Identification of Dual alpha 4beta 1 Integrin Binding Sites within a 38 Amino Acid Domain in the N-terminal Thrombin Fragment of Human Osteopontin*

Kayla J. Bayless and George E. DavisDagger

From the Department of Pathology and Laboratory Medicine, Texas A&M University Health Science Center, College Station, Texas 77843

Received for publication, December 18, 2000, and in revised form, January 19, 2001



    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Previous work from our laboratory demonstrates that the alpha 4beta 1 integrin is an adhesion receptor for OPN and that alpha 4beta 1 binding site(s) are present in the N-terminal thrombin fragment of osteopontin (OPN) (Bayless, K. J., Meininger, G. A., Scholtz, J. M., and Davis, G. E. (1998) J. Cell Sci. 111, 1165-1174). The work presented here identifies two alpha 4beta 1 binding sites within a recombinantly produced N-terminal thrombin fragment of human OPN. Initial experiments, using wild-type OPN containing an RGD sequence or an OPN-RGE mutant, showed identical alpha 4beta 1-dependent cell adhesive activity. A strategy to localize alpha 4beta 1 binding sites within the thrombin fragment of osteopontin involved performing a series of truncation analyses. Removal of the last 39 amino acids (130) completely eliminated adhesion, indicating all binding activity was present within that portion of the molecule. Combined mutation and deletion analyses of this region revealed the involvement of dual alpha 4beta 1 binding sites. Synthetic peptides for both regions in OPN, ELVTDFPTDLPAT (131) and SVVYGLR (162), were found to block alpha 4beta 1-dependent adhesion. The first peptide when coupled to Sepharose bound the alpha 4beta 1 integrin directly whereas a mutated ELVTEFPTELPAT peptide showed a dramatically reduced ability to bind. These data collectively demonstrate that dual alpha 4beta 1 integrin binding sites are present in a 38 amino acid domain within the N-terminal thrombin fragment of OPN.



    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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Integrins are a family of transmembrane heterodimeric cell adhesion receptors (1). The alpha 4beta 1 integrin (VLA-4) is predominantly expressed on leukocytes (2-4). It is capable of existing in multiple activation states (5) to mediate cell-cell and cell-extracellular matrix interactions (6-14). Known binding sites for alpha 4beta 1 include LDVP (8-10), IDAPS (15), REDV (16), QIDSPL (17-20), and under certain activating conditions, RGD (21). The alpha 4beta 1 integrin is intricately involved in trafficking of mononuclear leukocytes into tissues during the normal inflammatory response as well as in pathological situations (22), such as encephalomyelitis (23), diabetes (24), and graft rejection (25). Also, increased expression of alpha 4beta 1 is observed on smooth muscle cells within atherosclerotic plaques (26), and alpha 4beta 1 plays a role in tumor metastasis (12, 27). Collectively, the alpha 4beta 1 integrin appears to play a pathogenic role in inflammation, wound repair and tumor progression.

Osteopontin (OPN)1 is an extracellular matrix protein originally isolated from bone (28), and much evidence has accumulated as to its role in bone physiology (29). OPN is also secreted by many epithelial surfaces (30), and one early study supported a role for OPN in host-response to bacterial infection (31). Accumulating evidence also indicates OPN secretion is involved in inflammation and tumor progression. OPN has previously been found to be up-regulated in a variety of inflammatory, cardiovascular, and infectious diseases (32-37) and is a major secreted product of macrophages in inflammatory settings (32-36). It is also associated with tumors (38-41), particularly at the tumor-host interface (42). Recent data from knockout mice show that OPN facilitates wound healing (43), aids in host defense against viral (44) and bacterial infections (44, 45), and is involved in granuloma formation (44). Other studies have suggested OPN may facilitate tumor cell metastasis (46) and decrease complement-mediated tumor cell destruction (47). Based on these data, the presence of OPN in the wound environment likely plays an important role in regulating disease progression in inflammatory and other conditions. The molecular domains in OPN that mediate its effects in these phenomena remain to be defined.

The parallels between expression of the alpha 4beta 1 leukocyte integrin and expression of OPN in wounds prompted a previous study by our laboratory to define osteopontin as a ligand for the alpha 4beta 1 integrin (14). Here, we define two binding sites for the alpha 4beta 1 integrin in the recombinant N-terminal thrombin fragment of human OPN using deletion and mutation analyses.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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Preparation of Recombinant Osteopontin; Cloning Strategy-- A full-length cDNA clone of the human osteopontin gene was obtained from the American Type Culture Collection (ATCC, Manassas, VA) (48), and the sequence is shown in Fig. 1A. Sequences encoding the wild-type N-terminal thrombin fragment of osteopontin (rOPN-(17-168)) were amplified by polymerase chain reaction using the primers 5'-TAGGATCCATACCAGTTAAACAGGCTGATTCTGGAAG-3' and 5'-GTAAGCTTTTACCTCAGTCCATAAACCACACTATCACCTCGGCCA-3'(Genosys, The Woodlands, TX). Sequences encoding a mutated N-terminal fragment ([Glu161]rOPN-(17-168)) in which the single RGD sequence at residues 159-161 was changed to RGE were obtained by substituting 5'-TAAAGCTTTTACCTCAGTCCATAAACCACACTTTCACCTCGGCCA-3' for the downstream primer used to generate the wild-type fragment. Restriction digests of the polymerase chain reaction products and the pQE30 vector (Qiagen) were carried out overnight with BamHI and HindIII (Life Technologies, Inc.). Digested vector and insert were purified, quantitated, and ligated at an insert/vector ratio of 4.5:1 overnight at 14 °C (Roche Molecular Biochemicals). These constructs encode a modified version of rOPN-(17-168) and [Glu161]rOPN-(17-168) where the sequence RGSHHHHHHS replaces MRIAVICFCLLGITCA at the N terminus of wild-type osteopontin. All positive clones were confirmed by sequence analysis at Lone Star Labs (Houston, TX). All subsequent constructs studied contained the RGE mutation at amino acid 161. 


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Fig. 1.   Production of the N-terminal thrombin fragment of human OPN. A, amino acid sequence reported for the N-terminal thrombin fragment of OPN (48). Bracketed signal sequence representing amino acids 1-16 is not present on constructs generated here. This sequence is replaced by RGSHHHHHHS. The underlined sequences represent regions of OPN with affinity for alpha 4beta 1. B, combined truncation and mutation analyses generated various constructs listed by amino acid number. Asterisk indicates mutation of Asp to Glu.

Additional recombinant constructs of OPN (Fig. 1B) were produced using the following primer sets: rOPN-(55-168), 5'-TAAAGCTTTTACCTCAGTCCATAAACCACACTTTCACCTCGGCCA-3' and 5'-AGGGATCCCTAGCCCCACAGAATGCTGTGTCC-3'. The remaining constructs were constructed with a common upstream primer, 5'-TAGGATCCATACCAGTTAAACAGGCTGATTCTGGAAG-3' and different downstream primers: rOPN-(17-164), 5'-AGAAGCTTTTAAACCACACTTTCACC-3'; rOPN-(17-129), 5'-AGAAGCTTTTAAGATTCATCAGAATGGTGAGAC-3'; rOPN-(17-135), 5'-CGAAGCTTTTAATCAGTGACCAGTTCATCAG-3'; rOPN-(17-138), 5'-AGAAGCTTTTACGTGGGAAAATCAGTGACC-3'; [Glu135]rOPN-(17-138), 5'-AGAAGCTTTTACGTGGGAAACTCAGTGACCAGTTC3'; rOPN-(17-142), 5'-AGAAGCTTTTATGCTGGCAGGTCCGTGGGAAAATC-3'; [Glu139]rOPN-(17-142), 5'-AGAAGCTTTTATGCTGGCAGCTCCGTGGGAAAATC-3'; rOPN-(17-150), 5'-AGAAGCTTTTAGACAACTGGAGTGAAAACTTCGGTTGC-3'.

Cloning experiments were performed as described above, and positive clones were confirmed by sequence analysis at Lone Star Labs (Houston, TX).

Production and Characterization of Recombinant Wild-type and Mutated N-terminal Thrombin Fragments of Osteopontin-- Escherichia coli strain RY2840 [MC4100 lacIQ1 lac+ slyD Kmr] (49) was transformed with plasmids encoding the His6-tagged OPN derivatives. 2 ml of overnight cultures were inoculated into 200 ml of Luria-Bertani media (Life Technologies, Inc.) containing 50 µg/ml ampicillin (Sigma). Cultures were grown to an A600 of 1.0 (~2.5 h) before induction with 0.5 mM isopropylthiol-beta -D-galactoside (IPTG-Life Technologies, Inc.). Cultures were allowed to incubate for 3.5-4 h at 37 °C in a shaking incubator before being placed on ice for 15 min. Bacteria were pelleted, supernatants removed, and pellets frozen at -80 °C. Pellets were thawed at 25 °C for 10 min, resuspended on ice in 20 ml Hepes buffered saline, pH 8.1 containing 25 mM Hepes, 150 mM NaCl and 1 mM 4-(2-aminoethyl)benzene sulfonylfluoride, HCl (CalBiochem). Bacteria were lysed and debris pelleted (20,000 × g at 4 °C for 20 min) before adding supernatants to 2 ml of TALON metal ion affinity column (CLONTECH) equilibrated with Hepes buffer. Columns were incubated for a minimum of 20 min at 4 °C before washing with 20-column volumes of Hepes buffer. His-tagged proteins eluted with 0.2 M imidazole (Sigma) in Hepes buffer, and fractions were dialyzed (Mr cutoff 7,500) against 8 liters of phosphate-buffered saline. The purity of recombinant proteins was assessed by SDS-PAGE and Western blot analysis. Protein concentration was estimated according to the method of Pace et al. (50). Yields were ~6 mg per 200 ml of culture.

Cell Adhesion Assays-- Cell adhesion assays were performed to determine the ability of OPN to promote leukocyte adhesion. Polystyrene microwells (Corning-Costar, Cambridge, MA) were coated with 50 µl of bovine OPN purified as previously described (51) or recombinant fragments of OPN at a concentration of 20 µg/ml in TBS overnight at 4 °C. After blocking with 100 µl of 10 mg/ml BSA (Sigma, St. Louis, MO) in TBS, wells were rinsed with PSA (Life Technologies, Inc., Grand Island, NY). HL-60 promyelocytic leukemia cells and Ramos cells (ATCC) were grown in RPMI 1640 (Life Technologies, Inc.) and 10% fetal calf serum. Human umbilical vein endothelial cells were grown in M199 (Life Technologies, Inc.) supplemented with heparin (Sigma), bovine brain extract (52), and 20% fetal calf serum (Life Technologies, Inc.). Leukocytes were rinsed and resuspended in PSA at a density of 100,000 cells/well and endothelial cells at 35,000 cells per well. Media for adhesion in all leukocyte experiments contained a final concentration of 100 µg/ml BSA with physiological doses of CaCl2 (2 mM) and MgCl2 (1 mM). HL-60 cells were activated with the beta 1-activating antibody, 8A2 (53) at a concentration of 1 µg/ml and a phorbol ester, 12-0-tetradecanoyl phorbol 13-acetate at a concentration of 50 ng/ml. Endothelial cells were allowed to attach in the presence of 100 µg/ml BSA with 1.5 mM CaCl2 and 1.5 mM MgCl2. After plating, cells were allowed to adhere for 30-60 min at which time they were rinsed and fixed with formalin. Plates were stained with 0.1% Amido Black for 5 min and rinsed and solubilized with 2 N NaOH to obtain an absorbance reading at 595 nm, which corresponds directly to the number of cells stained in each well (54).

Peptide Synthesis and Adhesion Blockade-- To confirm the findings of the truncation studies, the SVVYGLR peptide (corresponding to C-terminal amino acids 162-168) was synthesized (Sigma-Genosys). Also generated were the wild-type peptide ELVTDFPTDLPATK and aspartate mutant ELVTEFPTELPATK, representing amino acids 131-143. The molecular weight of each peptide was confirmed by mass-spectral analysis (Sigma-Genosys). The synthetic peptides SVVYGLR, ELVTDFPTDLPATK, and ELVTEFPTELPATK were preincubated with cells at 250, 500, and 500 µg/ml, respectively under activating conditions in the presence of divalent cations for 15 min. Following the incubation period, cells were seeded, and the assay was performed as described above.

Direct Integrin Binding using Affinity Chromatography-- To illustrate the integrin-binding capacity of osteopontin, the synthetic peptides ELVTDFPTDLPATK and ELVTEFPTELPATK were coupled to cyanogen-bromide 4B (Sigma) at 5 mg/ml according to the manufacturer's instructions. Ramos cells (ATCC) were surface biotinylated as described (55), and a 50-µl pellet of cells was extracted with 1 ml of TBS containing 3% octylglucoside (ICN, Irvine, CA) in 1.5 mM Mg2+, 1.5 mM Mn2+, and 10-3 M phenylmethane sulfonic acid. The HL-60 cell extracts were agitated at 5-10 min intervals with Sepharose columns (0.5 ml) over a 2-h period at 0 °C. The columns were washed with 5 ml of TBS containing 3% octylglucoside, 1.5 mM Mg2+, and 1.5 mM Mn2+. This was followed with a 15-ml wash in TBS containing 1% octylglucoside, 1.5 mM Mg2+, and 1.5 mM Mn2+. Integrins were eluted with 2 ml of TBS with 1% octylglucoside + 10 mM EDTA (0.25-ml fractions). 40 µl of each fraction were loaded and run under nonreducing conditions on a 7% acrylamide gel and transferred to polyvinylidene difluoride membrane (Millipore). The membrane was blocked overnight at 4 °C with 5% milk in 0.1% Tween 20 saline containing 2.5 mM Tris-HCl, pH 7.5. Blots were washed and streptavidin alkaline phosphatase (Sigma) was added (1:1000) to 1% BSA in Tween 20 saline and incubated for 30 min followed by a 30-min wash in Tween 20 saline. The alkaline phosphatase activity was developed using alkaline phosphatase development kit (Bio-Rad) and stopped with water.

Integrin Immunoprecipitation-- Integrins that bound to the OPN-Sepharose column were identified using immunoprecipitation. Sepharose beads conjugated with goat anti-mouse IgG (Sigma) were rinsed and suspended 1:1 with 0.5% Triton X-100 in TBS. In 1.5-ml microcentrifuge tubes, 200 µl of the bead mixture was added to 5 µg of monoclonal antibodies against several human integrin subunits including alpha 4 (HP2/1, Immunotech) (56), beta 1 (mAb13, Becton-Dickinson) (57), and alpha 5 (IIA1, PharMingen) (58). These mixtures were then combined with 280 µl of pooled EDTA eluate from OPN-Sepharose and 700 µl of 0.5% Triton X-100 in TBS. This mixture was rotated continuously at 4 °C overnight after which time tubes were centrifuged and rinsed six times with 1 ml of 0.5% Triton X-100 in TBS. 75 µl of 2× sample buffer was added to the beads, and this mixture was boiled for 5 min. 30-µl samples were run on 7% SDS-PAGE under nonreducing conditions, and blots were developed as described above.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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To rule out the involvement of the RGD site in alpha 4beta 1-dependent adhesion to OPN, the wild-type, RGD-containing N-terminal thrombin fragment (rOPN-(17-168)) and an RGE mutant ([Glu161]rOPN-(17-168)), where Asp161 was mutated to Glu161, were produced. Each clone is described as rOPN followed by the amino acids coded for in the construct (e.g. 17-168) and finally the mutation incorporated into the clone (e.g. Glu161 for Asp161 mutated to Glu161). Clones were sequenced to confirm successful mutation, and recombinant proteins (>95% purity) were analyzed using SDS-PAGE (not shown). Western blotting experiments using a monoclonal antibody directed to the N-terminal histidine tag revealed a pattern exactly matching staining results (not shown). Proteins were tested for their ability to promote alpha vbeta 3-dependent attachment (Fig. 2A). Wild-type rOPN-(17-168) promoted endothelial cell attachment dose dependently, whereas no attachment occurred to the RGE mutant. This confirmed successful functional mutation of the RGD site in OPN. The ability of both recombinant OPN constructs to promote alpha 4beta 1-dependent adhesion was compared using the HL-60 promyelocytic cell line in the presence of physiologic divalent cations (2 mM Ca2+, 1 mM Mg2+). As shown in Fig. 2B, no differences were observed in the ability of either construct to promote HL-60 cell attachment. Additionally, both rOPN-(17-168) and [Glu161]rOPN-(17-168) were comparable with bovine OPN with respect to their ability to promote alpha 4beta 1-dependent cell attachment (Fig. 2C). Adhesion to both native OPN and recombinant constructs was completely inhibited by the alpha 4beta 1-specific LDV peptide. The control peptide, LEV, had lesser effects compared with control (no peptide). Minimal adhesion was observed to the BSA substrate. Collectively, these data indicate that the RGD site in the N terminus of OPN is not involved in alpha 4beta 1-dependent adhesion to recombinant OPN, as rOPN-(17-168) and [Glu161]rOPN-(17-168). Consequently, subsequent constructs described contain the RGE mutation (Glu161) to rule out any influence from the RGD site in OPN, although the presence of this mutation is not reflected to simplify nomenclature.


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Fig. 2.   Characterization of recombinantly produced N-terminal thrombin fragment of OPN. A, dose-response curves comparing the ability of wild-type rOPN-(17-168) (17-168) and [Glu161]rOPN-(17-168) (17-168E161) mutant to promote RGD-dependent attachment of human endothelial cells. B, dose-response curves comparing the ability of wild-type rOPN-(17-168) and [Glu161]rOPN-(17-168) mutant to promote alpha 4beta 1-dependent attachment of HL-60 cells. C, peptide-blocking data indicating HL-60 cell adhesion to native and recombinantly produced OPN occurs through the alpha 4beta 1 integrin. Experiments were conducted in the presence of 250 µg/ml of the LDV and LEV peptides. Experiments were performed as described under "Experimental Procedures." bOPN, bovine OPN; BSA, control substrate. The data shown are representative experiments (n = 3) performed in triplicate wells, and values shown are mean absorbance ± S.D.

Various deletions of the OPN molecule were introduced in an attempt to localize alpha 4beta 1 binding sites. SDS-PAGE analysis of the recombinant proteins revealed ~95% purity as visualized by Coomassie Blue staining, and all proteins contained an N-terminal histidine tag by Western blot analysis (data not shown). The rOPN-(55-168), rOPN-(17-129), rOPN-(17-138), rOPN-(17-142), rOPN-(17-164), and rOPN-(17-168) constructs were tested for the ability to promote alpha 4beta 1-dependent attachment of HL-60 cells (Fig. 3). These experiments revealed that removal of the first 39 amino acids (rOPN-(55-168)) did not reduce cell binding compared with the entire thrombin fragment, rOPN-(17-168). All binding activity was removed by truncation of amino acids 130-168 (rOPN-(17-129)), with partial activity returning in the presence of amino acid residues 130-142 (rOPN-(17-138) and rOPN-(17-142)). In addition, decreased adhesion occurred by deletion of the last 4 amino acids (rOPN-(17-164)), which was previously shown to eliminate the alpha 9beta 1 integrin binding site in OPN (59).


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Fig. 3.   Deletion analyses of the N-terminal thrombin fragment of OPN indicating the ability of constructs to promote alpha 4beta 1-dependent attachment of HL-60 cells. Proteins were coated at concentrations of 5 µg/ml, and cell-binding assays were conducted and quantitated as described under "Experimental Procedures." Data shown are from a representative experiment (n = 4) performed in triplicate wells. Data shown are absorbance readings normalized to control binding, rOPN-(17-168), ± S.D.

A more detailed analysis of binding activity was conducted with the constructs shown in Fig. 1B. Dose-response curves illustrating the ability of constructs to promote HL-60 cell attachment are shown in Fig. 4. No binding was observed with truncation of the last 38 amino acids (rOPN-(17-129)), and identical results were observed with the rOPN-(17-135) construct. Partial activity was restored with the addition of amino acids 130-138 (rOPN-(17-138)) indicating the presence of a potential binding site. Activity remained at similar levels with the addition of amino acids 139-164 (rOPN-(17-142), rOPN-(17-150), rOPN-(17-164)). Only in the presence of the last 4 amino acids did full activity return (rOPN-(17-168)). Thus, deletion of the last 4 amino acids on the C terminus of the thrombin fragment of OPN partially eliminated the ability of alpha 4beta 1-dependent cell attachment to occur. The remainder of binding activity was completely removed with further truncation of the molecule by ending at amino acid residue 129. These data strongly support the concept that two binding sites exist within residues 130-168. To examine in more detail whether the Asp135 and Asp139 residues located within the upstream binding region identified were important in alpha 4beta 1-dependent cell attachment, additional constructs incorporating mutations were generated (Fig. 4). The [Glu135]rOPN-(17-138) exhibited reduced adhesion compared with rOPN-(17-138). The same was true for [Glu139]rOPN-(17-142) versus rOPN-(17-142). Interestingly, neither of the mutations completely abolished adhesion, indicating that the conservative substitution of glutamate for aspartate did not remove all activity. Collectively, these data indicate that there are dual alpha 4beta 1 binding sites in the N-terminal thrombin fragment of OPN.


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Fig. 4.   Deletion analyses of the N-terminal thrombin fragment of OPN. Ability of constructs to promote alpha 4beta 1-dependent attachment of HL-60 cells. Proteins were coated at 20 µg/ml and serially diluted 2-fold. Cell binding assays were conducted and quantitated as described under "Experimental Procedures." Data shown are from a representative experiment (n = 4) performed in triplicate wells. Data shown are actual absorbance readings ± S.D.

To confirm both sequences in osteopontin were capable of binding the alpha 4beta 1 integrin, the synthetic ELVTDFPTDLPATK and SVVYGLR peptides, corresponding to amino acids 131-143 (with a C-terminal lysine residue added) and 162-168, respectively, were tested for their ability to block alpha 4beta 1-dependent attachment (Fig. 5). Also, the synthetic peptide ELVTEFPTELPATK was created containing two conservative Asp to Glu mutations to further examine whether the aspartate residues were involved in binding to alpha 4beta 1. In the presence of both the ELVTDFPTDLPATK and SVVYGLR peptides, alpha 4beta 1-dependent adhesion was inhibited significantly compared with control (p < 0.001). The ELVTEFPTELPATK peptide consistently had smaller effects compared with control, similar to that observed with the LEV peptide (see Fig. 2C).


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Fig. 5.   Peptide blocking data confirming the presence of dual alpha 4beta 1 integrin binding sites in OPN. Activated HL-60 cells were preincubated for 15 min at 37 °C with ELVTDFPTDLPATK (500 µg/ml), ELVTEFPTELPATK (500 µg/ml), and SVVYGLR (250 µg/ml) peptides, added at equimolar concentrations. Control indicates the absence of peptide. Experiments were performed as in Fig. 3. Values represent averaged absorbance readings compared with control from four separate experiments performed in triplicate wells ± S.D. *, p < 0.001 compared with control using Student's t test.

As further evidence for direct interaction of amino acids 131-143 in OPN with the alpha 4beta 1 integrin, affinity chromatography experiments were performed using surface-labeled Ramos cell extracts (Fig. 6A). Wild-type ELVTDFPTDLPATK-Sepharose, aspartate mutant ELVTEFPTELPATK-Sepharose and blank-Sepharose beads were incubated with labeled extracts, and EDTA elutions were collected and analyzed (E1-E4). Results show strong binding of the alpha 4beta 1 integrin to the wild-type peptide, whereas minimal binding occurred to the aspartate mutant. No binding of the alpha 4beta 1 integrin was observed using blank-Sepharose. Immunoprecipitation of fractions from both wild-type and mutated peptide columns revealed the presence of alpha 4 and beta 1 integrin subunits (Fig. 6B), although much greater binding occurred to the wild-type peptide. Mutation of aspartate residues resulted in a reduced ability of alpha 4beta 1 to bind but did not completely eliminate binding activity. In both experiments, control integrin antibodies failed to immunoprecipitate integrins. Similar results were observed using surface-labeled HL-60 cell extracts (data not shown).


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Fig. 6.   Affinity chromatography data illustrating binding of the alpha 4beta 1 integrin to synthetic OPN peptides. A, ELVTDFPTDLPATK-, blank-, and ELVTEFPTELPATK-Sepharose columns were incubated with surface-biotinylated Ramos cell extracts as described under "Experimental Procedures." Half-ml EDTA elution fractions were collected, analyzed using Western blots, and developed for alkaline phosphatase activity. Elution pattern for all columns is shown (E1-E4). Upper arrows denote the alpha  subunit, whereas lower arrows denote the beta  subunit. B, immunoprecipitations were performed with fractions from both ELVTDFPTDLPATK- and ELVTEFPTELPATK-Sepharose columns. Monoclonal antibodies directed to the integrin subunits tested for are indicated above each figure. These are empty  (no antibody), alpha 4-subunit (HP2/1), beta 1-subunit (mAb13) and alpha 5 (IIA1). Upper arrows denote the alpha 4-subunit, whereas lower arrows denote the beta 1-subunit.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Using sequential truncation analysis of the N-terminal thrombin fragment of OPN, we observed that dual alpha 4beta 1 integrin binding sites exist in a 38-amino acid C-terminal domain. Synthetic peptides encompassing either of these regions interfered with the ability of alpha 4beta 1-dependent attachment to occur, whereas a control peptide had lesser effects. Also, using affinity chromatography we were able to demonstrate direct binding of the alpha 4beta 1 integrin to the wild-type synthetic peptide coupled to sepharose, whereas a mutated peptide bound considerably less well. These results show that dual binding sites exist for the alpha 4beta 1 integrin in the N-terminal thrombin fragment of OPN.

Integrin Binding Sites in Osteopontin-- Numerous members of the integrin family have been reported to interact with OPN including alpha vbeta 3 (51, 60-62), alpha vbeta 1 and alpha vbeta 5 (63, 64), alpha 4beta 1 (14), alpha 5beta 1 (65), alpha 8beta 1 (66) and alpha 9beta 1 (67). The RGD site (68) has been reported to interact with the alpha vbeta 3, alpha vbeta 1, alpha vbeta 5 and alpha 5beta 1 integrins (51, 60-62). The alpha 9beta 1 integrin has been shown to bind the SVVYGLR amino acid sequence (59), which comprises the last 7 C-terminal amino acids in the thrombin fragment of OPN. These results are interesting based on sequence homology in that the alpha 9 integrin subunit is most closely related to the alpha 4 integrin subunit (69). Both the alpha 4beta 1 and alpha 9beta 1 integrins have the ability to interact with VCAM-1 (11, 70) and OPN (14, 59, 67), as well as sharing other common ligands (71). Here, we found that deletion of the four C-terminal amino acids of the N-terminal thrombin fragment of OPN reduced but did not eliminate alpha 4beta 1-dependent adhesion. Our result contrasts with those observed previously for alpha 9beta 1 interaction with OPN where deletion of the YGLR sequence completely eliminated alpha 9beta 1-dependent cell attachment, and the synthetic SVVYGLR peptide completely blocked alpha 9beta 1-dependent cell attachment (59). Previous work by Barry et al. (72) demonstrated that the synthetic SVVYGLR peptide also interfered with alpha 4beta 1-dependent cell adhesion, reporting that the SVVYGLR site in OPN is a binding site for the alpha 4beta 1 integrin. In the same study, using recombinantly produced peptide sequences of the OPN molecule coupled to GST, it was postulated that an additional binding site may exist for alpha 4beta 1, yet this was not demonstrated convincingly since the FPTDLPA synthetic peptide used in the study failed to block adhesion (72). The work presented here confirms that the SVVYGLR site in the N-terminal thrombin fragment of OPN is a binding site for the alpha 4beta 1 integrin. Truncation of the last 4 amino acids, YGLR, resulted in ~50% decrease in adhesion, and the synthetic SVVYGLR peptide significantly blocked alpha 4beta 1 cell attachment. We also define a second binding site from amino acids 131-143, ELVTDFPTDLPAT, as demonstrated by truncation analyses, peptide blockade, and direct integrin binding. Interestingly, this peptide contains a tandem sequence consisting of two copies of a consensus Asp-hydrophobic residue-Pro that is similar to the alpha 4beta 1 binding site LDVP seen in CS-1 FN (10). In addition, the related alpha 4beta 1 binding sequence hydrophobic residue-Asp-X-Pro is seen in other alpha 4beta 1 ligands such as QIDSPL in VCAM-1 and IDAPS in FN (15-20). The one common feature of these sequences is the presence of a proline residue two-amino acids downstream of an aspartate residue.

Additionally, we present evidence for the direct involvement of aspartate residues in the affinity of alpha 4beta 1 for ELVTDFPTDLPAT based on evidence that a mutant synthetic peptide, containing glutamate substituted for aspartate residues, was less effective at blocking cell adhesion. It also minimally bound the alpha 4beta 1 integrin in affinity chromatography experiments. Conservative substitution of glutamate for aspartate residues did not remove 100% activity in either peptide blocking studies or direct integrin binding experiments. These results are consistent with previous data from our laboratory where the control LEV peptide also had slight effects (Ref. 14 and Fig. 2C). These results correlate with previous evidence that the alpha 4beta 1 integrin recognizes a wide variety of motifs in FN, VCAM-1, OPN and denatured proteins (10, 15-21). As was previously suggested, this integrin shows a broader ligand binding specificity than most other members of the integrin family (13).

Evidence That the Thrombin Fragment of OPN Is a Matricryptin-- During tissue injury, considerable alterations occur in the ECM because of enzymatic breakdown, multimerization, adsorption, mechanical forces, or denaturation to expose matricryptic sites (73). Matricryptic sites are defined as biologically active sites that are not revealed in the mature secreted form of the ECM molecules, but become exposed after structural or conformational alterations, and matricryptins represent biologically active fragments of ECM that contain exposed matricryptic sites (73). Ample evidence exists for the presence of both OPN and thrombin in injury sites (38, 74), therefore increasing the likelihood that thrombin cleavage of OPN occurs in these settings (38, 74). Several studies support the contention that exposure of matricryptic sites occurs within OPN following thrombin cleavage (Refs. 38, 62, 65, 67, 75; Fig. 7). Senger et al. (62) have found the thrombin fragment of OPN is more potent at promoting RGD-dependent attachment than the native protein. The immobilized thrombin fragment of OPN also stimulated greater haptotactic migration of tumor cells compared with the intact molecule (75). The alpha 9beta 1 (67) and alpha 5beta 1 (65) integrins were unable to bind OPN without thrombin cleavage. These data support the concept that matricryptic sites liberated in OPN following thrombin cleavage may be important in regulating inflammatory cell interactions with OPN in the wound environment.


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Fig. 7.   Schematic diagram of the entire human OPN molecule illustrating the localization of known integrin binding sites. OPN contains a poly(aspartate) region, a thrombin proteolytic cut site, and integrin binding sites. The ELVTDFPTDLPAT, RGD, and SVVYGLR sites are located in a 38-amino acid region just upstream from the thrombin cut site. Sequences of human (48), bovine (76), porcine (77), and rat OPN (68) are provided. Shaded boxes indicate three known regions of OPN with affinity for integrins. The open boxes indicate areas of sequence homology in the intervening amino acids between integrin binding sites.

An interesting feature of the above findings is that all three known integrin binding sites are located within a very limited 38-amino acid region of the thrombin fragment of OPN. The ELVTDFPDLPAT (shown here), RGD (51, 60-62, 67) and SVVYGLR sites (shown here and in Refs. 59, 72) are all localized to a 38-amino acid region just proximal to the thrombin cleavage site (Fig. 7). These sequences are highly conserved, particularly in large species of mammals, whereas they are less conserved or absent in rodents, particularly concerning the upstream binding site. The thrombin fragment appears to have altered biological activity compared with intact OPN and contains matricryptic sites (62, 65, 67, 73, 75). Localization of these integrin binding sites directly adjacent to the thrombin cleavage site strongly implicate the physiological importance of this region of osteopontin in inflammatory and wound repair responses.

    ACKNOWLEDGEMENTS

We would like to thank Dr. Nicholas Kovach for the kind gift of 8A2 antibody, Dr. Doug Struck for assistance in production and purification of recombinant proteins, and Dr. Ry Young for the kind gift of slyD-deficient E.coli strain.

    FOOTNOTES

* This work was supported by National Institutes of Health Grant HL59971 (to G.E.D.).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.

Dagger To whom correspondence should be addressed: Dept. of Pathology and Laboratory Medicine, Texas A&M University Health Science Center, 255 Reynolds Medical Bldg., College Station, TX 77843-1114. Tel.: 979-845-0823; Fax: 979-862-1229; E-mail: gedavis@tamu.edu.

Published, JBC Papers in Press, January 25, 2001, DOI 10.1074/jbc.M011392200

    ABBREVIATIONS

The abbreviations used are: OPN, osteopontin; TBS, Tris-buffered saline; PSA, Puck's Saline A, BSA, bovine serum albumin; Tween 20, polyoxyethylene sorbitan monolaurate; PAGE, polyacrylamide gel electrophoresis.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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