Báo cáo khoa học: Inactivation of annexin II tetramer by S-nitrosoglutathione pot

Inactivation of annexin II tetramer by S -nitrosoglutathione Lin Liu 1,2 , Edward Enright 2 , Peng Sun 1 , Shwu Yar Tsai 1 , Pragna Mehta 2 , David L. Beckman 2 and David M. Terrian 3 1 Department of Physiological Sciences, Oklahoma State University, USA; Departments of 2 Physiology, and 3 Anatomy and Cell Biology, East Carolina University, USA We investigated the effect of nitric oxide (NO) donors on the activities of annexin II tetramer (AIIt), a member of the Ca 2+ - dependent phospholipid-binding protein family. Incubation of purified AIIt with S-nitrosoglutathione (GSNO) led to the inhibition of AIIt-mediated liposome aggregation. This effect was dose-dependent with an IC 50 of approximately 100 l M . Sodium nitroprusside, another NO donor also inhibited AIIt-mediated liposome aggregation, whereas reduced glutathione, nitrate, or nitrite had no effects. GSNO also inhibited AIIt-mediated membrane fusion, but not the binding of AIIt to the membrane. GSNO only has a modest effect on liposome aggregation mediated by annexins I, III or IV. The binding of AIIt to the mem- brane protected the reactive sites of GSNO on AIIt. GSNO did not inhibit AIIt-mediated liposome aggregation in the presence of dithiothreitol. Taken together, our results sug- gest that GSNO inactivates AIIt possibly via S-nitrosylation and/or the formation of disulfide bonds. Keywords: annexin; nitric oxide; S-nitrosoglutathione; lipo- some aggregation; membrane fusion. Annexins are a multigene family of Ca 2+ -dependent phospholipid-binding proteins and plays roles in many membrane-associated events including exocytosis, endocy- tosis, ion transport, inflammation, anticoagulation, inhi- bition of phospholipases, signal transduction, Ca 2+ homeostasis, cell-matrix, cell-cell or cell–virus interaction, etc. [1–7]. However, most studies were carried out in vitro. Physiological functions of annexins are still unclear although progress has been made in the past several years. Annexin VI and VII knock-out mice [8,9], and annexin VI over expression transgenic mice [10] have been generated and annexin-related diseases (annexinopathies) have recently been recognized [11]. Annexins share common structural features, i.e. a con- served core domain of four or eight repeats of approxi- mately 70 amino acids and a short variable N-terminal segment. The C-terminal core domain contains Ca 2+ - and phospholipid-binding sites. N-termini of annexins are regulatory and are subjected to various post-translational modifications including proteolysis and phosphorylation. Annexin II, a member of this family, exists as a monomer (p36) or a heterotetramer [(p36) 2 (p11) 2 ]. The latter consists of two annexin II monomers; each associated with p11 protein, member of S100 family of Ca 2+ -binding proteins. Annexin II binds to acidic phospholipids or biological membranes and causes them to aggregate and fuse [12–14]. The formation of annexin II tetramers (AIIt) markedly reduces the Ca 2+ requirement for its membrane aggregation activity compared to annexin II monomers [12,14]. How- ever, the N-terminal phosphorylation of annexin II tetra- mer by protein kinase C (PKC) or protein tyrosine kinase pp60 c–src inhibits its membrane aggregation activity without affecting its membrane binding activity [15,16]. In vitro incubation of annexin II tetramers with plasma membrane vesicles and chromaffin granules results in the formation of a plasma membrane vesicle-annexin II tetramer-chromaffin granule complex [16]. An annexin II bridge between the plasma membrane and secretory granules has been observed in chromaffin cells and anterior pituitary secretory cells using electron microscopy [17,18]. Reconstitution experi- ments have demonstrated that annexin II can enhance secretory activity from permeabilized chromaffin cells [19]. A role of annexin II in regulated exocytosis in pulmonary artery endothelial cells has been documented [20]. We have previously shown that annexin II tetramer promotes in vitro fusion of lamellar bodies with liposomes. This process is enhanced by arachidonic acid, a lung surfactant secreta- gogue and is inhibited by 4,4¢-diisothiocyanatostilbene-2, 2¢- disulfonic acid (DIDS) and phenothiazines, inhibitors of lung surfactant secretion [14,21]. Annexin II also partially restores surfactant secretion from permeabilized type II cells [22]. Furthermore, annexin II translocates from cytoplasm to the plasma membrane of type II cells upon stimulation [23]. These results suggest that annexin II is involved in membrane fusion during surfactant secretion. In addition to its well-studied membrane fusion activity in exocytosis and endocytosis, biological activities of annexin II extend to both intracellular and extracellular compartments. Annexin II may regulate the organization of cytoskeleton by binding to F-actin [24]. Heterodimer formation between annexin II and DNA polymerase a Correspondence to L. Liu, Department of Physiological Sciences, Oklahoma State University, 264 McElroy Hall, Stillwater, OK 74078, USA. Fax: + 1 405 744 8263, Tel.: + 1 405 744 4526, E-mail: liulin@okstate.edu Abbreviations: AIIt, annexin II tetramer; GSH, reduced glutathione; GSNO, S-nitrosoglutathione; NBD-PtdEtn, N-(7-nitro-2-1,3-ben- zoxadiazol-4-yl) diacyl PtdEtn; NO, nitric oxide; PtdEtn, phospha- tidylethanolamine; PtdSer, phosphatidylserine; Rh-PtdEtn, N-(lissamine rhodamine B sulfonyl) diacyl PtdEtn; SNP, sodium nitroprusside. (Received 13 May 2002, revised 11 July 2002, accepted 16 July 2002) Eur. J. Biochem. 269, 4277–4286 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03118.x indicates a role for annexin II in DNA replication [4]. Partitioning of annexin II between nuclei and cytoplasm is controlled by a nuclear export signal and p11 [25]. Annexin II also exists in extracellular cell surface and acts as a receptor for cytomegalovirus [26], tenascin C [27], and tissue plasminogen activator [28]. Annexin II tetramer has been identified as a plasmin reductase [29] and may be involved in cancer [30]. Nitric oxide (NO) is a membrane-permeable intracellular and intercellular messenger and plays an important role in vascular tone, neurotransmission and pulmonary functions. However, it can be toxic when generated in excess. Alveolar epithelium is constantly exposed to NO from two sources: inhaled air and endogenous production from lung cells inclu- ding macrophages, endothelial cells, vascular smooth muscle cells, and epithelial cells. NO is generated from L -arginine by NO synthase (NOS). Two types of NOS have been described. One is a Ca 2+ -dependent and constitutive form (cNOS), which is stimulated by agents that increase intracellular Ca 2+ . Another is a Ca 2+ -independent and inducible form (iNOS), which is induced by cytokines and/or endotoxins and is transcriptionally regulated. Both types of NOS are present in alveolar type II cells [31]. NO has been shown to alter lung surfactant metabolism [31]. We have previously shown that NO donors inhibit lung surfactant secretion from cultured type II cells at high concentrations [32]. There are five cysteine residues in each annexin II mononer (four in human) and two in the p11 subunit. However, the role of cysteine residues in AIIt’s functions has not been appreciated. We have previously shown that treatment of AIIt by N-ethylmaleimide resulted in the loss of its activity [33]. NO and its derivatives have been reported to react with the sulfhydryl groups of several cellular proteins including calpain [34], protein kinase C [35], low molecular weight phosphotyrosine protein phosphatase [36] and glyceraldehyde-3-phosphate dehydrogenase [37], and inactivate these proteins. We reasoned that NO donors might also inhibit AIIt’s activity, and this could be another mechanism of NO-mediated inhibition of lung surfactant secretion, in addition to the nitration of annexin II by peroxynitrite [38]. As nitrosothiols occur naturally in human airways [39], we chosen S-nitrosoglutathione (GSNO) as a NO donor. In this report, we determined: (a) whether GSNO influences annexin II’s activities including mem- brane aggregation, membrane fusion, and membrane bind- ing; (b) whether the GSNO effect is specific to annexin II; (c) whether Ca 2+ and phospholipid alter the GSNO effect on annexin II; and (d) whether the GSNO effect is due to the modification of cysteine residues of annexin II. MATERIALS AND METHODS Materials S-Nitrosoglutathione (GSNO) was purchased from Cayman (Ann Arbor, MI, USA). Dithiothreitol, reduced glutathione (GSH), sodium nitrate, sodium nitrite and sodium nitro- prusside (SNP) were from Sigma (St Louis, MO, USA). Phosphatidylserine (PtdSer), phosphatidylethanolamine (PtdEtn), N-(7-nitro-2-1,3-benzoxadiazol-4-yl) diacyl PtdEtn (NBD-PtdEtn) and N-(lissamine rhodamine B sulfonyl) diacyl PtdEtn (Rh-PtdEtn) were from Avanti Polar Lipids (Alabaster, AL, USA). DEAE-Sepharose CL 6B, Sephacryl S-300, Mono S and Mono Q columns were from Amersham Biosciences Corp. (Piscataway, NJ, USA). 1,1¢- bis(4-anillino) naphthalene-5, 5¢-disulfonic acid (bis-ANS) was from Molecular Probes (Eugene, OR, USA). Biospin 6 column was from Bio-Rad (Melville, NY, USA). Anti- annexin I, II and IV antibodies were from Zymed (San Francisco, CA, USA). Anti-annexin III antibodies were kindly provided by Dr J. D. Ernst (University of California San Francisco, USA). Purification of annexins I–IV Annexins were isolated from bovine lung tissue according to Khanna et al. [40] as previously described in detail [22]. The bovine lung tissue (300 g) was powdered in a blender at slightly above liquid nitrogen temperature. One litre of buffer A (10 m M imidazole, pH 7.4, 150 m M NaCl, 1 m M dithiothreitol, 100 lgÆmL )1 soybean trypsin, 1 m M PMSF, 5 lgÆmL )1 leupeptin and 2 m M EGTA) was added to the powder. Once dissolved, the mixture was centrifuged at 650 g for 10 min. Ca 2+ concentration in the supernatant was then adjusted to 2 m M by the addition of 0.1 M Ca 2+ stock solution. The membrane fraction was collected by centrifugation at 24 000 g for 40 min and washed three timesinbufferB(10m M imidazole, pH 7.4, 150 m M NaCl, 1 m M dithiothreitol and 1 m M Ca 2+ ). The final pellet was resuspended in buffer C (buffer B plus 5 m M EGTA) and centrifuged at 100 000 g for 1 h. The supernatant containing all annexins was dialyzed against buffer D (10 imidazole, pH 7.4, 0.5 m M dithiothreitol and 1m M EGTA) for 2 days with three changes of buffer D. The dialyzate was centrifuged at 100 000 g for 1 h. The supernatant (the crude annexin preparation) was loaded on a DEAE-sepharose column (2.5 · 20 cm) and eluted using a linear salt gradient (0–0.3 M NaCl in buffer D). Three peaks were resolved: peak A (10–35 m M NaCl) contained annexins I and II; peak B (45–60 m M NaCl) contained annexins III and IV and peak C (160–190 m M NaCl) contained annexins V and VI. Annexins were identified by Western blot using specific antibodies. Fraction A was concentrated and applied to a Sephacryl S-300 column (1.5 · 150 cm) equilibrated with buffer E (40 m M Tris/HCl, pH 7.4, 150 m M NaCl, 0.5 m M dithio- threitol and 1 m M EGTA). Two peaks containing annexin I plus annnexin II monomer, and annexin II tetramer were collected separately. The low molecular weight peak was dialyzed against buffer F (25 m M Mes, pH 6.0 and 0.5 m M dithiothreitol) and applied to an FPLC Mono S column at a flow rate of 1 mLÆmin )1 .The column was developed with a gradient of 0–0.4 M NaCl in buffer F. Annexin I and annexin II monomer were eluted at 0.125 M and 0.225 M NaCl, respectively. The higher molecular weight peak (annexin II tetramer) was also purified by Mono S column chromatography as decribed above. Similarly, peak B, from the DEAE column, was concentrated and chromatographed on a Sephacryl S 300 column. The major peak containing annexins III and IV was dialyzed against buffer G (40 m M Tris, pH 8.5 and 0.5 m M dithiothreitol) and applied to an FPLC Mono Q column. Annexins III and IV were eluted at 0.114 M and 0.077 M NaCl when a gradient of 0–0.15 M NaCl in buffer G was applied. All annexins were homogenous as revealed by SDS/ PAGE and staining with coommassie Brilliant Blue. 4278 L. Liu et al.(Eur. J. Biochem. 269) Ó FEBS 2002 Preparation of liposomes Liposomes were prepared by the extrusion method [41]. Phospholipids dissolved in chloroform were dried in a test tube under a stream of nitrogen gas. The lipid film was hydrated with liposome buffer (40 m M Hepes, pH 7.0, 100 m M KCl) by vigorously vortexing. The resulting suspension was passed through a 0.1-lm-filter membrane three times using an Extruder (Lipex Biomembrane, Vancouver, Canada). Preparation of lamellar bodies Lamellar bodies were isolated from male Sprague–Dawley rat lung tissue by upward flotation [42] on a discontinuous sucrose gradient (1.0, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, and 0.2 M ). The lamellar bodies enriched at the interface between 0.4 and 0.5 M sucrose were collected and resuspended in 0.2 M sucrose, 10 m M Hepes/Tris buffer (pH 7.4). Treatment of annexin with NO donors The following standard procedure was used unless stated otherwise. Annexin II tetramer (5 lg) was mixed in 50 lL of 40 m M Hepes, pH 7.4 with GSNO or other agents. After a 30 min incubation at room temperature, the treated annexin was then tested for its ability to aggregate and fuse membrane. In some experiments, the unreacted GSNO was removed by gel filtration using a Biospin 6 chromatography column according to the manufacture’s instructions. In this case, in order to improve the recovery of annexin protein, 1mgÆmL )1 of BSA was added to the reaction mixture before loading on the column. Liposome aggregation and binding assays Liposome aggregation activity was determined by monit- oring the changes in turbidity as previously described [14]. Liposomes (PtdSer, 100 lg of lipid) were mixed in 1 mL of Ca 2+ -EGTA buffer (40 m M Hepes, pH.7.0, 100 m M KCl, 2m M MgCl 2 ,1m M EGTA and various concentrations of Ca 2+ ). After recording the zero time value (A° 540nm )of absorbance at 540 nm, annexin was added to initiate liposome aggregation and incubation continued for 30 min. At the end of incubation, the absorbance at 540 nm (A 30 540nm ) was read again. The aggregation activity was expressed as (A 30 540nm ) A° 540nm ). For the time-depend- ence of AIIt-mediated liposome aggregation, the absorb- ance at 540 nm was read every 2 min. At the end of the liposome aggregation assay, the sample was centrifuged at 100 000 g for 1 h. The pellet was analyzed on 10% SDS/ PAGE to determine the amount of AIIt bound to liposomes. The bands were quantitated by densitometry (GS-710 Calibrated Imaging Densitometer, Bio-Rad, Hercules,CA).Ca 2+ -EGTA buffer was prepared according to the method of Bers [43] and the free Ca 2+ concentration was verified using a Ca 2+ -selective electrophode (Orion Research, Inc, Boston, MA). Membrane fusion assay Membrane fusion between lamellar bodies and liposomes mediated by AIIt was measured, as described previously [14], according to the method of Struck et al.[44].Fusion was monitored by following the decrease in the efficiency of resonance energy transfer between two fluorescent-labeled phospholipid probes: NBD-PtdEtn (donor) and Rh-PtdEtn (acceptor), due to the dilution of the probes upon membrane fusion. Liposomes were composed of PtdSer/ PtdEtn/NBD-PtdEtn/Rh-PtdEtn (24.5 : 74 : 0.75 : 0.75). Labeled liposomes (4 l M in lipid) were mixed with lamellar bodies (20 lgÆmL )1 ) in 0.5 mL of the assay buffer (40 m M Hepes, pH 7.0, 100 m M KCl, 2 m M MgCl 2 ,1m M EGTA and 2 m M CaCl 2 ). After a 1 min incubation, AIIt was added to initiate the reaction. NBD-PtdEtn fluorescence (lambda Ex ¼ 450 nm and lambda Em ¼ 530 nm) was monitored as a function of time. Fusion was expressed as a percentage of the maximal NBD-PtdEtn fluorescence, which was determined after disrupting the membrane with 0.1% Triton X-100. Because Trition X-100 causes the fluorescence quenching, the maximal fluorescence was corrected by a factor of 1.3 [13]. Other methods Protein concentration was determined by the method of Bradford [45], using bovine plasma gamma globulin as a standard. SDS/PAGE was carried out according to Laemmli [46], using a Bio-Rad mini-protean II apparatus. RESULTS Effect of NO donors on AIIt-mediated liposome aggregation To determine whether NO donors affect AIIt’s functions, we exposed AIIt to GSNO and measured AIIt-mediated liposome aggregation activity as assessed by monitoring the changes in absorbance at 540 nm. Figure 1A shows the time course of AIIt-mediated liposome aggregation in the presence of various concentrations of GSNO. Figure 1B depicts a dose-dependence of GSNO inhibition of AIIt- mediated liposome aggregation. The concentration effecting 50% inhibition (IC 50 ) was approximately 100 l M .Ca 2+ (1 m M ) and/or GSNO (2 m M ) did not cause liposome aggregation in the absence of AIIt under our assay conditions. Sodium nitropresside (SNP), another NO donor structurally different from GSNO, also inhibited AIIt- mediated liposome aggregation (Fig. 1B) although the IC 50 (approximately 2 m M ) was higher than that of GSNO. To exclude the effect of the unreactive GSNO on liposome aggregation, we removed these small molecules from annexin II protein by gel filtration using a Biospin 6 chromatography column at the end of preincubation and measured its liposome aggregation. We observed a similar inhibition to these without column purification (data not shown). Furthermore, GSH, nitrite and nitrate had no effects (Fig. 2). GSNO inhibited AIIt-mediated liposome aggregation at all the AIIt concentrations and all the Ca 2+ concentrations tested (Figs 3 and 4). At a higher concen- tration of AIIt (10 lg) less inhibition was observed. Effect of GSNO on AIIt-mediated membrane fusion Although the mechanisms by which AIIt mediates mem- brane fusion are still unclear, at least three steps are Ó FEBS 2002 Annexin II and nitric oxide (Eur. J. Biochem. 269) 4279 involved: (a) the binding of AIIt to membrane; (b) membrane aggregation and (c) membrane fusion. We have previously shown that AIIt promotes the fusion of liposomes with lamellar bodies, the secretory granules of lung alveolar type II cells [14,22]. We therefore examined whether GSNO also blocks this process. Membrane fusion was monitored by a lipid mixing assay [44]. The addition of AIIt caused a rapid fusion of lamellar bodies with liposomes. Pre-treatment of AIIt with 0.1 and 1 m M GSNO resulted in 70 ± 4% and 83 ± 10% inhibition of AIIt- mediated membrane fusion, respectively (Fig. 5). It was noted that AIIt-mediated membrane fusion was more sensitive to GSNO compared to the membrane aggregation. This is probably because GSNO not only affects the membrane aggregation step, but also the membrane fusion step. Fig. 1. NO donors inhibit annexin II tetramer (AIIt)–mediated liposome aggregation in a dose-dependent fashion. Purified AIIt (5 lg) was incubated in 50 lLof40m M Hepes (pH 7.4) buffer containing varying concentrations of S-nitrosoglutathione (GSNO) or sodium nitroprusside (SNP) at room temperature for 30 min. Liposome aggregation activity was measured by monitoring the turbidity change (A 540nm ). The aggregation assay was carried out in 1 mL of Ca 2+ -EGTA buffer (40 m M Hepes, pH 7.0, 100 m M KCl, 1 m M EGTA, and 2 m M Ca 2+ ) containing 100 lg phosphatidylserine liposomes. AIIt was used to initiate liposome aggregation. (A) A representative time course curve of AIIt-mediated liposme aggregation in the presence of various concentrations of GSNO. (d)0m M (j) 0.001 m M (m)0.1 m M (.)1m M (r)10m M . (B) Dose-dependence of NO donor- mediated inhibition of AIIt-mediated liposome aggregation. The activity was expressed as the increase in absorbance at 540 nm after a 30 min incubation over the initial value. The results were expressed as percentage control. The control was treated the same way as other samples except that no other reagents were added. The data shown are mean ± SE from three experiments (GSNO, d) or mean from two experiments (SNP, j). Fig. 2. Sodium nitroprusside and S-nitrosoglutathione inhibit AIIt- mediated liposome aggregation, whereas reduced glutathione, nitrate or nitrite has little effect. AIIt (5 lg) was incubated with sodium nitro- prusside (SNP, 2 m M ), S-nitrosoglutathione (GSNO, 2 m M ), reduced glutathione (GSH, 2 m M ), nitrate (2 m M ) or nitrite (2 m M )in50lLof 40 m M Hepes buffer (pH 7.4) for 30 min. The treated AIIt was tested for its ability to mediate liposome aggregation. The results were expressed as a percentage of the control. The control was treated the same way as other samples except that no other reagents were added. The data shown are mean ± SE from three experiments. wP <0.05 vs. control. Fig. 3. A dose-dependence of AIIt-mediated liposome aggregation in the presence or absence of GSNO. Various concentrations of AIIt (0–10 lg) were incubated with or without 2 m M GSNO in 50 lLof 40 m M Hepes buffer (pH 7.4) for 30 min. Liposome aggregation was determined and expressed as the increase in absorbance at 540 nm after a 30 min incubation over the initial value. 4280 L. Liu et al.(Eur. J. Biochem. 269) Ó FEBS 2002 Effect of GSNO on the binding of AIIt to membrane As the binding of AIIt to membrane would be the first step of AIIt-mediated membrane fusion, we also investigated whether GSNO inhibits the binding of AIIt to liposomes. We treated AIIt with GSNO for 30 min and mixed the samples with liposomes and 1 m M Ca 2+ . After a 30 min incubation, we pelleted liposomes by centrifugation and analyzed AIIt associated with lipo- somes on 10% SDS/PAGE. As shown in Fig. 6A, GSNO had no effect on the amount of AIIt associated with liposomes. In the absence of Ca 2+ , little AIIt was bound to liposomes. The results indicate that the modification of annexin II by GSNO does not affect the binding of AIIt to membrane. When different amounts of AIIt were treated with GSNO, no inhibition were observed for the binding of AIIt to liposmes at all the AIIt concentrations tested (Fig. 6B). Effect of GSNO on liposome aggregation mediated by annexins I, III and IV Annexins are a large gene family. In mammals, so far, 12 members have been identified and in other organisms more than 60 and over 200 isoforms [47]. Annexins share common structural features and some biochemical pro- perties. All annexins bind to phospholipids in the presence of Ca 2+ . Some annexins (I, II, III, IV and VII) are able to mediate liposome aggregation although their Ca 2+ sensitivities differ [22]. We therefore examined whether GSNO inhibits liposome aggregation mediated by various annexins. As shown in Fig. 7, GSNO only has a modest effect on liposome aggregation mediated by other annexins. Effect of Ca 2+ and phospholipid on GSNO inhibition Ca 2+ causes protein conformational changes in annexin II [48] and may alter the environment of reactive sites of annexin II by GSNO. We tested whether such changes influence the GSNO inhibition of the activity of annexin II. AIIt (5 lg)waspreincubatedin50lLof buffer containing 1 m M Ca 2+ for 30 min to induce protein conformational changes. The mixture was then added to 1 mL of the assay buffer containing 100 lg liposome for measuring liposome aggregation. As shown in Fig. 8 (two bars with minus liposome during the preincubation), a similar inhibition was observed when AIIt was pretreated with EGTA or Ca 2+ , suggesting that the conformational changes caused by Ca 2+ had no effect on the GSNO inhibition. After binding to the membrane, some residues in AIIt may be hidden due to the polymerization of AIIt on the membrane or a protein conformational change, and are no longer accessible to GSNO. To test this possibility, AIIt (5 lg) was preincubated with 50 lg liposomes in 50 lL of buffer containing 1 m M Ca 2+ to allow AIIt binding to liposomes and then treated with 1 m M GSNO. At the end of preincubation, the mixture was added to 1 mL of assay buffer containing 50 lg liposomes for measuring liposome aggregation. As expected, GSNO still inhibited AIIt-mediated liposome aggregation when no Ca 2+ existed and thus AIIt did not bind to the membrane during the preincubation. However, in the presence of Ca 2+ and liposomes during the preincubation, AIIt was bound to the membrane before the addition of GSNO. In this case, no significant inhibition was observed (Fig. 8). Fig. 4. Ca 2+ -dependence of AIIt-mediated liposome aggregation in the presence or absence of GSNO. AIIt (5 lg) was incubated with or without 2 m M GSNO in 50 lLof40m M Hepes buffer (pH 7.4) for 30 min. Liposome aggregation was measured in various concentra- tions of Ca 2+ -EGTA buffer and expressed as the increase in absorb- ance at 540 nm after a 30 min incubation over the initial value. Fig. 5. GSNO inhibits AIIt-mediated fusion of lamellar bodies with liposomes. AIIt (5 lg) was incubated with 0.1 m M or 1 m M GSNO in 50 lLof40m M Hepes(pH7.4)for30minandAIIt-mediated membrane fusion was measured. Lipid (4 l M ) in labeled liposomes (PtdSer/PtdEtn/NBD-PtdEtn/Rh-PtdEtn, 24.5 : 74 : 0.75 : 0.75) were mixed with 20 lgÆmL )1 lamellar bodies in 0.5 mL Ca 2+ -EGTA buffer (1 m M free Ca 2+ ). After a stable baseline was established, AIIt was added to initiate the reaction. Fusion was monitored by following the increase in NBD-PtdEtn fluorescence (Ex ¼ 450 nm, Em ¼ 530 nm). The data shown are a representative from three experiments. Ó FEBS 2002 Annexin II and nitric oxide (Eur. J. Biochem. 269) 4281 In an additional experiment, GSNO (1 m M )wasdirectly added to liposome aggregation assay medium before the addition of AIIt. Under these conditions, a 48% inhibition was observed. However, if GSNO was added 5 min or 10 min after the addition of AIIt, less inhibition (26% or 17%) was seen (data not shown). Presumably, this is due to the binding of AIIt to liposomes. These results indicate that the reactive sites on AIIt were protected by the binding of AIIt to the membrane. Fig. 6. GSNO does not affect the binding of AIIt to liposomes. (A)AIItwasincubatedwithGSNO(1 m M ) for 30 min. At the end of incubation, AIIt was mixed with liposomes in the presence of 1 m M EGTA or Ca 2+ . After a 30 min incubation, liposomes were sedimented by centrifugation and AIIt associated with liposomes was analyzed by 10% SDS/PAGE. The data shown are a representative from three experiments. (B) A dose dependence of AIIt binding to liposomes in the presence or absence of GSNO. The conditions were the same as in the figure legend of Fig. 3. At the end of the aggregation assay, liposomes were sedimented by centrifugation. AIIt associated with liposomes were analyzed by SDS/PAGE and quantitated by densitometry. The results were expressed as the percentage of the maximal binding (i.e. 10 lg AIIt without GSNO). Fig. 7. A dose-dependence of liposome aggregation mediated by ann- exin I, III and IV in the presence or absence of GSNO. Various amounts of annexins I, III and IV (0–10 lg) were incubated with or without 2m M GSNO for 30 min. Liposome aggregation was determined and expressed as the increase in absorbance at 540 nm after a 30 min incubation over the initial value. For the comparison, the results were expressed as percentages of the maximal activity (i.e. 10 lg of annexins without GSNO treatment). The latter values were 0.28, 0.32, and 0.23 for annexins I, III, and IV, respectively. (d)AI(s) AI + GSNO (.) AIII (,) AIII + GSNO (j)AIV(h)AIV+GSNO. Fig. 8. Ca 2+ -induced protein conformational change in AIIt has no effect on GSNO inhibition of AIIt-mediated liposome aggregation. However, GSNO does not inhibit AIIt-mediated liposome aggregation once AIIt binds to membrane. For the first two bars, AIIt (5 lg) was incubated with or without 1 m M GSNO in 50 lLof40m M Hepes (pH 7.4) containing 1 m M EGTA or 1 m M Ca 2+ for 30 min. The samples were added to 1 mL of the assay buffer containing 100 lg liposome for aggregation activity determination. For the last two bars, AIIt (5 lg)waspreincubatedwith50lg liposome in the presence or absence of 1 m M GSNO in 50 lLof40 m M Hepes (pH 7.4) containing 1m M EGTA or Ca 2+ for30min.Thesampleswerethenaddedto 1 mL of assay buffer containing 50 lg liposome for aggregation activity determination. The zero time absorbance was recorded sepa- rately using 1 mL of the assay buffer containing 100 lg liposomes. The results were expressed as percentage control (i.e. activity of GSNO- treated AIIt/activity of untreated AIIt · 100%). The data shown are mean ± SE from three experiments. 4282 L. Liu et al.(Eur. J. Biochem. 269) Ó FEBS 2002 Effect of dithiothreitol on GSNO inhibition of AIIt-mediated liposome aggregation To evaluate whether the GSNO inhibition of AIIt-mediated liposome aggregation is involved in the formation of disulfide bonds, we incubated purified AIIt (5 lg) with GSNO (1 m M ) in the presence of the reducing agent, dithiothreitol. As shown in Fig. 9, when dithiothreitol (0.5 m M ) was included in the incubation medium, the inhibition of AIIt-mediated liposome aggregation by GSNO was no longer observed, suggesting that the inactivation of AIIt may be due to the formation of disulfide bridges. However, no intermolecular disulfide bonds between annexin molecules were formed, because when GSNO-treated AIIt was resolved on nonreduced SDS/PAGE, no extra-bands were seen (data not shown). However, we cannot rule out the possibility of disulfide bond formation between AIIt and glutathione because of the resolution of SDS/PAGE. Conformational changes To detect possible conformational changes of AIIt treated with GSNO, we used the hydrophobic fluorescent probe, bis-ANS. This dye binds to hydrophobic sites of proteins and causes an increase of intensity in fluorescence with a concomitant shift to the lower wavelength [49]. As expected, the addition of AIIt to the bis-ANS aqueous solution, the fluorescence increased and maximal emission wavelength was shifted from 510 nm to 490 nm (data not shown). Those changes are less, compared to annexin I [50]. GSNO- treated AIIt had a similar increase of fluorescence and wavelength shifts. GSNO itself had no effect on either fluorescence or maximum wavelength. The results suggest that GSNO does not cause a major conformational change of AIIt as detected by the fluorescent dye, bis-ANS. However, it is possible that the method used here may not be able to detect small conformational changes. DISCUSSION Annexins are subjected to various post-translational modi- fications. Although the phosphorylation of tyrosine or serine/threonine residues in annexin has been extensively studied, the relationship between other residues and annex- in’s activity attracted less attention. When AIIt was treated with N-ethylmaleimide, a sulfhydryl agent, AIIt’s activity was reduced [33]. However, modification of annexin by reactive nitrogen species has not been reported. Our recent study has shown that AIIt can be nitrated by peroxynitrite to form nitrotyrosine and such modification inhibited AIIt- mediated liposome aggregation [38]. In the present study, we, for the first time, showed that NO donors, GSNO and SNP, also inhibit annexin II’s activities including membrane aggregation and fusion. This modification was abolished in the presence of dithiothreitol. Although physiological significance of this in vitro observation remains to be determined, it might imply a new post-translational modi- fication and possibly a regulatory mechanism for annexin II in cells. Recently, Fas-induced caspase-3 de-nitrosylation was observed in lymphocyte cells, but the factors responsible for the de- nitrosylation was not identified [51]. Because NO inhibits surfactant secretion from alveoar type II cells [32] and AIIt is a criticial component for the secretion of lung surfactant in type II cells [14,22], NO inhibition of AIIt’s activity may provide an alternative mechanism of NO-mediated reduction of lung surfactant secretion. Annexins including annexin II have also shown to be associated with oxidative stress [52–56], NO modification may also have implications in this process as well as other biological activities of annexin II. NO and its derivatives can attack protein targets involved in many physiological processes and thus modifies their functions. Interaction of NO with the heme or nonheme iron of proteins leads to activation of soluble guanylyl cyclase [57] and inactivation of cyclooxygenase [58] or mitochondrial complexes I and II [59]. NO also regulates protein functions by covalent attachment of the NO group to cysteine residues in proteins via S-nitrosylation, which may involve other nitrogen species such as NO + .Increasing amounts of evidence demonstrate that this post-transla- tional modification may represent an important cellular regulatory mechanism [34–37]. Depending on different proteins, S-nitrosylation may be followed by secondary modification. For example, for glyceraldehyde-3-phosphate dehydrogenase, S-nitrosylation of four active site cysteines in the tetramer ultimately results in S-ADP-ribosylation and inactivation [37]. If vicinal thiols in the protein are S-nitrosylated, a more stable disulfide may be formed. One of the examples is the N-methyl- D -asparate receptor [60]. Our present study has shown that the GSNO inhibition of annexin II-mediated liposome aggregation is no longer observed in the presence of dithiothreitol, suggesting that, most likely, the modification of annexin II’s activity by Fig. 9. Effect of ditiothreitol on the inhibition of AIIt-mediated liposome aggregation caused by GSNO. AIIt (5 lg) was incubated with 1 m M GSNO in the presence of the reducing agent, dithiothreitol (0, 0.1, 0.5, 1.0 m M ). After a 30 min incubation, liposome aggregation activity was determined. The results were expressed as a percentage of the control. The control was treated as the same way as other samples except that no GSNO and dithiothreitol was added. The data shown are mean ± SE from three experiments. Ó FEBS 2002 Annexin II and nitric oxide (Eur. J. Biochem. 269) 4283 GSNO is through S-transnitrosylation and the formation of disulfide bond(s). As no dimers or polymers in GSNO- treated AIIt were observed on nonreduced SDS/PAGE, the disulfide bonds could be formed either within the AIIt molecules or between annexin II thiol and GSNO [61]. UV and fluorescence studies of annexin II revealed a Ca 2+ -induced conformational change in which the aroma- tic amino acids, tyrosine and tryptophan, expose more to the aqueous phase [48]. For annexin V, Ca 2+ causes conformational changes in domain III that leads to the formation of an additional Ca 2+ -binding site and exposure of Trp187 to the solvent [62,63]. These conformational changes appear not to affect the GSNO reaction with annexin II, as a similar inhibition of AIIt-mediated lipo- some aggregation by GSNO was observed whether AIIt was pretreated with Ca 2+ or not prior to the liposome aggregation assay. Probably, because Ca 2+ only induced a modest conformational change circular dichroism studies failed to detect major changes in secondary structure of Ca 2+ -bound annexin II [48]. The present study indicated that GSNO no longer inhibits AIIt-mediated liposome aggregation once the protein binds to the membrane. This is consistent with the finding that some annexins are accessible to quenchers in the solution more than in the membrane-bound state [64]. There are several possibilities: (a) after the binding of annexin II, the reactive sites were hidden by membrane; (b) the binding of AIIt to membrane causes a conformational change [65], such changes may bury the reactive sites of GSNO more deeply in the protein matrix therefore rendering them inaccessible to GSNO; (c) annexins V and XII has been shown to form trimers or hexamers on membrane [66,67]. We have previ- ously shown that AIIt can self-associate in the presence of Ca 2+ [23]. Therefore, it is possible that AIIt forms polymers on membrane, thus hiding the reactive sites. Nitrosothiols occur naturally in human plasma mainly as the nitrosothiol of human serum albumin [68]. S-nitroso- glutathione has been identified on normal airways [39] and in neutrophils [69]. S-nitrothiol concentrations in inflamed and transplanted lungs were much higher than normal subjects. The half-life of GSNO in the lavage fluid is approximately 3 h, much longer than NO [39]. 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