples. Elution of bound material was achieved by adding 0.5 M NaCl to the starting buffer. All fractions were analyzed by 15% SDS-PAGE. For peptide inhibition, 50 µl of the resin was incubated with 5 µg of HepV for 30 min at room temperature and washed with 1 ml of starting buffer. Bound material was then incubated with various concentrations of the synthetic peptide HepP, and the released material was recovered by gentle centrifugation. The resin was then washed with starting buffer, and the elution of the remaining bound material was achieved by adding 1 M NaCl to the starting buffer. Fractions were analyzed by 15% SDS-PAGE.Analytical Methods-- Amino acid compositions were determined after hydrolysis under vacuum (6 N HCl, 115 °C, 24 h) in presence of 2-mercaptoethanol in a Pico Tag system (Waters) with a Beckman amino acid analyzer. For NH2-terminal amino acid sequencing, proteins were electrotransferred onto polyvinylidene difluoride membrane (Problott, Applied Biosystems) for 4 h at 60 V in 10 mM CAPS, 5% methanol, pH 11, and the band of interest was excised from the membrane after brief staining with 0.2% Ponceau S in 1% acetic acid (Sigma). Amino acid sequence analysis was performed by automated Edman degradation using an Applied Biosystems 473A protein sequencer.
Rotary Shadowing-- Collagen solutions (collagen V samples and collagen I as control) were dialyzed overnight against PBS at 4 °C and then incubated with heparin-BSA-gold 10-mm particles (Sigma) for 3 h at room temperature. Samples were dialyzed against 1 M ammonium acetate and finally diluted to 10 µg/ml with the same buffer. After the addition of an equal volume of glycerol, the solutions were sprayed onto freshly cleaved mica sheet and were placed immediately on the holder of a MED 010 evaporator (Balzers). Rotary shadowing was carried out as described previously (19). Observations of replicas were performed with a Philips CM120 microscope at the CMEABG (Centre de Microscopie Electronique Appliquée à la Biologie et à la Géologie, Université Claude Bernard, Lyon I).
Cell Adhesion Assays and Inhibition Assays-- Chinese hamster ovary (CHO) cells were maintained in monolayer cultures in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, glutamine, nonessential amino acids, and a mixture of antibiotics. Before the adhesion assay, cells were harvested with 1% EDTA in PBS or with 0.05% trypsin, 0.02% EDTA in PBS, pH 7.4, when indicated.
Multiwell plates (96-well tissue culture plates, Costar, France) were coated by overnight adsorption at 4 °C with substrates at concentrations ranging from 0 to 40 µg/ml (100 µl/well). After saturation of the wells with 1% bovine serum albumin, freshly suspended cells in serum-free Dulbecco's modified Eagle's medium (4 × 105 cells/ml) were plated onto coated wells (100 µl/well) and allowed to attach for 30-40 min at 37 °C. The attached cells were washed with PBS, fixed in 1% glutaraldehyde, and stained for 25 min with 0.1% crystal violet in water. After extensive washing, the dye absorbed to the cells was solubilized with 0.2% Triton X-100, and optical density was read with an enzyme immunosorbent assay reader (Dynex MRX) at 570 nm. Each assay point was carried out in triplicate. For inhibition assays, the coated wells were preincubated with 10 µg/ml glycosaminoglycans (heparin, heparan sulfate, or chondroitin sulfate) for 1 h at 37 °C. Freshly suspended cells were then added, and the wells were incubated for 30-40 min. For synthetic peptide inhibitions, cells were first mixed with 500 µM RGDS peptide before being seeded onto the coated wells. The assays were then continued as described above.Two-dimensional Representation and Molecular Modeling--
The
two-dimensional projection used to represent the three-dimensional
structure of -helices is called a helical wheel. It allows, in the
case of heparin binding consensus sequences, visualization of clusters
of basic residues (20). The helical wheel representation for collagen
helix differs from a true
helix and thus was represented as
described previously (21). For the representation of collagen V triple
helices ([
1(V)]2
2(V) and [
1(V)]3),
the helical wheels were positioned with the glycyl residues at the
center of the molecule.
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RESULTS |
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Expression and Characterization of HepV--
We designed a
fragment, referred to as HepV, which encompasses the complete
NH2-terminal part of the 30-kDa CNBr peptide defined by
Yaoi et al. (15) and the sequence around the endoproteinase Glu-C cleavage site which was found to be determinant for heparin binding (Fig. 1). The resulting heparin
binding site was thus narrowed down from the COOH-terminal end of the
30-kDa fragment to a 12-kDa polypeptide, HepV. An expression vector
pT7-7, which encodes amino acids Ile824 to
Pro950 of human 1(V) chain was constructed as described
under "Experimental Procedures." After induction with
isopropyl-
-D-thiogalactopyranoside, SDS-PAGE analysis of
the soluble and insoluble cellular extracts showed the presence of an
additional protein band of 17 kDa in soluble extracts which was not
found in transformed E. coli cells before induction. The
difference between the apparent molecular mass and the predicted
polypeptide mass of 12 kDa was attributed to the peculiar structure of
collagen chains migrating slower than the globular standards, as well
as the additional NH2-terminal amino acid residues
originating from the construct. Passage of the supernatant over a Mono
S column separated the HepV domain from most of the contaminating
bacterial proteins. Final purification was achieved by rechromatography
on a Mono Q column (Fig. 2A). Analysis of the NH2-terminal amino acid sequence of the
purified band indicated the sequence XRIPIKGD, which agreed with that
of the HepV construct. The four first amino acid residues correspond to
NH2-terminal extension added for cloning purposes.
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Binding of 1(V) Chain Fragments to Heparin--
The binding
interaction of heparin with collagen V fragments was studied using
their affinities on a heparin-Sepharose column. We showed that HepV
bound to the heparin-Sepharose column at physiological pH and ionic
strength. Moreover, HepV was eluted from the column at 0.35 M NaCl (Fig. 2B), which is exactly the
concentration required to elute isolated native
1(V) chains. In
addition, binding to heparin is inhibited by incubation of HepV with
free heparin or heparan sulfate but not with chondroitin sulfate before
loading on the heparin-Sepharose column (Fig.
3A).
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Binding of Heparin to the Different Collagen V Molecular Forms and
Its Constitutive Chains--
We showed that isolated 1(V), but not
2(V) and
3(V) chains, was able to bind heparin at physiological
pH and ionic strength and was eluted from the column at exactly 0.35 M NaCl (Fig. 4A). To evaluate whether the binding site present in
1(V) is available when incorporated into the triple helix, different collagen V molecular
forms were passed through a heparin column: the native heterotrimeric molecular form [
1(V)]2
2(V) from
bovine bone and from human placenta, also containing in a 1:1 ratio the
molecular form
1(V)
2(V)
3(V). To circumvent the problem of the
1(V) homotrimer, which is difficult to obtain from tissue, we used
the recombinant homotrimer expressed and purified as described
elsewhere (14). The results demonstrate that the affinities of each
molecular form for heparin in terms of NaCl concentrations necessary
for elution followed this order:
1(V)
2(V)
3(V) (0.28 M) < [
1(V)]2
2(V) (0.35 M) < [
1(V)]3 (0.45 M) (Fig. 4B).
These results indicate that the affinity of the different molecular
forms could be modulated in relation to the number of
1(V) chains
present in each subtype, two
1(V) chains in the triple helix being
necessary to obtain the same affinity determined with isolated
1(V)
chain.
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Mapping of the Heparin Binding Site on Collagen V
Molecules--
Because an active site might be available in small
fragments or individual chains but masked in the context of the whole
molecule, we then examined, by electron microscopy, the location of the heparin binding site on triple helices using heparin as a probe (according to Ref. 5). For this purpose, collagen V, and collagen I as
control, were complexed with heparin-BSA-gold and prepared for rotary
shadowing as mentioned under "Experimental Procedures." Pepsinized [1(V)]2
2(V) collagen V-heparin gold
complexes were often observed as large intermolecular aggregates for
which it was difficult to identify the precise location of heparin
binding. However, selected areas contained a sufficient proportion of
individual molecules to determine the position of the gold particles on
collagen V molecules. The binding site was located at about 100 nm from one of the extremities of the molecules (Fig.
5A). This corresponds exactly
to the
1(V) binding site position if we consider that the distance
is measured from the NH2-terminal extremity of the collagen
molecule. This binding site is specific to collagen V molecules since
it was not observed on collagen I molecules labeled with heparin-gold
(data not shown). Attempts to determine the polarity of collagen V ends
using the recombinant [
1(V)]3 homotrimer encompassing
the entire N-propeptide were not successful. Indeed, the
presence of the N-propeptide seemed to enhance the formation of intermolecular aggregates associated with heparin-gold, and individual labeled molecules were rarely observed. Therefore mapping experiments were undertaken with a 200-nm fragment product of the
recombinant [
1(V)]3 homotrimer (Fig. 5B).
We have shown previously that this fragment resulted from a proteolytic
cleavage occurring close to a flexible region present in the triple
helical domain of the homotrimer (14). Interestingly, the
NH2-terminal sequence of this fragment was found to start
at residue Asp759, which is 65 amino acid residues upstream
from the heparin binding site we have determined (Ile824 to
Pro950) (14). This would correspond to about 20 nm from the
NH2-terminal end of the fragment and allow the orientation
of the molecules. As expected, when this fragment was applied to
heparin-Sepharose it was retained on the column and was eluted in the
same conditions as the entire recombinant homotrimer, thus confirming
the presence of the heparin binding site (data not shown).
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CHO Cell Adhesion in Response to HepV Fragment-- CHO cells were shown previously to interact with collagen V likely via a cell surface heparan sulfate proteoglycan (7). We show that not only intact collagen V but also recombinant HepV fragments were equally efficient in promoting cell attachment of EDTA-released CHO (Fig. 6). CHO cells inspected by inverted phase microscopy remained round in shape on a HepV substrate, whereas on a collagen V substrate, even though most of cells are round, a more flattened morphology was also observed (Fig. 6, upper panel). The glycosaminoglycan contribution to CHO adhesion was examined by plating the cells on collagen V and HepV substrates in the presence of heparin, heparan sulfate, and chondroitin sulfate. We show that heparin and heparan sulfate, but not chondroitin sulfate, are potent inhibitors of cell interaction with HepV and to a less extent with intact collagen V, whereas chondroitin sulfate and the irrelevant synthetic peptide RGDS have no effect (Fig. 6). Interestingly, binding of trypsin-released cells drastically affects cell adhesion to HepV, whereas adhesion to intact collagen V is only partially decreased. These results indicate that CHO cells possess at least two distinct cell surface receptors for collagen V: a trypsin-labile receptor that binds specifically to the heparin binding site, HepV, and a trypsin-resistant receptor that binds to another region of the molecule. Altogether, the data support the conclusion that the interaction between CHO cells and the collagen V heparin binding site is mediated by a cell surface heparan sulfate proteoglycan receptor.
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Representation of Heparin Binding Sequence Folded into
Collagen V Triple Helices--
We showed that HepV binds heparin as
tightly as the parent 1(V) chains. However, even though the cluster
of basic amino acids present in HepV contains conspicuously the
sequence responsible for heparin binding activity, the synthetic
peptide encompassing this sequence has no affinity for heparin.
Furthermore, no amino acid sequence was found in HepV which fits one of
the postulated consensus motifs identified in other heparin-binding
proteins (XBBXBX and
XBBBXXBX, where B designates a basic
amino acid and X any other amino acid). This probably means
that the secondary structure of collagen holds the sequence of interest
in a particular spatial pattern that fits more to heparin or that
residues flanking this sequence contribute to the charge density of the
binding site. The spatial pattern of the putative binding site was
assayed by analyzing the amino acid distribution of residues 905-921
(KPGPRGQRGPTGPRGER) in a helical wheel representation. The
representation of a collagen helix known as a polyproline II helix has
been shown to differ from a true
helix in the sense that proline
residues induce a more extended structure, and thus the helical wheel
contains 17 residues instead of 18 for an
helix (21).
Interestingly, the representation of the peptide sequence segregates
the basic residues to one side of the helical face and forms a cluster
of 5 basic residues within a stretch of 9 amino acids. It is clear that
the spatial distribution of the basic amino acids promotes the
formation of a high positive charge region. The helical wheel representations for peptides flanking this sequence, even if they do
correspond to basic amino acid-rich regions, do not allow the observation of positive charge clusters (data not shown). Because HepP
encompasses this region, this indicates that the designed peptide,
although it did not bind to an heparin column, corresponds to the
sequence that fits the best the postulated criteria for heparin
recognition.
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DISCUSSION |
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Collagen V was shown to bind heparan sulfate proteoglycans
through its heparin binding site (7, 15). The same site might mediate
interaction with syndecan-1 (4, 5). A first approach to study these
interactions in terms of function and possible involvement in
physiological events is to characterize better the heparin binding site
on individual chains and also on collagen V triple helix molecules. In
the present study we have narrowed the sequence involved in the
interaction on the individual 1(V) chains and identified the heparin
binding site on collagen V molecules. Indeed, experiments with
individual chains or small fragments cannot solve the question of
whether the same specific domain is required for heparin binding to
triple helical molecules. As a matter of fact collagen I binds to
heparin, but it was shown that the triple helical structure is strictly
required for heparin binding (4, 25, 26). This suggests that triple
helical formation can generate new combinations of basic amino acid
residues from the association of the two distinct constitutive chains
creating a heparin binding site. Therefore, it appears essential to
determine the level of structural organization of collagen V required
for binding to heparin.
The results presented here strongly suggest that the recombinant 12-kDa (HepV) fragment is necessary and sufficient for heparin/heparan sulfate binding activity, whereas a more restricted sequence (HepP) including a cluster of basic amino acid residues is not sufficient to mediate binding to heparin. The lack of heparin binding activity of this peptide might be the result of the need for neighboring amino acid residues for the interaction to occur or the failure of the peptide to assume a correct secondary structure consistent with its configuration in the folded domain.
It is now clear that specific structural motifs rather than just ionic
interactions are required for protein binding to heparin (20, 27). This
is supported by studies where the use of heparin-binding synthetic
peptides led to a reduction of affinity (28, 29). As a matter of fact,
HepV was shown to fold into the characteristic collagenous structure,
the polyproline II helix, by circular dichroism, whereas the peptide
tested in the same experimental condition was not (data not shown). A
large range of extracellular proteins interacts specifically with
heparin, and a common motif emerged from comparison of the various
heparin binding sequences (20). Such sequences were not found in the
entire 1(V) chain. However, from the research on the identification
of a common motif in heparin binding sequences, Margalit et
al. (27) defined the motif KPGPRGQR as the sequence responsible
for heparin recognition. Even though this sequence was considered to
fold into a
strand, which appears to be unlikely since this
sequence is included within the triple helical domain known as a
polyproline II helix, analysis of the basic residue distribution in the
helical wheel representation adapted to this peculiar structure ensures
the segregation of the basic amino acid residues on one side of the
helix. This representation allowed us to observe that all of the basic
residues contained in the synthetic peptide sequence (HepP) form a
cluster of positive charge which likely interacts with heparin. Because
the formation of this cluster is dependent on the correct folding of
the peptide, we suggest that the peptide does contain the information
to bind heparin but fails to adopt the correct conformation.
Thus it appears that heparin binding motif of collagenous structure
exhibits the same characteristics already described for other proteins
(folded into helix or
strand): a spatial distribution of the
basic amino acid forming an amphipatic structure (20, 27). Although it
is not possible to generalize to the other collagens containing heparin
binding sites within the triple helical domain, this is at least
confirmed for the heparin binding sites present on the
acetylcholinesterase collagenous tail (21).
A preferred site of attachment of heparin gold on collagen triple
helices was positioned at about 100 nm from the
NH2-terminal extremity of the collagenous domain,
indicating that the heparin binding site occurs at the same position on
individual chains and on triple helical molecules. Nevertheless the
level of affinity for heparin depends on the chain composition
constituting the collagen V molecules. Collagen V can associate into at
least three different molecules: [1(V)]2
2(V), found
in most tissues;
1(V)
2(V)
3(V), described only in human
placenta; and the homotrimer [
1(V)]3, produced by
hamster lung cells and present in some embryonic tissues (11-13).
Indeed, a different apparent affinity for the two heterotrimers for
heparin has been described recently by others as a way to purify the
two molecular forms from tissues (26). In principle, one might expect
collagen V containing one
1(V) chain, i.e. the heterotrimer
1(V)
2(V)
3(V), to exhibit an affinity similar to that of individual
1(V) chain for heparin. However, we show that the
presence of two
1(V) chains, which is the case for the heterotrimer [
1(V)]2
2(V), is necessary to achieve the same
apparent affinity as an individual
1(V) chain. This suggests that
the formation of the triple helix constrains the
1(V) chains in a
more restricted conformation that is not as suitable for heparin
binding as the individual chains are. Our molecular modeling reveals
that only the homotrimer allowed the formation of a real ring of basic
amino acid residues on the surface of the molecule. Concerning the
heterotrimer containing one
3(V) chain, the sequence facing the
1(V) heparin binding being unknown (30), the molecular
representation cannot be drawn. The lower affinity obtained for this
heterotrimer can be however explained by the fact that neither
2(V)
nor
3(V) binds to heparin at physiological ionic strength.
The clustering of positive charges through the formation of dimers and tetramers can increase the affinity for heparin as has been described for the platelet factor-4 protein (31). Therefore, we propose that the positively charged ring observed for the homotrimer can reinforce the interaction with heparin and thus explain the higher affinity obtained. Considering the various associations described for collagen V molecules, the difference in affinities we have observed for the three molecules represents the only data showing a difference in their behavior and subsequently in their role in the interaction with heparan sulfate proteoglycans in the matrix and on cell surface. Interestingly, our experiments establish that recombinant HepV fragments mediate CHO cell attachment that was blocked by the addition of heparin/heparan sulfate. Inhibitory experiments and the loss of cell adhesion subsequent to trypsin treatment strongly suggest that a heparan sulfate cell surface proteoglycan may act as a receptor for the heparin binding site we have mapped. These results point to a physiological role in cell adhesion for the collagen V heparin binding site.
The approach described here is an essential step in the elucidation of
the function of the collagen V heparin binding site in cell and matrix
interactions. Moreover, the basic amino acid residue(s) crucial for
heparin binding activity can be investigated further by mutagenesis
experiments on the recombinant 1(V) fragment and particularly on the
recombinant homotrimer. As we have shown that collagen V binds to cell
surface proteoglycans and integrins (9, 10), it is tempting to
speculate that these sites can act cooperatively as described already
for other extracellular matrix proteins such as laminin (32, 33) and
fibronectin (34).
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ACKNOWLEDGEMENTS |
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We are indebted to Dr. K. Takahara (Takara
Shuzo Co., Shiga, Japan) for the generous gift of 1(V) clones and to
Dr. J.-C. Cortay and Dr. D. Nègre for advice and helpful
discussion concerning the prokaryotic expression. We thank Dr. F. Letourneur for providing CHO cells, M.-M. Boutillon for protein
sequencing and amino acid analysis, and D. Ficheux for peptide
synthesis. We are grateful to A. Bosch and C. Van Herreweghe for expert
artwork and Dr. P. Carroll for assistance with English.
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FOOTNOTES |
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* This work was supported in part by a Bonus Qualité Recherche grant from the Université Claude Bernard, Lyon; a Program Emergence grant from the Région Rhône-Alpes; and Contract BIO-4-CT96-0537) from the European Community.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 atomic coordinates and structure factors (codes 1a89 and 1a9a) have been deposited in the Protein Data Bank, Brookhaven National Laboratory, Upton, NY.
Recipient of a fellowship from the program Emergence de la
Région Rhône-Alpes.
§ To whom correspondence should be addressed. Tel.: 33-4-7272-2657; Fax: 33-4-7272-2602; E-mail: f.ruggiero{at}ibcp.fr.
1 The abbreviations used are: PAGE, polyacrylamide gel electrophoresis; HPLC, high performance liquid chromatography; PBS, phosphate-buffered saline; CAPS, 3-(cyclohexylamino)propanesulfonic acid; CHO, Chinese hamster ovary.
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REFERENCES |
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