(Received for publication, October 23, 1995; and in revised form, December 19, 1995)
From the
The region in human osteonectin (ON) responsible for binding to
type V collagen has been identified as the first 17
NH-terminal residues. This conclusion is based upon binding
studies with deletion mutants of ON produced in Escherichia
coli, in which parts of the first 17 amino acids have been
removed. Wild-type ON from E. coli and mammalian cell-derived
nonglycosylated ON bind identically to type V collagen and at least
twice as effectively as mammalian cell-derived N-glycosylated
ON. In previous studies, it was shown that N-glycosylation at
residue 99 significantly reduces the capacity of ON to bind to type V
collagen. Results reported in this communication demonstrate that the
actual binding site on ON for type V collagen is distal from the site
of N-glycosylation in terms of amino acid sequence but may be
proximal in the folded, fully glycosylated, three-dimensional
structure. Consistent with this conclusion is the ability of a
synthetic peptide consisting of amino acids 1-17 to specifically
inhibit the binding of ON to type V collagen.
Osteonectin
(ON)()/SPARC/BM-40(1, 2, 3) , a
secreted, single-chain, acidic, Ca
-binding
glycoprotein, is a major noncollagenous extracellular matrix protein in
bone and dentine (1, 4) as well as in many normal and
neoplastic human soft tissues (5) and cultured
cells(6, 7) . It is also synthesized, stored, and
secreted by human blood platelets(8, 9) . Earlier
studies have suggested several functions for ON in the extracellular
matrix, including the regulation of bone
mineralization(1, 10) , the control of cell
shape(11) , tissue remodeling or repair(12) , cell
migration(6) , proliferation, and
differentiation(12, 13, 14, 15, 16) .
Osteonectin from different sources binds differentially to different
type collagens. Osteonectin from bovine bone binds to type I, III, and
V collagen(1, 15, 17) , whereas that from
mouse parietal yolk sac cells binds only to type III and V
collagen(18) , and that from human platelets has no affinity
for any of the three collagen types(17) . The difference
between bovine bone and human platelet ON binding to collagen has been
attributed to differences in N-glycosylation(17) .
Osteoblast- and megakaryocyte-derived mRNA encoding ON are identical in
size and restriction enzyme fragmentation patterns(19) ,
lending further support to the hypothesis that differences in structure
and collagen binding between bone and platelet-derived ON reside at the
level of N-glycosylation. Osteonectin (BM-40) from mouse
Engelbreth-Holm-Swarm tumor binds to type IV collagen but shows
markedly reduced binding to type I, III, V, and VI
collagen(20) . The region of BM-40 binding to type IV collagen
has been identified as the EF-hand and
-helical domains in the
carboxyl-terminal half of BM-40 (21) .
Mature human ON consists of 286 amino acids and contains two potential Asn-X-(Thr/Ser) N-gycosylation sites, located at positions 71 and 99(22, 23) . Recently, we reported that the binding site in human ON for type V collagen resides in the amino-terminal half (amino acids 1-146) of ON and that the capacity for type V collagen binding is significantly reduced by N-glycosylation at residue 99(24) . Residue 71 appears to have no, or very little, attached carbohydrate(24) . In the present study, employing a set of deletion mutants, the binding region in ON has been further localized to the first 17 amino acids of the mature protein and appears to involve a tertiary structure comprised of several amino acids.
Figure 1:
Construction and expression of
recombinant osteonectin in E. coli. Part A, schematic
diagram of pUC19-derived wild-type truncated osteonectin expression
vector. The mammalian cell expression vector tHON/pD5, described in (24) , was used for PCR template. PCR mutagenic primers A and B
are described under ``Experimental Procedures.'' RI, EcoRI; H3, HindIII; MCS, multiple
cloning site. Segment lengths are not shown to scale. The relevant
coding strand nucleotide and amino acid sequences in the expression
vector obtained by ligation is shown. Lower case letters represent flanking segments of the -galactosidase (lacZ) gene (broad, open arrow) in pUC19, and upper case
letters represent the ends of the osteonectin portion of the
resulting recombinant protein. Dashed line represents the
internal position of ON (amino acids 8-140). Amino acid numbering
is in reference to the NH
-terminal Ala residue of mature
osteonectin (+1). Mutants were generated by the same scheme, using
primers A containing amino acid deletions, as described in the text. Part B, schematic representation of chimeric proteins. Dots
represent deleted amino acids. Zig-zag lines represent
flanking lacZ amino acids. Osteonectin amino acids 18-146 are
represented with a broad horizontal line. Amino acids are
shown in single-letter code. Name designations are
described in the text. Part C, purified recombinant proteins
resolved by SDS-PAGE. Purified recombinant proteins (
5 µgm)
were run on a reducing SDS-PAGE gel (12%) according to Laemmlii (33) and stained with Coomassie Blue. Lanes 1 and 10, molecular markers; lanes 2 and 9,
wild-type tHON; lane 3,
; lane 4,
; lane 5,
; lane
6,
; lane 7,
; lane 8,
.
PCR conditions were the same as described previously(25) .
After PCR, each product was digested with HindIII and EcoRI restriction enzymes, and the resulting fragment was
resolved on a 1% agarose gel (TAE buffer), isolated by electroelution,
and ligated into pUC19 from which the corresponding HindIII/EcoRI MCS fragment had been removed. The
ligated mixture was then used to transform E. coli strain
DH5 cells according to the supplier's directions and plated
on 5-bromo-4-chloro-3-indoyl
-D-galactoside/isopropyl-1-thio-
-D-galactopyranoside/ampicillin/LB
agar plates. Isolated white colonies were grown in liquid culture, and
plasmid DNA was isolated by the alkaline lysis/Qiagen column procedure
according to the supplier's directions. Resulting plasmids were
analyzed by digestion with HindIII, EcoRI, and PvuII, and the region between the HindIII/EcoRI sites was sequenced (Applied Biosystems
Inc., model 373 A DNA sequencer) to confirm the desired deletion and to
exclude inadvertent mutations introduced from PCR. Sequencing reactions
were performed with the TaqDye Deoxy(TM) Terminator cycle sequencing
kit according to the supplier's instructions (Applied Biosystems
Inc., Foster City, CA).
Figure 2:
Affinity of monoclonal antibody
IIIAA
for different ONs. Osteonectin-antibody
complexes were detected immunologically as described in the text. tHON
(
),
, (
),
(
),
(
),
(
),
(
), and
(
). Error bars represent the range in values from three
independent determinations.
Figure 3:
Assessment of the binding capacity of
equimolar tHON from E. coli to collagen V as compared with
tHON and NQ from 293 human kidney cells. Type V collagen,
at 10 µgm/ml in 50 mM NaHCO
, pH 9.7, was
applied to plastic microtiter wells for 3 h. After washing and blocking
(2% bovine serum albumin) steps, solutions of
tHON
(
), tHON
(
), and
N
Q
(
) in 20 mM Tris-HCL (pH
7.4), 150 mM NaCl, 2 mM CaCl, 0.05% (v/v) Tween 20
were applied to the wells and incubated for 18 h at room temperature.
After washing, ON-collagen complexes were detected with the ELISA assay
described under ``Experimental Procedures.'' Error bars represent the range of duplicate
assays.
Figure 4: Binding of deletion mutants to type V collagen. Samples were prepared as described under ``Experimental Procedures'' and assayed as described for Fig. 3. The absolute absorbance at 490 nm for wild-type tHON (lane 1) was 1.35, as shown in Fig. 3. Measured binding of all the protein species at concentrations indicated in Fig. 3was equal or less than that at 1 µM (data not shown).
Figure 5:
Competitive inhibition of ON binding to
type V collagen by aa 1-17 peptide. Type V collagen, at 10
µgm/ml in 50 mM NaHCO (pH 9.7), was applied to
plastic microtiter wells for 3 h. After washing and blocking steps,
solutions of tHON and
in
TBS/Tween/CaCl
buffer were applied to the wells and
incubated with different concentrations of peptide for 18 h at 4
°C. Final concentration of ON in each well is 0.125
µM. After washing, ON-collagen complexes were detected by
the ELISA assay as described under ``Experimental
Procedures.'' tHON + 1-17 peptide (
); tHON +
control peptide (
);
+ 1-17 peptide
(
); 1-17aa peptide alone (
); and control peptide
alone (
).
Earlier studies indicate that bovine bone and human bone ON bind to type V collagen(4, 17) . Osteonectin from mouse parietal yolk sac cells also binds to type V collagen(18) . In addition, studies with a truncated form of human ON containing only the amino-terminal half of the protein effectively binds to type V collagen, demonstrating that the binding region of ON resides within amino acid residues 1-146 (24) . Based upon differences in glycosylation patterns as well as lectin binding properties, it was proposed that the collagen binding specificity of bone and platelet ON is related to differences in glycosylation(16) . In order to further understand the region of ON involved in collagen binding and the effects of specific N-glycosylation sites on biological activity, we have demonstrated that after removal of oligosaccharide chain structures from bovine bone and human platelet ON by N-glycanase, their ability to bind to type V collagen is increased to an equal level. In addition, the results of site-specific mutagenesis at each of the two potential Asn-X-Thr glycosylation sites (amino acids 71 and 99) in ON indicate that only glycosylation at residue 99 affects type V collagen binding activity(24) . Further studies using deletion mutants reported here demonstrate that the binding site in ON for type V collagen is in the first 17 amino acids of the protein. This segment is distal from the N-glycosylation site in regard to the amino acid sequence, but due to protein folding, and the spacial umbrella of the carbohydrate, may be conformationally proximal to the site of N-linked carbohydrate. No information relating to the three-dimensional structure of ON is available to further interpret our results. A second important conclusion reached from this study involving a family of related deletion mutants is that it does not appear that a unique amino acid residue or short segment of amino acids is capable by itself of directing effective ON binding to type V collagen, but rather, several small segments and/or an overall conformational state in the first 17 residues is necessary.
As
indicated in Fig. 3and reported in our earlier
studies(24) , tHON achieves half-maximum binding at 100
nM, representing approximately 100-fold greater affinity for
type V collagen than the blocking peptide (K
10 µM). Differences of this order of
magnitude are commonly observed for synthetic peptides versus the native protein and are thought to be largely due to the
greater flexibility and conformational heterogeneity of the synthetic
peptide. Despite the difference in affinity, the fact that complete
inhibition can be accomplished by the peptide alone and the absence of
binding in the presence of peptide plus
ON mutant
protein strongly suggest that the binding of ON to type V collagen is
recapitulated by the peptide.
Type V collagen is particularly abundant in vascular tissue, primarily due to its synthesis in smooth muscle cells. Smooth muscle cells and their protein products, including possibly type V collagen, are believed to play an important role in the development of atherosclerotic plaque. In human atherosclerotic lesions, the ratio of type V collagen to other types is elevated relative to the normal situation(28, 29) . Additionally, two reports indicate that type V collagen can have a procoagulant effect(30, 31) . This might result from the disruption or destruction of vascular endothelial cells at sites of tissue injury or remodeling, and consequential exposure of type V collagen by underlying smooth muscle cells. Kelm et al.(32) have reported that osteonectin binds to plasminogen and enhances tissue plasminogen activator conversion of plasminogen to plasmin. Both plasmin and tissue plasminogen activator are important thrombolytic agents. Kelm and co-workers (32) also reported the mediation of plasminogen binding to type V collagen by bovine bone osteonectin(32) . Consequently, osteonectin, by serving as a bridge between type V collagen and thrombolytic agents, may play an important role in hemostasis in the absence of functional endothelium.