(Received for publication, January 17, 1995; and in revised form, May 26, 1995)
From the
In this study we demonstrate that the binding region of
recombinant truncated human bone osteonectin (tHON) for type V collagen
resides between amino acids 1 and 146. After removal of oligosaccharide
chain structures from tHON, bovine bone osteonectin (BBON) and human
platelet osteonectin (HPON) by N-glycanase, their ability to
bind to type V collagen is increased, and HPON affinity to collagen V
is the same as that of BBON. These data suggest that glycosylation of
osteonectin has a direct or regulatory effect on osteonectin binding to
collagen V and that the increase in tHON binding upon removal of
carbohydrate is the result of a loss of a down-regulation site or
direct interference of the carbohydrate at the binding site. To
determine the specific role of each N-glycosylation site in
tHON, Asn and Asn
were mutated to Gln (N71Q,
N99Q) and Thr
and Thr
mutated to Ala (T73A,
T101A) to selectively inhibit oligosaccharide attachment. The binding
affinity of N99Q and T101Q to collagen V is markedly increased over
wild-type tHON, whereas N71Q and T73A are the same as wild-type tHON.
The doubled mutant (N71,99Q) binds identically to collagen V as N99Q
and T101A. These data suggest that only the position 99 glycosylation
site (Asn
-X-Thr
) in tHON is
important in the reduction of binding of osteonectin to collagen V.
Consistent with the binding data is the observation that both the N71Q
and T73A mutant proteins migrate on SDS-polyacrylamide gel
electrophoresis gels identically to wild-type tHON, suggesting that
there is little or no N-glycosylation of residue 71 in
wild-type osteonectin.
Osteonectin (ON), ()a single chain acidic,
Ca
-binding glycoprotein, was initially identified as
an integral component of the noncollagenous matrix of
bone(1, 2, 3) . Later studies indicated that
it occurs widely in extracellular matrices and some other body
compartments, as well as within cells including blood
platelets(4, 5) . As the endothelial and endodermal
cell product it has been called SPARC (6) and as protein BM-40
obtained from the matrix of a basement membrane mouse
tumor(7) . It is now apparent, from cDNA sequencing analyses of
human (8, 9) and bovine (10) ON that the
mature protein consists of 286 or 287 amino acids, respectively, and
contains two potential Asn-X-Thr/Ser N-glycosylation
sites, located at positions 71 and 99. The amino acid sequence of mouse
SPARC is 92% identical to the cDNA-derived amino acid sequence of human
ON but lacks the potential N-glycosylation site at human
position 71(11) .
Structural analysis of
osteonectin/BM-40/SPARC indicated that it consists of four distinct
domains with potentially different ligand binding
properties(10, 12, 13) . Earlier studies
indicate that bovine bone osteonectin binds to types I, III, and V
collagen(1, 2, 14) . SPARC from mouse
parietal yolk sac cells binds to types III and V collagen(15) .
BM-40 from the mouse Engelbreth-Holm-Swarm tumor binds to type IV
collagen but shows markedly reduced binding to types I, III, V, and VI
collagen(16) . The binding site of BM-40 for type IV collagen
has been located at the EF hand and the -helical domains in the
carboxyl-terminal half of BM-40(17) . Both the amino-terminal
Glu-rich domain and two potential EF hands in the carboxyl-terminal
half of the molecule have been implicated in Ca
binding. Early studies with proteolytic fragments indirectly
suggested Ca
binding sties in carboxyl-terminal
domains III and IV (16) . A more recent report (17) using deletion and site-specific mutagenesis confirms this
earlier finding. Osteonectin also binds specifically to plasminogen and
enhances the conversion of plasminogen to plasmin by tissue plasminogen
activator and may play a role during tissue remodeling or
repair(18) .
Kelm and Mann previously demonstrated that bone (M 31,000) and platelet (M
33,000) osteonectin differ in apparent molecular weight when
analyzed on SDS-polyacrylamide gels(5) . Subsequent studies
demonstrated that the differences in mobility can be attributed to
differences in N-glycosylation(14) . In the same
study, a comparison of the binding capacity of bone and platelet
osteonectin for immobilized collagen revealed that platelet osteonectin
had no apparent affinity for collagen in a concentration range in which
bone osteonectin binding was observed(14) . Osteoblast- and
megakaryocyte-derived mRNA-encoding osteonectin are identical in size
and restriction enzyme fragmentation pattern, thus lending further
support to the hypothesis that differences in structure and collagen
binding between bone and platelet-derived osteonectin reside at the
level of N-glycosylation(19) .
Full-length human osteonectin (BM-40) has been expressed in stably transfected human kidney 293 cells(20) . The purified protein was very similar to mouse Engelbreth-Holm-Swarm tumor derived BM-40 in its structural and functional properties, including glucosamine and galactosamine content(20) . We have now constructed for expression in 293 cells a SV40/adenovirus-derived expression vector containing the leader peptide and amino-terminal half of truncated human bone osteonectin (amino acids -17 to +146) followed by a termination codon, and successfully created site-specific mutations relating to position 71 and 99 N-glycosylation sites of tHON using the PCR-enabled method of Nelson and Long(21) . In this study we describe the binding properties of tHON to type V collagen before and after removal of oligosaccharide both by N-glycanase treatment and site-directed mutagenesis.
Figure 1:
Affinity of
IIIAA
to different osteonectins.
Osteonectin-antibody complexes were detected immunologically as
described in the text.
, BBON;
, HPON;
, tHON;
, N71Q;
, N99Q;
, no osteonectin. Error bars represent the range in values from three independent
determinations.
Figure 2:
Assessment of the binding capacity of
equimolar BBON and tHON for type V collagen. Type V collagen, at 10
µg/ml in 50 mM NaHCO, pH 9.7, was applied to
plastic microtiter wells for 3 h. After washing and blocking steps,
solutions of BBON (
), tHON (
), and BSA (
) 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, osteonectin-collagen complexes were
detected with an ELISA assay as described under ``Experimental
Procedures.'' Error bars represent the range of duplicate
assays.
Figure 3:
N-Glycanase digestion of tHON,
BBON, and HPON. tHON, BBON, or HPON (5 µg) in 30 µl of 20
mM Tris-HCl, pH 7.6, 150 mM NaCl, 0.5% (w/v) SDS, 50
mMo-phenanthroline, 50 mM
-mercaptoethanol, and 1.25% (v/v) Nonidet P-40 were incubated with N-glycanase, 0.5 unit, for 18 h at 37 °C. The enzyme was
inactivated by the addition of a 5-fold concentrated stock of SDS-gel
sample preparation buffer and boiling for 5 min. The proteins were
resolved on a 8-18% gradient SDS-PAGE gel and visualized by
staining with Coomassie Blue. Lane 1, the size of molecular
mass standards is indicated in kilodaltons; lanes 2 (HPON), 4 (BBON), and 6 (tHON) represent the untreated
proteins. Lanes 3 (HPON), 5 (BBON), and 7 (tHON) represent the enzyme-treated proteins. Western blotting
with antibody IIIA3A8 also resulted in visualization of the above bands
(data not shown).
Figure 4:
Binding of BBON, HPON, and tHON to type V
collagen after deglycosylation. Type V collagen at 10 µg/ml in 0.05 M NaHCO, pH 9.7, was applied to plastic microliter
wells for 3 h. After washing and blocking steps solutions of either N-glycanase-digested or untreated BBON, HPON, or tHON 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,
osteonectin-ligand complexes were detected as described under
``Experimental Procedures.'' A, BBON (
) and
HPON (
) represent the N-glycanase-treated proteins;
untreated BBON (
), HPON (
), and BSA (
) control were
also studied for comparison. B, treated tHON (
),
untreated tHON (
) in the above
conditions.
Figure 5: Wild-type tHON and N-glycosylation mutants. Wild-type tHON, N71Q, T73A, N99Q, T101A, and N71,99Q (3 µg) following immunoaffinity purification were run on an 8-18% SDS-PAGE gradient gel and visualized by staining with Coomassie Blue. Lane 1, sizes of protein standards are indicated in kilodaltons; lanes 2 and 8, tHON; lane 3, N71Q; lane 4, T73A; lane 5, N99Q; lane 6, T101A; and lane 7, N71,99Q. Immunoblotting revealed identical bands as shown in the figure (data not presented).
Figure 6:
Binding of N-glycosylation
mutants to type V collagen. Samples were prepared as described under
``Experimental Procedures''and assayed as described for Fig. 4. A, wild-type tHON (), N71Q (
), and
T73A (
). B, wild-type tHON (
), N99Q (
),
T101A (
), and N71,99Q double mutant
(
).
Enzymatic deglycosylation studies suggest that bone osteonectin possesses a high mannose-type oligosaccharide structure, whereas platelet osteonectin likely possesses a complex-type structure (14) and may be due to differential expression of glycosyltransferases by osteoblasts and megakaryocytes(26, 27) . Based upon differences in glycosylation patterns as well as lectin binding properties, it was proposed that the collagen binding specificity of bone and platelet osteonectin is related to differences in glycosylation(14) . In the present study we demonstrate that after removal of oligosaccharide chain structures from bovine bone and human platelet osteonectin by N-glycanase, their ability to bind to collagen V is increased and indistinguishable from one another. These results indicate that glycosylation of ON has a direct or regulatory effect on ON binding to collagen and that the glycosylation pattern dramatically affects binding to collagen. In addition, studies with a truncated form of human osteonectin 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. As in the case of BBON and HPON, N-glycanase treated truncated ON displays increased binding to type V collagen.
In order to further define the region of ON involved in collagen binding and the effects of specific N-glycosylation sites on biological activity, we have systematically altered, by site-specific mutagenesis, each of the two potential Asn-X-Thr glycosylation sites in osteonectin. Both sites are within the amino-terminal half of the mature protein, affording the use of the truncated ON cDNA as template for mutagenesis as well as for production of wild-type counterpart recombinant protein. Expression vectors for wild-type tHON, one double and four single mutants of tHON (N71,99Q and N71Q, T73A, N99Q, T101A) were constructed using the PCR-enabled method of Nelson and Long(21) . Each expression vector was then used to transiently express recombinant protein in human kidney 293 cell cultures, and secreted protein was subsequently purified from conditioned media and characterized.
Initial studies revealed that SDS-PAGE migration of both the N99Q and T101A mutant recombinant proteins is identical and significantly faster than wild-type protein, suggesting a similar reduction in glycosylation for both mutants. Estimates based on mobility differences suggest a loss of about 4-5-kDa mass for both mutants. The mass difference is similar to that observed by Pottgiesser et al.(17) when the protein was expressed in the presence of tunicamycin or treated with N-glycosidase F. Surprisingly, both the N71Q and T73A mutants migrated identically to wild-type protein, suggesting that there is little or no N-glycosylation in the wild-type protein at this position. Consistent with these results is the observation that the N71,99Q double mutant migrates identically to the N99Q and T101A single mutant proteins. The interpretation of little or no glycosylation at human position 71 is also consistent with the report of Nischt et al.(20) , indicating that mouse tumor-derived osteonectin (BM-40) lacking a potential N-glycosylation site at residue 71 and recombinant human osteonectin have the same carbohydrate content and composition.
Differential glycosylation at the two potential N-glycosylation sites suggested by the above structural
analysis is also implicated by functional studies relating to type V
collagen binding. Data presented above indicate that mutation at either
residue 71 or 73 has no effect on collagen binding compared with either
wild-type tHON or BBON. However, mutation at residue 99 or 101, thereby
abolishing N-glycosylation at residue 99, results in protein
with significantly enhanced collagen binding capacity and which mimics
enzymatically deglycosylated truncated or full-length ON. Pottgiesser et al.(17) , using deletion and site-specific
mutagenesis, have also studied some of the ligand binding properties of
293 cell-derived recombinant osteonectin (BM-40). Their studies
focusing on the carboxyl-terminal half of the protein suggest that this
portion of the molecule is involved in type IV collagen binding as well
as a high affinity (K
80 nM)
Ca
binding interaction(17) . Deletion of
residues 149-200 or 202-245 each result in the loss of
Ca
-dependent type IV collagen binding(17) .
Deletion of residues 8-67 in the amino-terminal half of
osteonectin had no effect on type IV collagen binding(17) . In
an earlier report (20) they also observed that deglycosylation
of recombinant osteonectin had no effect on type IV collagen binding.
These results are consistent with ours, showing the effects of N-glycosylation on the binding of the amino-terminal half of
osteonectin to type V collagen. These data derived from studies with
mutated forms of human ON taken together strongly indicate that residue
99 is the major, and possibly sole, site of N-glycosylation.
Furthermore, the results indicate that the region of ON interacting
with type V collagen involves residue 99. It is unclear, however, from
our studies whether residue 99 and/or adjacent residues are involved
directly in collagen binding and are consequentially masked by the
presence of N-linked carbohydrate at this position.
Alternatively, N-glycosylation of residue 99 may have an
indirect distal allosteric regulatory effect.
Saturation binding
curves presented in this communication suggest that the presence of N-linked carbohydrate decreases the capacity of osteonectin
for type V collagen but does not have a measurable effect on binding
affinity. Under the conditions of the binding assay used in our
studies, all species of osteonectin achieved half-maximum binding at
0.07-0.10 µM, which is close to the apparent
dissociation constant (K = 0.2
µM) for bovine bone osteonectin derived by Kelm and Mann
from a Scatchard plot(14) .
The negative effect of N-linked carbohydrate on ligand binding is not unique to
osteonectin. Grinnel et al.(28) , for example, report
that abolition of an N-glycosylation site in protein C by
mutagenesis (N313Q) results in a protein having about a 3-fold decrease
in apparent K (6.1 µM
1.9
µM) for thrombin-thrombomodulin activation. Wittwer et
al.(29) observed a 3-4-fold reduction in fibrin
binding and clot lysis activity by tissue-type plasminogen activator
(t-PA) synthesized by Bowes melanoma and human colon fibroblasts in the
presence of tunicamycin. Similar results for t-PA were obtained with
tunicamycin in Chinese hamster ovary cells by Hansen et
al.(30) . In the latter report, mutation of all three
potential Asn N-glycosylation sites in t-PA also resulted in a
marked increase in fibrin binding(30) . Lijnen et al.(31) have also reported that the apparent dissociation
constant for binding of plasminogen to its natural protein inhibitor
-antiplasmin is affected by the presence of N-linked carbohydrate. Both Glu- and Lys-plasminogen K
values (4.2 and 1.9 µM,
respectively) are reduced upon removal of carbohydrate (2.9 and 0.2
µM, respectively).
Type V collagen is particularly abundant in vascular tissue, primarily due to synthesis in smooth muscle cells(32) . Smooth muscle cells and their protein products are believed to play an important role in the development of atherosclerotic plaque. The ratio of type V collagen to other types is increased in human atherosclerotic lesions(33, 34) . In addition to its possible role in atherosclerosis, type V collagen may also be important in hemostasis. It has been reported to not stimulate platelet aggregation (35) and prevent platelet adhesion (36) and consequentially would have an anti-thrombotic effect. This effect may be mediated through its binding to the cytoadhesive molecule thrombospondin(37) . Recently a 19-kDa fragment from bovine thrombospondin has been identified which binds to both type V collagen and heparin(38) . Type V collagen has been reported to also inhibit the activation of the intrinsic clotting path factor XII(39) .
On the other hand, procoagulant activity by type V collagen has also been reported(40) . Coating of vascular prostheses surfaces with type V collagen inhibits endothelial cell attachment and growth (41) and as a result may also contribute to a prothrombotic state. A similar prothrombotic state, due to the absence of endothelial cells, may exist at sites of tissue injury and remodeling. Kelm et al. have recently reported that osteonectin also binds to plasminogen and enhances t-PA conversion of plasminogen to plasmin(18) . Both plasminogen and t-PA are important antithrombotic agents. The authors also reported the mediation of plasminogen binding to type V collagen by bovine bone osteonectin, but not by human platelet-derived osteonectin(18) . Consequently, osteonectin, by serving as a bridge between exposed type V collagen and antithrombotic agents such as plasminogen and t-PA, may play an important compensatory role in preventing unwanted clot formation in the absence of functional endothelium.