extfieldsofstudy: Medicine Chemistry

text:
The cell adhesion domain in plasma vitronectin is cryptic.

Vitronectin (Vn) is a major adhesive glycoprotein in blood. However, many of the functions of Vn are regulated by its conformational state and degree of multimerization. Here, the ability of native and denatured Vn to bind to integrin adhesion receptors was compared. Three lines of evidence suggest that the native, plasma form of Vn is not an adhesive glycoprotein. (i) Antibodies that bind in close proximity to the cell adhesion domain of Vn fail to bind to native Vn present in unfractionated plasma. (ii) Denatured Vn binds to both glycoprotein IIb/IIIa and alphavbeta3 in a dose-dependent manner. In contrast, native Vn is unable to bind either integrin. (iii) Thermal denaturation of native Vn, or its complexation with type 1 plasminogen activator inhibitor, exposed the cell adhesion domain of Vn. Thus, while plasma Vn is unable to bind integrins and is not an adhesive glycoprotein, the conformationally altered from of the protein binds avidly to both alphavbeta3 and glycoprotein IIb/IIIa. The data presented here indicate that such conformational changes in Vn are likely to occur in areas of tissue injury and thrombosis.

Vitronectin (Vn) is a major adhesive glycoprotein in blood. However, many of the functions of Vn are regulated by its conformational state and degree of multimerization. Here, the ability of native and denatured Vn to bind to integrin adhesion receptors was compared. Three lines of evidence suggest that the native, plasma form of Vn is not an adhesive glycoprotein. (i) Antibodies that bind in close proximity to the cell adhesion domain of Vn fail to bind to native Vn present in unfractionated plasma. (ii) Denatured Vn binds to both glycoprotein IIb/IIIa and ␣ v ␤ 3 in a dose-dependent manner. In contrast, native Vn is unable to bind either integrin. (iii) Thermal denaturation of native Vn, or its complexation with type 1 plasminogen activator inhibitor, exposed the cell adhesion domain of Vn. Thus, while plasma Vn is unable to bind integrins and is not an adhesive glycoprotein, the conformationally altered from of the protein binds avidly to both ␣ v ␤ 3 and glycoprotein IIb/IIIa. The data presented here indicate that such conformational changes in Vn are likely to occur in areas of tissue injury and thrombosis.
Vitronectin (Vn), 1 an adhesive glycoprotein present in blood and in a variety of tissues, belongs to a group of molecules that contain the RGD cell adhesion sequence and play key roles in the attachment of cells to their surrounding matrix (1,2). In addition, Vn has several other functions in the complement, coagulation, and fibrinolytic system. For example, Vn is identical to the S protein of the complement system and thus inhibits complement-mediated cell lysis (3,4). Vn competes with heparin binding to antithrombin III, thereby preventing the rapid inactivation of thrombin and factor Xa by this protease inhibitor (5,6). Vn also binds to and stabilizes the biological activity of type 1 plasminogen activator inhibitor (PAI-1), the physiological inhibitor of both tissue-and urinary-type plasminogen activators (7,8). Thus, Vn provides unique regulatory links between cell adhesion and proteolytic enzyme cascades, although it is not clear how the multiple functions of Vn are coordinately regulated.
Vn is a conformationally labile molecule, and this lability is likely to play a large role in regulating the functions of the protein. This notion is based on the observation that different preparations of Vn exhibit differing abilities to bind to the glycosaminoglycan heparin. Only 2% of the Vn in plasma is capable of binding to heparin-Sepharose, whereas this fraction is increased to 7% by the generation of serum (9). In addition, the formation of complexes of Vn with PAI-1 (10), thrombinantithrombin III (11), or complement C5b-C9 (12) induces conformational changes in the molecule, which expose the heparin binding domain. Moreover, conformational changes are also induced by denaturation with chaotropic agents, heat-treatment and acidification. These changes are accompanied by the spontaneous formation of disulfide-linked multimers of Vn. Using a panel of conformationally sensitive antibodies, we provided evidence that conformational changes within Vn are not limited to the heparin binding domain, but also occur in the N-terminal half of the molecule, including the N-terminal somatomedin B (SMB) domain (13), a site that encompasses the RGD sequence.
Understanding the interrelationship between the binding domains of Vn has been complicate, because the exposure and function of these sites depends on the conformational state of this molecule. For example, a number of ligands interact preferentially with the conformationally altered and multimeric (i.e. denatured form) of Vn. These include collagen (14), glycosaminoglycans (15,16), ␤-endorphin (17), PAI-1 (18), and urokinase receptor-urokinase complex (19,20). In contrast, thrombin-antithrombin III complexes appear to preferentially interact with native, plasma Vn (21). The function of native and denatured Vn in promoting cell adhesion is even more complicated. Native Vn was reported to bind specifically and saturably to glycoprotein (GP) IIb/IIIa on stimulated platelets (22)(23)(24). The binding of denatured Vn to platelets are conflicting (25). In addition, Vn has been reported to both block (26) and stimulate (23) platelet aggregation. The reasons for these discrepancies remain unclear.
Vn is one of many adhesive proteins that bind to cell surface receptors called integrins (27,28). Each integrin is a noncovalently associated ␣␤ heterodimer that spans the plasma membrane. To date, nine integrin ␣ subunits and 12 ␤ subunits have been identified. Since the ligand binding specificity of each receptor is conferred by its subunit composition, functional diversity within the family of integrins is achieved by heterologous pairing between subunits. Three different integrins have been identified as receptors for Vn, GPIIb/IIIa (also known as ␣II␤ 3 (29)), ␣ v ␤ 3 , expressed by endothelial cells and implicated in angiogenesis (30), and ␣ v ␤ 5 (31). Vn contains a single RGD sequence located adjacent to the SMB domain (3,4). Site-directed mutagenesis of the RGD motif shows that this sequence is required for cell adhesion and can not be compensated for by other parts of the molecule (32,33). A number of reports indicated that the cell adhesion domain is exposed on both native and denatured Vn (1,34). Consequently, the adhesive capacity of the protein is not thought to be regulated by the conformational lability of the molecule. However, this concept is challenged by two observations. First, only denatured Vn induces tyrosine phosphorylation in endothelial cells by binding to the ␣ v ␤ 3 integrin on the cell (35). Second, ␣ v ␤ 5 -dependent endocytosis of Vn by skin fibroblasts was only observed using denatured Vn, the native form of Vn is not internalized (36,37).
In the present report, we compare the binding of native and denatured Vn to purified GPIIb/IIIa and ␣ v ␤ 3 . Evidence is provided that native Vn is incapable of binding to integrin adhesion receptors and that its RGD sequence is not exposed. Conformational changes in Vn, induced by chemical or thermal denaturation, or by complexation with PAI-1, expose the integrin binding site on Vn, allowing it to bind tightly to both ␣ v ␤ 3 and GPIIb/IIIa.

MATERIALS AND METHODS
Proteins and Antibodies-Denatured Vn was purified by heparin affinity chromatography in the presence of 8 M urea (38). Vn was also isolated under nondenaturing conditions (i.e. native Vn) according to published procedures (39). Protein concentrations were determined by the bicinchoninic acid method (Pierce). The final Vn preparations were devoid of fibrinogen and fibronectin immunoreactivity as judged by immunoblotting using rabbit anti-human fibrinogen or fibronectin (Calbiochem). Denatured Vn was biotinylated using NHS-LC-biotin (Pierce). Briefly, 1.5 mg of Vn was dissolved in 50 mM sodium phosphate buffer, pH 8.5, at 20 M and incubated with 3 mg of biotinylation reagent dissolved in 100 l of Me 2 SO for 3 h at 37°C, followed by extensive dialysis against phosphate-buffered saline (PBS). GPIIb/IIIa was obtained from Enzyme Research Laboratories, and ␣ v ␤ 3 was isolated from human placenta extracts by affinity chromatography (40). Human fibrinogen was obtained from Calbiochem. PAI-1 was purified as described (41), activated with 4 M guanidinium hydrochloride (42), dialyzed against 5 mM sodium phosphate, 1 mM EDTA, 0.345 M NaCl, pH 6.5, to stabilize the biological activity of PAI-1 (43), and the specific activity was determined by titration against urinary-type plasminogen activator (Calbiochem). Prior to each experiment, PAI-1 was dialyzed into ice-cold PBS. Latent PAI-1 was prepared by incubation of activated PAI-1 at 37°C for 24 h. After this incubation step, the structural integrity of the resulting PAI-1 preparation was confirmed by SDSpolyacrylamide gel electrophoresis followed by staining with silver nitrate. The remaining PAI-1 activity after this incubation was determined by binding to immobilized tissue-type plasminogen activator (Calbiochem) (44) and was less than 0.5% of the starting material (not shown). Monoclonal antibodies (mAbs) 611, 153, and 1244 were obtained by standard hybridoma techniques using denatured Vn as immunogen (41). IgG was produced in mice as ascites fluid and purified by using protein A-Sepharose. The generation of rabbit anti-human Vn was described previously (41). Biotin-labeled secondary antibodies, streptavidin alkaline phosphatase, and substrate were from Zymed. Conformational changes in the Vn molecule were quantified by competitive enzyme-linked immunosorbent assay (13). Briefly, microtiter wells were coated with denatured Vn (1 g/ml in PBS, 4°C, 16 h), and nonspecific binding sites on the plastic dishes were blocked by incubating the washed wells for 1 h at 37°C in PBS containing 3% casein and 0.05% Tween 20. The wells were co-incubated for 1 h with a constant amount of IgG (50 ng/ml final concentration) and the indicated concentration of Vn in PBS containing 0.1% casein and 0.1% Tween 20. Bound IgG was detected with biotin-conjugated goat anti-mouse IgG, followed by streptavidin alkaline phosphatase conjugate and the chromogenic substrate p-nitrophenyl phosphate (Zymed). The change of absorbance at 405 nm of duplicate wells was determined and the absorbance of wells not incubated with antibodies but otherwise treated identically was subtracted.
Effect of mAbs to Vn on Cell Adhesion-The human fibrosarcoma cell line HT 1080 was obtained from ATCC and maintained in Dulbecco's modified Eagles's medium/F-12 medium containing 10% fetal bovine serum as described (45). Semiconfluent cultures were labeled for 16 h with [ 35 S]methionine (Amersham Corp; 100 Ci/ml in 80% methioninedeficient medium, 20% complete growth medium). After washing with PBS, the cells were detached by treatment with PBS containing 0.2 mM EDTA for 5 min and washed three times in adhesion buffer (1 ϫ Hanks' balanced salt solution, 50 mM Hepes, 1 mg/ml bovine serum albumin, 1 mM CaCl 2 , 1 mM MgCl 2 , 1 mM MnCl 2 , pH 7.4) and resuspended at 2 ϫ 10 5 cells/ml in adhesion buffer. Flat-bottomed microtiter wells were coated with 100 ng/well denatured Vn. Following an 18-h coating period at 4°C, the plates were blocked with PBS containing 10 mg/ml bovine serum albumin. Antibodies (50 l) were prebound to the wells at 37°C for 1 h. Cells (1 ϫ 10 4 ) were added to the antibody containing wells in triplicate and allow to adhere for 1 h. The wells were washed gentle three times in PBS to remove nonadherent cells, and the bound cells were solubilized in 1% SDS and quantified by ␤-counting. The radioactivity associated with bovine serum albumin coated wells was subtracted from that of the Vn-coated wells. Results are expressed as percentage cell binding in the absence of antibodies.
Protein Binding Analysis-The binding of purified Vn preparations to immobilized integrins was analyzed with the methods essential as described previously (46). Briefly, integrins were immobilized at 50 ng/well (␣ v ␤ 3 ) or 250 ng/well (GPIIb/IIIa) for 18 h at 4°C. Subsequently, nonspecific protein binding sites on the plates were blocked with 50 mM Tris, pH 7.4, containing 150 mM NaCl, 1 mM MgCl 2 , 1 mM CaCl 2 , 1 mM MnCl 2 (binding buffer), and 35 mg/ml bovine serum albumin. After washing with binding buffer, unlabeled Vn was added for 1 h at 37°C in binding buffer containing 0.1% bovine serum albumin. Bound Vn was detected using rabbit anti-human Vn IgG (10 g/ml), followed by biotinlabeled anti-rabbit IgG, streptavidin alkaline phosphatase, and substrate. The change of absorbance (405 nm) of duplicate wells was determined. Specific binding was determined by subtracting the binding to wells incubated with the same concentration of Vn in the presence of 10 mM EDTA. For competitive ligand binding studies, biotinlabeled denatured Vn (1 g/ml; a concentration required to obtain halfmaximal binding) was co-incubated (3 h, 37°C) with unlabeled native Vn, denatured Vn, or fibrinogen on integrin-coated plates (see above). After washing, bound Vn was detected using streptavidin alkaline phosphatase and substrate. Specific binding was determined by subtracting the binding of biotin-labeled wells incubated in the presence of 10 mM EDTA from that incubated in the presence of divalent cations. Results are expressed as percentage binding in the absence of soluble competitor.

Characterization of mAbs That Block Cell Adhesion to Vn-
The effects of three different anti-Vn mAbs on cell adhesion to immobilized Vn were determined (Fig. 1A). Microtiter wells were coated with denatured Vn and incubated with the indicated concentration of antibody and metabolically labeled HT 1080 cells. Cells were allowed to attach for 1 h. Bound cells were quantified by ␤-counting. mAb 153 and 611 inhibited cell adhesion to Vn in a dose-dependent manner, whereas mAb 1244 was significantly (approximately 20-fold) less effective. The observed difference was apparently not due to a lower affinity of mAb 1244 for denatured Vn, since this antibody and clone 153 differ in the affinity for denatured Vn only by factor 2 (41). In contrast, no inhibition was observed using normal mouse IgG (data not shown). The mAbs 153 and 611 bind to Vn polypeptides encompassing amino acids 1-51, a fragment of Vn containing the RGD sequence, whereas the control antibody interacts with amino acids 52-239 (41) (data not shown).
We previously provided evidence that an immunoepitope (mAb 153) located in the SMB domain of Vn (amino acids 1-51) is cryptic in plasma Vn (13). Experiments were performed to understand whether lack of reactivity with native plasma Vn is a general feature of antibodies derived to this region of the Vn molecule. Competitive enzyme-linked immunosorbent assays were employed to determine the reactivity of mAB 611 with purified preparations of native and denatured Vn and Vn present in unfractionated plasma (Fig. 1B). Results were compared with mAb 153. Microtiter wells coated with denatured Vn were incubated with a constant amount of anti-Vn IgG and increasing concentrations of different types of Vn (Fig. 1B). Denatured Vn competes with the binding of both antibodies to immobilized denatured Vn in a dose-dependent manner, whereas neither antibody reacted well with Vn present in unfractionated plasma. The reactivity with purified native Vn with these two conformationally sensitive antibodies was at least 50-fold lower than that of denatured Vn.
Binding of Native and Denatured Vn to GPIIb/IIIa and ␣ v ␤ 3 -Based on the strong reactivity of the inhibitory antibodies with denatured Vn, and the inability of these antibodies to bind native Vn, we hypothesized that the RGD sequence in native Vn is cryptic. This hypothesis was tested by measuring the binding of native and denatured Vn to purified integrins (Figs. 2 and 3). To measure binding, microtiter wells were coated with purified integrin and then incubated with a range of either native or denatured Vn. Bound Vn was detected with an polyclonal antibody against Vn. Denatured Vn bound to GPIIb/IIIa in a dose-dependent manner and approached saturation at 5 g/ml. Specific binding did not further increase using 10 g/ml denatured Vn. In the concentration range presented in Fig. 2A, specific binding was more than 85% of total binding. In contrast, little binding of native Vn was observed under the same conditions.
To exclude the possibility that the lack of binding of native Vn to GPIIb/IIIa was due to reduced reactivity of the rabbit anti-Vn IgG with native Vn, competitive binding assays were employed to confirm the results of the direct binding studies (Fig. 2B). In this assay, immobilized GPIIb/IIIa was co-incubated with a constant amount of biotinylated denatured Vn and a range of competing native or denatured Vn. Fibrinogen was also used as a competitor, because it is the physiological ligand for GPIIb/IIIa. Fibrinogen was 10 -50-fold more efficient in competing with the binding of labeled Vn for GPIIb/IIIa than denatured Vn. More importantly, no competition was observed using native Vn, confirming the inability of the native form of Vn to bind to GPIIb/IIIa. GPIIb/IIIa is only one of several integrins that are reported to bind Vn. The ␣ v ␤ 3 integrin is also considered a receptor for Vn and was originally named the "vitronectin receptor." To determine whether the ␣ v ␤ 3 integrin also exhibited preferential binding to the denatured form of Vn, similar binding studies were performed using immobilized ␣ v ␤ 3 (Fig. 3A). Denatured Vn bound to ␣ v ␤ 3 in a dose-dependent manner and again approached saturation at 5 g/ml Vn. In contrast, native Vn showed little binding. Again, results were confirmed in competitive binding studies and compared with fibrinogen. Native Vn failed to compete with the binding of denatured Vn for binding to ␣ v ␤ 3 . Both denatured Vn and fibrinogen competed for the binding of biotinylated Vn to ␣ v ␤ 3 . Little difference was observed in the extent of competition between the latter two ligands.
Denaturation of Native Vn Exposes the RGD Sequence-Only about 7% of the total Vn present in plasma is recovered using a purification procedure that maintains the protein in its native conformation (39). This raised the possibility that a nonintegrin-binding subpopulation of native Vn could have been purified and employed in the binding studies. To address this issue, a heat denaturation procedure was employed to convert native Vn into conformationally altered, multimeric Vn (Fig. 4). Heat treatment of native Vn resulted in the exposure of the mAb 611 epitope (Fig. 4A) and mAb 153 epitope (not shown) to a similar extent as in denatured Vn. In addition, the electrophoretic mobility of heat-treated native Vn on native polyacrylamide gel electrophoresis was indistinguishable from denatured Vn (i.e. heat treatment resulted in the formation of high molecular weight multimers that failed to enter the separating gel) (not shown). The interaction of heat-treated native Vn with ␣ v ␤ 3 was compared with that of purified denatured Vn (Fig.  4B). Heat-treated Vn binds to immobilized ␣ v ␤ 3 in a dose-dependent manner similar to denatured Vn (Fig. 4B). In addition, heat-treated native Vn competes with labeled denatured Vn for binding to this integrin to an extent comparable with that of denatured Vn (Fig. 4C). Similar results were obtained when binding to GPIIb/IIIa was examined (not shown). In addition, an alternative denaturation procedure (i.e. treatment of native Vn with 8 M urea) also induced integrin binding (not shown). These observations indicate that denaturation of native Vn results in the exposure of the cryptic cell binding domain and that the inability of native Vn to bind to ␣ v ␤ 3 or GPIIb/IIIa was not due to the purification of a nonintegrin-binding subpopulation of Vn from plasma.
Effects of PAI-1 on the Adhesive Functions of Native and Denatured Vn-Vn binds to a number of plasma proteins, and these interactions alter the conformation of this molecule (10 -12). Experiments were performed to test whether physiological ligands of native Vn can induce the exposure of the cell adhesion domain. The binding of native Vn to active PAI-1 results in the formation of Vn multimers that express epitopes for conformationally sensitive antibodies like mAbs 153 (10) and 611 (data not shown). Interestingly, PAI-1⅐Vn complexes are relatively labile and PAI-1 readily dissociates from Vn, but Vn remains multimeric and conformationally altered (10). We reasoned that the binding of PAI-1 to native Vn is likely to expose the integrin binding site on Vn. Native Vn was incubated with a 2-fold molar excess of active PAI-1 for 16 h at 37°C. After this incubation period, more than 99% of the PAI-1 was converted into the latent conformation as based by binding to immobilized tissue-type plasminogen activator (not shown). Little competition of native Vn (Fig. 5A, left closed bar) for mAB 153 binding to immobilized denatured Vn was observed. Incubation of native Vn with active PAI-1 (Fig. 5A, left open bar) resulted in the exposure of the conformationally sensitive mAb 153 epitope. In contrast, no effects of latent PAI-1 on the exposure of this epitope was observed (Fig. 5A, left hatched bar). The mAb 153 does not detect Vn in complex with PAI-1 (41), con- , and compared with denatured Vn (closed circles). Results are expressed as percentage binding of control wells in the absence of soluble competitor. B and C, heat-treatment of native Vn induces the binding to ␣ v ␤ 3 . Native Vn was heat-treated as in A. In B, the binding of unlabeled Vn forms to ␣ v ␤ 3 was determined as in Fig. 3A. Results are expressed as mOD/min (change of absorbance at 405 nm). In C, the competition of unlabeled Vn conformers for the binding of biotinylated denatured Vn to ␣ v ␤ 3 was determined as in Fig. 3B. Results are expressed as percentage binding in the absence of soluble competitor. The designation of the symbols in B and C is as in A.
firming that the majority of the Vn was present in an uncomplexed, conformationally altered form. In parallel experiments, the effects of pretreatment of native Vn with PAI-1 on its ability to bind to ␣ v ␤ 3 (Fig. 5A, right bars) was analyzed. Again, while little binding of native Vn (Fig. 5A, right closed bar) to ␣ v ␤ 3 was observed, PAI-1 induced Vn multimers (A, right open bar) demonstrated increased binding to ␣ v ␤ 3 . In contrast, latent PAI-1 (Fig. 5A, right hatched bar) had failed to induce ␣ v ␤ 3 binding of native Vn.
The PAI-1 and integrin binding sites in denatured Vn are structurally distinct but located in close proximity, raising the possibility that PAI-1 may modulate the adhesive functions of denatured Vn. To test this hypothesis, microtiter wells were co-incubated with a constant amount of denatured Vn (which binds to ␣ v ␤ 3 ) and increasing concentrations of either active or latent PAI-1 in this binding assay (Fig. 5B). Active PAI-1 blocked the binding of denatured Vn to immobilized ␣ v ␤ 3 in a dose-dependent manner. At the highest concentration of PAI-1, approximately 80% inhibition was observed. The competition reached a plateau at a 10-fold molar excess of active PAI-1 over denatured Vn. In contrast, latent PAI-1 has little effect on the binding of Vn to ␣ v ␤ 3 . These findings are consistent with the observation that latent PAI-1 lacks high-affinity binding to denatured Vn (41). However, when Vn⅐␣ v ␤ 3 were preformed on microtiter wells, active PAI-1 failed to dissociated these complexes (data not shown). DISCUSSION The observations in this report demonstrate that native Vn, present in blood, is not an adhesive glycoprotein. This conclusion is based on the findings that antibodies against Vn that block its adhesive function fail to bind to the native form of Vn. These blocking antibodies are capable of binding to denatured Vn or PAI-1-induced Vn multimers. In addition, direct binding studies prove that denatured Vn binds to both GPIIb/IIIa and ␣ v ␤ 3 , but that native Vn is unable to bind to these integrins. Moreover, native Vn, which is not adhesive, could be converted into an adhesive glycoprotein by chemical and thermal denaturation. It should be noted that at high concentration, binding of native Vn to integrins was observed. This finding is probably related to the presence of some denatured Vn in the native Vn preparation. This conclusion is supported by the observation that unfractionated plasma was unreactive with conformationally sensitive antibodies, whereas the native Vn employed in this study showed exposure, although limited in comparison with denatured Vn, of conformationally sensitive epitopes. Based on the observations presented here, the majority of Vn present in vivo (i.e. in plasma) is not expected to bind to integrins. A number of consideration suggest that the adhesive functions of Vn are mediated through the N-terminal SMB domain rather then the C-terminal glycosaminoglycan binding domain. First, site-directed mutagenesis studies revealed that the RGD motif located between amino acids 45 and 47 is required for cell adhesion to Vn (32,33). Second, heparin does not effect the adhesion of HT 1080 or a number of other cell lines to immobilized Vn. 2 Similarly, heparin was without effect in the purified receptor binding assay. Third, recent studies from our laboratory indicate that native Vn binds heparin, implying that the glycosaminoglycan binding domain is exposed in the native conformation. 3 Interestingly, PAI-1 can have opposing effects on the adhesive properties of Vn. First, active PAI-1 blocked the binding of denatured Vn to ␣ v ␤ 3 in a dose-dependent manner. The binding site for active PAI-1 has been localized to amino acids 1 to 40 (41), a region in close proximity to the RGD sequence at residues 45-47 of Vn (3,4). RGD-containing peptide has no effect on the binding of PAI-1 to Vn (41), suggesting that PAI-1 is not binding directly to the cell adhesion domain in Vn. The close proximity of both sides makes it likely that the competition of PAI-1 is due to steric hinderance. In contrast, latent PAI-1, which does not bind to the N-terminal high affinity PAI-1 binding site in Vn (41), failed to compete with integrin binding of Vn. In addition, PAI-1 failed to dissociate pre-existing Vn⅐␣ v ␤ 3 complexes. Thus, PAI-1 is not expected to detach anchored cells, but may modulate biological processes dependent on de novo formation of adhesive contacts between Vn and ␣ v ␤ 3 , including cell migration. These observations have been confirmed in a recent publication (47).
Second, active PAI-1 had an additional affect on Vn binding to integrins. PAI-1 induced the formation of conformationally altered Vn multimers, and these multimers present, after dissociation from PAI-1, an exposed cell adhesion domain. The Vn concentration in plasma is between 2. Results are expressed as percentage inhibition (mAb 153) or binding (␣ v ␤ 3 ) in comparison with denatured Vn (10 g/ml). Identical results were obtained in three independent experiments with a standard error of less than 10%. B, effects of PAI-1 on the binding of denatured Vn to ␣ v ␤ 3 . Increasing concentrations of active (closed circles) or latent (open circles) PAI-1 were co-incubated with denatured Vn (500 ng/ml) on ␣ v ␤ 3 -coated plates. Bound Vn was detected with rabbit anti-human Vn IgG, followed by biotin-labeled anti-rabbit IgG, streptavidin alkaline phosphatase, and substrate, Results are expressed as percentage binding in the absence of PAI-1. Closed squares, ␣ v ␤ 3 -coated wells were preincubated with denatured Vn (500 ng/ml), washed, and then incubated with the indicated concentration of active PAI-1 for 1 h at 37°C. Bound Vn was detected as above. tions, and for example endotoxinemia results in a dramatic up-regulation of plasma PAI-1 levels to 20 nM (48). However, these concentrations are still 100-fold lower than that of Vn. Using this ratio of PAI-1 and Vn, we were unable to detect PAI-1 induced Vn multimers (10). Thus, PAI-1 induced Vn multimerization in plasma is expected to have little affects on the adhesive properties of Vn. The scenario may be quite different in tissues and in platelets. PAI-1 and Vn are both contained within platelet ␣-granules (49,50), and platelets contain 26 nM Vn and 12 nM PAI-1 per 10 9 platelets (49,51,52). Interestingly, platelet Vn is present in high molecular weight multimers that express the mAB 153 epitope (50). It should be noted that the expression of the cryptic mAB 153 epitope appears to correlate with the adhesive properties of Vn. These observation suggest that platelet Vn is adhesion competent. Platelet-derived proteins concentrate up to 200-fold in areas of thrombosis, suggesting that platelet-derived adhesion competent Vn will accumulate in areas of tissue injury and thrombosis. Similarly, increased tissue-associated levels of PAI-1 have been reported in a number of pathophysiological conditions, including endotoxinemia (48), raising the possibility that PAI-1 will regulate the adhesive properties of Vn in inflamed tissues. Taken together, these observation support the concept that plasma Vn is in a latent form. Interactions with either free proteases inhibitors or inhibitors in complex with the respective target protease results in the exposure of the cryptic RGD motif.


extfieldsofstudy: Biology Medicine

s2fieldsofstudy: Biology Chemistry

text:
Presenilins are processed by caspase-type proteases.

Presenilin 1 (PS1) and presenilin 2 (PS2) are endoproteolytically processed in vivo and in cell transfectants to yield 27-35-kDa N-terminal and 15-24-kDa C-terminal fragments. We have studied the cleavage of PS1 and PS2 in transiently and stably transfected hamster kidney and mouse and human neuroblastoma cells by immunoblot and pulse-chase experiments. C-terminal fragments were isolated by affinity chromatography and SDS-polyacrylamide gel electrophoresis and sequenced. The processing sites identified in PS1 and PS2 (Asp345/Ser346 and Asp329/Ser330, respectively) are typical for caspase-type proteases. Specific caspase inhibitors and cleavage site mutations confirmed the involvement of caspase(s) in PS1 and PS2 processing in cell transfectants. Fluorescent peptide substrates carrying the PS-identified cleavage sites were hydrolyzed by proteolytic activity from mouse brain. The PS2-derived peptide substrate was also cleaved by recombinant human caspase-3. Additional processing of PS2 by non-caspase-type proteases was also observed.

Mutant presenilin 1 and 2 (PS1, PS2) 1 genes are linked to early-onset familial Alzheimer's disease (1)(2)(3)(4)(5). The PS gene products are integral membrane proteins localized in Golgi and ER compartments (6 -8). They share ϳ65% sequence identity and similar structural features, i.e. 6 -8 transmembrane domains, hydrophilic regions at their N and C termini, and a large hydrophilic loop C-terminal to transmembrane domain 6 (6,9,10). The loop and the N terminus are the regions of significant sequence divergence between PS1 and PS2. More than 30 mutations in PS1 and 2 mutations in PS2 have been genetically linked to early-onset FAD (3,4). The biological functions of PS currently remain unknown. Studies on the structural homologue in Caenorhabditis elegans, SEL-12, sug-gest a role in protein trafficking and in the Notch signaling pathway (11). Human carriers of PS FAD mutations show a small but significant increase (approximately 1.5-3-fold) in plasma levels of A␤ 1-42 compared with normal controls, whereas levels of A␤  are unchanged (12)(13)(14). A specific effect on ␥-secretase processing of ␤-amyloid precursor protein has been proposed, and this enhanced misprocessing of ␤-amyloid precursor protein is thought to be crucial for FAD pathogenesis. Recent experiments have indicated a regulatory role for PS2 in cell apoptosis. C-terminal fragment of mouse PS2 reportedly protected T-cells or PC12 cells from different apoptosis-inducing challenges, whereas the susceptibility to the same challenges was enhanced when full-length PS2 was overexpressed (15,16).
Extensive proteolytic cleavage of PS1 resulting in a 27-28-kDa N-terminal fragment and a 16 -17-kDa C-terminal fragment was reported for PS1-transfected COS-1 cells, mice transgenic for human PS1, and endogenous PS1 in human and murine tissues (12,(17)(18)(19). Proteolytic processing has also been reported for human PS2 resulting in a 35-kDa N-terminal fragment and a 20-kDa C-terminal peptide (3,10,13). In the present study we have identified proteolytic processing sites in PS1 and PS2 and demonstrate that processing occurs through caspase-type protease(s). We also observed additional, caspaseindependent cleavage products suggesting alternative pathways of presenilin processing.

EXPERIMENTAL PROCEDURES
Antibodies-Amino acids 1-80 of human PS2 (N terminus) and 270 -387 of murine PS2 (large hydrophilic loop between transmembrane domains 6 and 7) were expressed in Escherichia coli as (NANP) 19 -fusion proteins and used for immunization of mice. PST20 is a monoclonal antibody specific for the hydrophilic N-terminal region of human and mouse PS2. PST18, PST8, and PST10 react with the PS2 hydrophilic loop region, PST18 having preference for murine PS2. The rabbit antiserum 53217 is directed against the hydrophilic loop of PS2. The M2 is a commercially available anti-FLAG antibody (Kodak Scientific Imaging Systems Products).
Cloning and Expression of Mouse and Human Presenilins-Mouse and human PS1 and PS2 cDNAs were amplified by PCR from a mouse brain cDNA library in pcDNA1 and a human brain cDNA library in pACT2 (CLONTECH) and inserted into pcDNA3 as BamHI/XbaI fragments. FLAG epitopes (DYKDDDDK) were added at the C terminus using appropriate primers and PCR amplification. The respective PS1 and PS2 fragments were cloned into pcDNA3 (Invitrogen) and pSFV1 (Life Technologies, Inc.) for stable expression in mouse Neuro2a and transient expression in BHK21, respectively. BHK cells were transfected by electroporation (20 g of RNA per 10 7 cells) and harvested 20 -24 h later. For stable tetracycline-controlled expression in SK-N-MC cells, mouse PS2-FLAG and mouse PS2-N141I-FLAG were placed under control of the tTA-dependent promoter and cloned into * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ptetREP, a pREP9 (Invitrogen) derivative containing a modified tTA under control of the tTA dependent promoter 2 (20). Clones were selected in medium containing 1.5 mg/ml G418. For expression experiments, cells were grown with or without 1 g/ml tetracycline for 3 days.
Immunoblotting-BHK cells expressing the various PS2 or PS1 constructs were collected, washed in phosphate-buffered saline, and dissolved without heating in SDS sample buffer containing 8 M urea and 0.2 M dithiothreitol. The proteins were separated by SDS-PAGE and electrophoretically transferred to a nitrocellulose membrane. Blocking, incubation with the various antibodies, and ECL detection were performed according to the recommendation of the manufacturer (Amersham).
Isolation of the C-terminal Fragment and Sequencing-ϳ5 ϫ 10 8 BHK cells transfected with PS2-FLAG or PS1-FLAG constructs were washed in 5 mM Hepes, pH 7.2, 250 mM sucrose, 1 mM EDTA, 10 mM benzamidine, 100 units/ml aprotinin, 10 g/ml leupeptin, 10 g/ml pepstatin A, 1 mM phenylmethylsulfonyl fluoride (breaking buffer) and mechanically disrupted in a mini-bead-beater using 0.1-mm glass beads. After removing nuclei and large cell debris by low speed centrifugation a crude membrane fraction was collected at 100,000 ϫ g for 45 min and resuspended in breaking buffer. The suspension was adjusted to 0.5% SDS and incubated for 10 min at 37°C. The solution was then diluted 1:5 with 1.25 ϫ RIPA buffer, not containing SDS (25 mM Tris, pH 7.4, 171 mM NaCl, 12.5% glycerol, 0.625% sodium deoxycholate, 1.25% Triton X-100, 2.5 mM EDTA), and clarified by centrifugation at 100,000 ϫ g for 30 min. The clear supernatant was applied to an M2 anti-FLAG immunoaffinity column (0.5-ml bed volume) equilibrated in 1 ϫ RIPA buffer. After extensive washing with 1 ϫ RIPA buffer and phosphate-buffered saline containing 0.1% octyl glucoside the column was eluted with 100 mM glycine, pH 2.6, 0.1% octyl glucoside. Fractions containing the C-terminal fragment were concentrated, subjected to SDS-PAGE, and electrophoretically transferred to an Immobilon-P SQ membrane (Millipore). The membrane was stained with 0.1% Amido Black in 45% methanol, 7% acetic acid for 10 min, and the band corresponding to the PS2 fragment was cut out and N-terminally sequenced by Edman degradation in an ABI 490cLC sequencer. The same procedure was applied to isolate human PS2 from ϳ10 9 Neuro-2a cells except that a mixture of immobilized antibodies PST8, PST10, and PST18 was used to affinity purify the C-terminal fragment.
Expression of Recombinant Caspase-3-The active form of human caspase-3 was amplified by PCR from Ser 29 to the end of the coding region and inserted as a BamHI fragment into pET15b (Novagen). Transformants in E. coli BL21(DE3)pLysS (Novagen) were grown in 5 ml of LB medium, induced at A 600 ϭ 0.6 with 0.2 mM isopropyl-1-thio-␤-D-galactopyranoside for 2 h before extracts were prepared by sonification in 0.5% Nonidet P-40, 20 mM Hepes (pH 7.4), 100 mM NaCl (21).
Caspase Activity Assay-5 g of E. coli extract containing recombinant caspase-3 or 0.5 mg of mouse brain homogenate were incubated with 20 M aminomethylcoumarin (AMC) tetrapeptide substrates in 100 mM Hepes, pH 7.0, 5 mM dithiothreitol, 0.5 mM EDTA, 0.1% CHAPS, 20% glycerol at 25°C. Total volume was 1 ml. The release of AMC as a function of time was measured in a fluorescence spectrophotometer using 380 nm and 460 nm as excitation and emission wavelengths, respectively. Background reagent fluorescence was determined and subtracted. The relative fluorescence intensities were correlated with AMC concentrations by standard curve titrations. In the inhibition experiments the release of AMC from Ac-DSYD-AMC was measured in the presence of different concentrations of the tetrapeptide aldehydes after incubation for 3 h (E. coli extract) or 4.5 h (brain homogenate) at 25°C.

RESULTS AND DISCUSSION
Analysis of PS2 Processing Pattern-We have studied the expression and processing of murine and human PS2 and PS1 constructs tagged with a C-terminal FLAG epitope in transient and stable transfectants. Immunoblot experiments with the M2 anti-FLAG antibody confirmed expression of full-length murine PS2 in Semliki Forest virus (SFV) transfected BHK cells migrating at around 55 kDa and the presence of a major C-terminal fragment at around 18 kDa (Fig. 1A). This fragment was recognized by the monoclonal antibody PST18 directed against the C terminus of PS2. Using monoclonal antibody PST20 directed against the N terminus of PS2, a major Nterminal fragment migrating at ϳ34 kDa and a weaker band at ϳ31 kDa were detected by immunoblotting. The 18-kDa Cterminal fragment together with a double band at 22 and 24 kDa were detected using the rabbit antiserum 53217. The poor but occasionally clearly visible immunoreactivity of the 22-and 24-kDa double band with the anti-FLAG and PST18 antibodies probably reflects a rather low abundance of these fragments. The processing pattern of murine PS2 carrying the FAD mutation N141I was similar to that of wild-type PS2 as revealed by the antiserum 53217. Likewise, processing of human PS2 lacking the C-terminal FLAG epitope was identical to that of murine PS2-FLAG, indicating that the introduction of the FLAG sequence into the C terminus of PS2 did not affect its processing pattern (Fig. 1A).
Transient expression of the murine PS2-FLAG construct in CHO cells under the control of a cytomegalovirus promoter (data not shown) or in stably transfected human neuroblastoma SK-N-MC cells under a tetracycline-regulated promoter yielded, similar to the SFV/BHK expression system, full-length murine PS2 migrating at 55 kDa and a major C-terminal fragment at 18 kDa when probed with the anti-FLAG antibody (Fig. 1B). A faint band migrating at 22 kDa could also be seen which most likely corresponds to the 22-kDa fragment easily detected by the antiserum 53217 in BHK cells. Introduction of the N141I FAD mutation did not result in a different processing pattern of murine PS2 stably expressed in SK-N-MC cells.
The precursor-product relation for full-length PS2 and its fragments was established by a pulse-chase experiment using BHK cells transfected with murine PS2. Over a 6-h chase mPS2.1F, murine PS2 with the N141I FAD mutation and C-terminal FLAG; hPS2, human PS2 without a FLAG epitope. A control vector with a cDNA insert coding for the green fluorescent protein (GFP) was included as a negative control. The blot was probed with the various antibodies as indicated and described in the text. Panel B, SK-N-MC cells harboring a ptetREP vector with murine PS2 with the N141I mutation and C-terminal FLAG epitope (mPS2.1F) or wild-type murine PS2 with C-terminal FLAG (mPS2F) were grown in the presence or absence of 1 g/ml tetracycline (tet) for 3 days, and extracts were analyzed by immunoblotting using the M2 anti-FLAG antibody. The arrows indicate the 18-kDa and, as a faint band, the 22-kDa C-terminal fragments. Numbers on top indicate different clones; C represents a vector control.
period, an increase in the 34-kDa N-terminal fragment was clearly observed in immunoprecipitations with the PST20 antibody (data not shown). Together, these experiments proved the processing of PS2 into N-and C-terminal fragments in transient and stable expression systems and suggested a slow proteolytic processing of the full-length protein to yield the processing products.
Determination of the Proteolytic Cleavage Site(s)-To determine the sites of proteolytic cleavage, the 18-kDa C-terminal fragment of murine PS2-FLAG was isolated from a BHK cell lysate by M2 anti-FLAG affinity chromatography and SDS-PAGE. N-terminal sequencing of the fragment indicated cleavage between Asp 329 /Ser 330 (Table I). In a second independent experiment an additional minor sequence was determined starting at Ser 327 and representing about 5% of the material that started at Ser 330 . Cleavage of PS2 between Asp 329 and Ser 330 results in two processing products with predicted molecular masses of 37 kDa and 14 kDa (including the FLAG epitope) which is in reasonable agreement with the observed mobilities on SDS-PAGE/immunoblots. An 18-kDa C-terminal fragment was also isolated from mouse Neuro-2a cells stably expressing the human PS2 cDNA under the control of the cytomegalovirus promoter by immunoaffinity chromatography using a mixture of PST8, PST10, and PST18 antibodies. The fragment was shown to start at the same N terminus as its mouse homologue (Table I).
A similar approach was chosen to map the cleavage site in PS1. We isolated and expressed the murine PS1 cDNA, purified its 15-kDa C-terminal fragment from BHK cell lysates, and determined the proteolytic processing site ( Table I). The Nterminal residue of this fragment corresponded to cleavage between Asp 345 /Ser 346 of the full-length protein.
Both the PS2 and PS1 cleavage sites yielding the 18-kDa and 15-kDa fragments, respectively, are located within the predicted hydrophilic loop C-terminal of transmembrane domain 6 but in a region where the PS1 and PS2 primary structures are highly divergent. However, the mapped cleavage sites shared the same residues in the P1 position (Asp) and in the P1Ј position (Ser). Interleukin-1␤-converting enzyme-type proteases (or caspases, following a recently introduced nomenclature) cleave their substrates at positions following Asp residues (22,23). In particular the PS2 cleavage site (Asp-Ser-Tyr-Asp/ Ser) corresponds closely to sites which are recognized and cleaved by caspase-3 (CPP32 or apopain). Such sites are found in poly(A)DP-ribose polymerase and the Huntington protein and follow the general structure Asp-X-X-Asp for the P1 to P4 positions (24,25).
Podlisny et al. (26) reported the isolation and radiosequencing of a C-terminal fragment from human PS1 transfected into HEK-293 cells. They mapped a major fragment starting at Ala 299 and two minor fragments starting at positions Met 292 and Val 293 . These sites are clearly distinct from the position Asp 345 /Ser 346 mapped by us and, moreover, bear no similarities to caspase-type cleavage sites. This indicates that PS1 can be processed at multiple sites and by presumably different proteases. For PS2 we also have identified larger C-terminal fragments of about 22 and 24 kDa (see Fig. 1A). While the exact identity of these fragments remains to be determined, they clearly contain the C-terminal domain of PS2 and correspond in size to the larger C-terminal fragment of PS1. Thus, there may be alternate processing pathways also for PS2 with possibly different functional properties.
Cleavage Site Inhibitors-Substrate mimetics of naturally occurring caspase cleavage sites have been described which act as specific caspase inhibitors in vitro and in vivo (25,27). We tested the activity of a caspase-3-type inhibitor (Ac-DEVD-CHO) and of a caspase-1-type inhibitor (Ac-YVAD-dbmk) on PS2 and PS1 processing in BHK cells transfected with SFV constructs. The caspase-3-selective inhibitor reduced the accumulation of the 18-kDa C-terminal fragment of PS2 almost completely in a concentration-dependent manner, whereas the caspase-1-selective inhibitor had only a minor effect ( Fig. 2A). The processing of PS1 was also affected by both types of inhibitors although the extent of inhibition was less pronounced (Fig. 2B). The clear inhibition of PS2 and PS1 processing by the general serine and cysteine protease inhibitor TLCK suggests a common pathway of PS1 and PS2 fragmentation. TLCK may interfere with serine-type protease(s) acting upstream in a cascade of proteolytic events which finally leads to the activation of caspases selective for PS2 and PS1.
Cleavage Site Mutations-The cleavage site in murine PS2 was further characterized by analyzing the effects of mutations around the cleavage site upon expression in the SFV/BHK cell system. Deletion of residues 326 -331 completely abolished processing as did the mutation of the P1 residue Asp 329 3 Arg (Fig. 2C). Mutant Asp 326 3 Asn showed some residual activity whereas mutations of Ser 327 3 Phe or Tyr 328 3 Ala had little effect on PS2 processing (not shown). The mutants thus confirmed the importance of an aspartic acid residue in the P1 and, to a lesser extent, in the P4 position for PS2 processing. Interestingly, the caspase cleavage site mutations did not induce a significant increase of the 22-24-kDa PS2 fragments strongly suggesting independent processing pathways. The slightly smaller size of the caspase-independent processing product of the ⌬326 -331 PS2 mutant might reflect a subtle structural change or a secondary modification induced by the deletion.
In Vitro Caspase Activity Assay-The proteolytic activity of caspase-type proteases can be assayed in vitro by fluorogenic peptide substrates. We have tested homogenates of mouse brains for proteolytic activity on fluorogenic substrates based on the PS2 and PS1 cleavage sites. The strongest endopeptidase activity was measured with the caspase-3-specific substrate DEVD-AMC followed by the PS2-peptide DSYD-AMC. We also observed a weak but significant activity on the PS1 substrate peptide AQRD-AMC (Fig. 3A). Thus, caspase-type activities are present in mouse brain which also accept, in addition to the caspase-3 substrate, peptide substrates derived from both PS2 and PS1. Caspase substrate peptides, if derivatized with an aldehydic or dimethylbenzoyloxymethyl ketone function at the C-terminal Asp, act as potent caspase inhibitors. Indeed, the aldehyde derivatives of all three tetrapeptides used here were found to inhibit DSYD-AMC cleavage by mouse brain homogenates in a concentration-dependent fashion, the most potent inhibitor being the caspase-3 inhibitor followed by the PS2-and PS1-derived peptides (Fig. 3C). Several caspases, including caspase-3, show activity when expressed in E. coli as an N-terminally truncated protein (21). To further substantiate the role of caspases in PS processing and to characterize the caspase-type activity found in mouse brain we inserted the human caspase-3 cDNA (from Ser 29 to the end of its open reading frame) into a pET15 vector and prepared crude cell lysates from selected transformants. The lysates were assayed for activity on the fluorogenic peptides based on the PS1 or PS2 cleavage sites and on the prototype caspase-3 substrate peptide to confirm expression of a functional enzyme. We observed a time-dependent cleavage activity with the PS2-derived substrates whereas, in contrast to the mouse brain homogenate, no activity was measured with the PS1 peptide (Fig. 3B). Inhibition of recombinant caspase-3 activity was most pronounced with the caspase-3-type inhibitor DEVD-aldehyde with an IC 50 of about 2 nM (Fig. 3D). The same inhibitor was 50 -100 times less efficient in inhibiting the endopeptidase(s) from mouse brain indicating that the caspase involved in the hydrolysis of the PS2-derived substrate might be different from caspase-3. Although the PS1-derived tetrapeptide AQRD-AMC was not hydrolyzed by recombinant caspase-3, the corresponding aldehyde derivative was capable of inhibiting DYSD-AMC cleavage by caspase-3 similar to the DSYD-aldehyde analogue (Fig. 3D). This apparent discrepancy might reflect improper binding and processing of the AQRD-AMC substrate peptide at the active site of caspase-3, whereas the inhibitory function of the aldehyde derivative is maintained by forming a thiohemiacetal with the catalytic cysteine.
In conclusion, our results demonstrate that PS2 and PS1 are substrates for caspase-type proteases. Alternate processing by non-caspase-type proteases yields fragments of different sizes. Full-length PS and C-terminal fragments are indicated by arrowheads and arrows, respectively. The strong band at around 32 kDa represents a BHK cellular protein cross-reacting with the M2 antibody. Panel C, wild-type murine PS2 with a C-terminal FLAG (mPS2F), the deletion mutant ⌬326 -331, and the point mutation D329R were expressed in the BHK/SFV system. The PS2 processing was analyzed by immunoblotting using the M2 anti-FLAG antibody. 21 kDa and 18 kDa were detected in mouse tissues indicating alternative processing pathways for PS2 also in vivo. 3 A Cterminal PS2 fragment of about 10 kDa protected T-cells against apoptotic challenge whereas overexpression of PS2 restored their sensitivity (15,16). Cleavage of PS2 by a caspasetype protease generating a similar fragment suggests a role of PS2 processing in the cellular response to apoptotic signals. Elucidating the significance of presenilin processing will help to understand apoptotic or neurodegenerative phenomena in Alzheimer's disease. 4