Calcium-dependent interaction of annexin I with annexin II and mapping of the interaction sites.

Annexins are multifunctional intracellular proteins with Ca2+- and phospholipid-binding properties. Their structures consist of four conserved repeat domains that form the core and a diverse N-terminal tail, from which their functional differences may arise. We searched for cellular proteins that interact with the N-terminal tail plus domain I of annexin I (ANX1) by using the yeast two-hybrid method. Screening of a HeLa cell cDNA library yielded annexin II (ANX2) cDNA. The interaction between ANX1 and ANX2 also occurred in vitro in a Ca2+-dependent manner. Mapping of the interaction sites revealed that interaction between domain I of ANX1 and domain IV of ANX2 was stronger than the other combinations.

Differential effects of annexins I, II, III, and V on cytosolic phospholipase A2 activity: specific interaction model.

Annexins (ANXs) are a family of proteins with calcium-dependent phospholipid binding properties. Although inhibition of phospholipase A2 (PLA2) by ANX-I has been reported, the mechanism is still controversial. Previously we proposed a 'specific interaction' model for the mechanism of cytosolic PLA2 (cPLA2) inhibition by ANX-I [Kim et al., FEBS Lett. 343 (1994) 251-255]. Here we have studied the cPLA2 inhibition mechanism using ANX-I, N-terminally deleted ANX-I (DeltaANX-I), ANX-II, ANX-II(2)P11(2), ANX-III, and ANX-V. Under the conditions for the specific interaction model, ANX-I, DeltaANX-I, and ANX-II(2)P11(2) inhibited cPLA2, whereas inhibition by ANX-II and ANX-III was negligible. Inhibition by ANX-V was much smaller than that by ANX-I. The protein-protein interactions between cPLA2 and ANX-I, DeltaANX-I, and ANX-II(2)P11(2) were verified by immunoprecipitation. We can therefore conclude that inhibition of cPLA2 by specific interaction is not a general function of all ANXs, and is rather a specific function of ANX-I. The results are consistent with the specific interaction model.

Solution structure and membrane-binding property of the N-terminal tail domain of human annexin I.

The conformational preferences of AnxI(N26), a peptide corresponding to residues 2-26 of human annexin I, were investigated using CD and NMR spectroscopy. CD results showed that AnxI(N26) adopts a mainly alpha-helical conformation in membrane-mimetic environments, TFE/water and SDS micelles, while a predominantly random structure with slight helical propensity in aqueous buffer. The helical region of AnxI(N26) showed a nearly identical conformation between in TFE/water and in SDS micelles, except for the orientation of the Trp-12 side-chain, which was quite different between the two. The N-terminal region of the AnxI(N26) helix showed a typical amphipathic nature, which could be stabilized by the neighboring hydrophobic cluster. The helical stability of the peptide in SDS micelles was increased by addition of calcium ions. These results suggest that the N-terminal tail domain of human annexin I interacts with biological membranes in a partially calcium-dependent manner.

Annexin-I inhibits phospholipase A2 by specific interaction, not by substrate depletion.

Annexin-I is a calcium dependent phospholipid binding and phospholipase A2 (PLA2) inhibitory protein. A 'substrate depletion' model has been proposed for the mechanism of PLA2 inhibition by annexin-I in studies with 14 to 18 kDa PLA2s. Herein, we have studied the inhibition mechanism using 100 kDa cytosolic PLA2 from porcine spleen. The inhibition has been measured at various substrate and calcium ion concentrations. The pattern of PLA2 inhibition by annexin-I was consistent with a 'specific interaction' mechanism rather than the 'substrate depletion' model. Apparent contraction with previous studies can be explained by the calcium-dependent binding of annexin-I to the substrate.

NMR analyses of the interactions of human annexin I with ATP, Ca2+, and Mg2+.

Human annexin I is a member of the annexin family of calcium-dependent phospholipid binding proteins. The structure of an N-terminally truncated human annexin I (delta-annexin I) and its interactions with Ca2+, Mg2+, and ATP were studied at the atomic level using nuclear magnetic resonance (NMR) spectroscopy. Since delta-annexin I is a large protein, with a molecular weight of 35 kDa, a site-specific (carbonyl-13C, amide-15N) labeling technique was used to determine the interaction sites of delta-annexin I with Ca2+, Mg2+, and ATP. The 13C NMR study focused on the carbonyl carbon resonances of the histidine residues of delta-annexin I. We found that ATP binds to delta-annexin I, and that the ATP binding site is located in the 1-domain of annexin I. We also found that histidine-52 is involved in that site, and that the binding ratio of ATP to delta-annexin I is 1:1.

Annexin-I inhibits PMA-induced c-fos SRE activation by suppressing cytosolic phospholipase A2 signal.

Annexin-I (ANX-I) is a 37-kDa protein with a calcium-dependent phospholipid-binding property. Previously we have observed the inhibition of cytosolic phospholipase A2 (cPLA2) by ANX-I in the studies using purified recombinant ANX-I, and proposed a specific interaction model for the mechanism of cPLA2 inhibition by ANX-I [Kim et al. (1994) FEBS Lett. 343, 251-255]. Here we have studied the role of ANX-I in the cPLA2 signaling pathway by transient transfection assay. The stimulation of Rat2 fibroblast cells with phorbol 12-myristate 13-acetate (PMA) induced the c-fos serum response element (SRE). The SRE stimulation by PMA was dramatically reduced by (1) pretreatment with a cPLA2-specific inhibitor, arachidonyltrifluoromethyl ketone, or (2) co-transfection with antisense cPLA2 oligonucleotide, indicating that the SRE activation was through cPLA2 activation. Co-transfection with an ANX-I expression vector also reduced the SRE stimulation by PMA, suggesting the inhibition of cPLA2 by ANX-I. The active domain of ANX-I was mapped using various deletion mutants. ANX-I(1-113) and ANX-I(34-346) were fully active, whereas ANX-I(114-346) abolished the activity. Therefore the activity was in the amino acid 34 to 113 region, which corresponds to the conserved domain I of ANX-I.

Translocation of lipocortin (annexin) 1 to the membrane of U937 cells induced by phorbol ester, but not by dexamethasone.

1. Induction of lipocortin 1 secretion by dexamethasone has been demonstrated, although the secretory mechanism is still unknown. We have studied the effects of 12-tetradecanoyl phorbol 13-acetate (TPA) and/or dexamethasone on the expression, translocation, and secretion of lipocortin 1 in U937 cells. 2. The expression of lipocortin 1 and its mRNA increased during TPA-induced differentiation of U937 cells to a maximum of 1.9 fold and 8.2 fold, respectively, after 48 h. Both the protein and the mRNA levels decreased after 48 h. 3. TPA caused the translocation of lipocortin 1 from the cytosol to the membrane of U937 cells in a time-dependent manner, as determined by Western blot analysis. The translocation was concurrent with the differentiation of the cells. After 48 h of TPA treatment, 82.6 +/- 6.5% of lipocortin 1 was present in the membrane fraction compared to 41.6 +/- 1.7% in untreated cells. 4. The amount of lipocortin 1 that was externally bound (associated) with the membrane increased to 3.2 fold as the cytosol to membrane translocation of lipocortin 1 increased. 5. Dexamethasone decreased the externally bound lipocortin 1, but had no effect on the cytosol to membrane translocation. 6. This offers a model system with which the function and the secretion mechanism of lipocortin 1 can be studied. Our data is consistent with the hypothesis that the secretory mechanism is through an unknown pathway, involving the translocation of lipocortin 1 from the cytosol to the internal membranes, and then, its secretion to the external membrane.

Determination of enzymatic activity and properties of secretory phospholipase A2 by capillary electrophoresis.

A capillary electrophoretic (CE) system coupled with a diode array UV detector was used for the assay of secretory phospholipase A2 (sPLA2) activity. This method is based on monitoring both the breakdown of substrates and the formation of products simultaneously using micellar electrokinetic chromatographic techniques. Under our developed separation conditions, we analyzed the substrates and products quantitatively, and investigated enzyme activity as a function of reaction time and presence of enzyme activator or inhibitor. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry was also utilized to confirm the phosphatidylcholine, a substrate of sPLA2. In order to test the feasibility of the developed method for measurement of enzymatic activity, we compared it to the conventional radioactive assay method for sPLA2. On the basis of our results, the conventional method can be complemented, or even replaced, by this new CE method which possesses the advantages of short analysis time, use of non-radiolabeled and inexpensive substrates, simple measurement of enzymatic activity, and exact quantitation of substrate and product.

Detection of Mycobacterium leprae DNA in formalin-fixed, paraffin-embedded samples from multibacillary and paucibacillary leprosy patients by polymerase chain reaction.

BACKGROUND: Diagnosis of paucibacillary leprosy is often difficult. A method that could confirm the diagnosis is the polymerase chain reaction (PCR) of M. leprae DNA. This reaction was applied to biopsied tissues of leprotic patients to determine the suitability and sensitivity of the reaction. METHODS: Biopsy samples were taken from previously untreated patients with multibacillary (5 patients) and paucibacillary (3 patients) leprosy, fixed in formalin, and embedded in paraffin. DNA was extracted from paraffin blocks and PCR applied. The sensitivity of the PCR method was tested by using the serially diluted DNA sample as the template. RESULTS: All eight patients showed a positive PCR for M. leprae DNA. The sensitivity was such that a single organism of M. leprae, as counted by staining of the acid-fast bacilli was identified by the PCR. CONCLUSIONS: The PCR method is simple, sensitive, specific, and does not require the use of radioisotopes. It can be applied to the unequivocal diagnosis of paucibacillary leprosy which is difficult by other means. The diagnosis can be obtained within 10 hours.

Detection of Salmonella typhi in the blood of patients with typhoid fever by polymerase chain reaction.

A polymerase chain reaction (PCR)-based test was developed for the detection of Salmonella typhi in the blood specimens from patients with typhoid fever. Two pairs of oligonucleotide primers were designed to amplify a 343-bp fragment of the flagellin gene of S. typhi. Amplified products were analyzed by agarose gel electrophoresis and Southern blot hybridization by using a 32P-labeled 40-base probe internal to the amplified DNA. The nested PCR with two pairs of primers could detect 10 organisms of S. typhi as determined by serial dilutions of DNA from S. typhi. The peripheral mononuclear cells from 11 of 12 patients with typhoid fever confirmed by blood culture were positive for DNA fragment of the flagellin gene of S. typhi, whereas 10 blood specimens of patients with other febrile diseases were negative. With the nested PCR, S. typhi DNAs were detected from blood specimens of four patients with suspected typhoid fever on the basis of clinical features but with negative cultures. We suggest that the PCR technique could be used as a novel diagnostic method of typhoid fever, particularly in culture-negative cases.

Lipocortin-1 inhibits proliferation of cultured human mesangial cells.

Lipocortin-1, a 37-kDa member of the annexin family of proteins, originally evoked interest as one of the second messengers for the anti-inflammatory actions of glucocorticoids. Studies showed that glucocorticoids inhibited the proliferation of various cell types and lipocortin-1 mediated growth inhibition of glucocorticoids in a human lung adenocarcinoma cell line. The presence of specific lipocortin-1-binding sites (receptor-like molecules) on monocytic cells has been demonstrated. This study was performed to evaluate the effects of hydrocortisone and recombinant human lipocortin-1 on cultured human mesangial cells (CHMC), and the effects of anti-lipocortin-1 antibody on the hydrocortisone-induced inhibition of CHMC proliferation. The existence of specific binding sites for lipocortin-1 was also investigated. Lipocortin-1 inhibited CHMC proliferation in a dose-dependent manner as determined by [3H]thymidine uptake and cell count. Growth of CHMC was inhibited to 18% of the control in the presence of 5 micrograms/ml of lipocortin-1. Similar growth-inhibitory activity by lipocortin-1 was observed in CHMC activated by platelet-derived growth factor. Hydrocortisone also inhibited cell proliferation in a dose-dependent manner. One to 5,000 dilution of anti-lipocortin-1 antibody reversed hydrocortisone-induced inhibition of CHMC proliferation partially, whereas concentrations over 1:1,000 reversed the inhibition completely. Flow cytometry analysis as well as indirect immunofluorescent microscopy revealed specific binding sites on the surface of CHMC. These results support the hypothesis that corticosteroids act by inducing CHMC to synthesize or secrete lipocortin-1, and that lipocortin-1 generates proliferation-suppressive signal(s) through specific binding sites on CHMC.

Chaperone-like function of lipocortin 1.

Lipocortin 1 (LC1) is a 37 kDa member of the annexin family of proteins. It has been proposed to act as a mediator of some of the actions of glucocorticoids in anti-inflammatory and immune suppressive functions. LC1 has been shown to play a role in cell proliferation, apoptosis, and differentiation. However, the exact biological functions of LC1 still remain obscure. Here it is shown that LC1 displays a chaperone-like function. Stoichiometric amounts of LC1 suppressed thermally induced inactivation and aggregation of the test enzymes citrate synthase and glutamate dehydrogenase. LC1 was also effective in refolding guanine hydrochloride-denatured glutamate dehydrogenase, as judged by circular dichroism spectroscopy.

Annexin I is a stress protein induced by heat, oxidative stress and a sulfhydryl-reactive agent.

Annexin I (also called lipocortin 1) is a 37-kDa member of the annexin family of proteins. It has been proposed to be involved in the regulation of cell growth and differentiation, apoptosis, and inflammation. Previously, we have reported that annexin I displays a chaperone-like function (Kim, G.Y., Lee, H.B., Lee, S.O., Rhee, H.J. & Na, D.S. (1997) Biochem. Mol. Biol. Int. 43, 521-528). To determine the possibility that annexin I is a stress protein, we examined whether expression of annexin I and annexin I mRNA increases in response to stresses in A549 and HeLa cells. Treatments of cells with heat, hydrogen peroxide or sodium arsenite resulted in (a) an increase in annexin I and annexin I mRNA and (b) translocation of annexin I from the cytoplasm to the nucleus and perinuclear region. The annexin I gene promoter region, cloned upstream of a reporter gene, was inducible in response to heat, hydrogen peroxide, and sodium arsenite. These results indicate that annexin I serves as a stress protein and annexins may constitute a new class of stress proteins.

Bcl3, an IkappaB protein, as a novel transcription coactivator of the retinoid X receptor.

We have recently shown that the IkappaB protein IkappaBbeta interacted with the retinoid X receptor (RXR) and inhibited the 9-cis-retinoic acid (RA)-dependent transactivations (Na, S.-Y., Kim, H.-J., Lee, S.-K., Choi, H.-S., Na, D. S., Lee, M.-O., Chung, M., Moore, D. D., and Lee, J. W. (1998) J. Biol. Chem. 6, 3212-3215). Herein, we show that a distinct IkappaB protein Bcl3 also interacts with RXR, as shown in the yeast two-hybrid tests and glutathione S-transferase pull-down assays. The Bcl3 interaction involved two distinct subregions of RXR, i.e. constitutive interactions of the N-terminal ABC domains and 9-cis-RA-dependent interactions of the C-terminal DEF domains. In contrast to IkappaBbeta, Bcl3 did not interact with the AF2 domain of RXR. Bcl3 specifically interacted with the general transcription factors TFIIB, TBP, and TFIIA but not with TFIIEalpha in the GST pull-down assays. TBP and TFIIA, however, were not able to interact with IkappaBbeta. Accordingly, Bcl3 coactivated the 9-cis-RA-induced transactivations of RXR, in contrast to the inhibitory actions of IkappaBbeta. In addition, coexpression of SRC-1 but not p300 further stimulated the Bcl3-mediated enhancement of the 9-cis-RA-induced transactivations of RXR. These results suggest that distinct IkappaB proteins differentially modulate the 9-cis-RA-induced transactivations of RXR in vivo.

Inhibition of cytosolic phospholipase A2 by annexin I. Specific interaction model and mapping of the interaction site.

Annexins (ANXs) display regulatory functions in diverse cellular processes, including inflammation, immune suppression, and membrane fusion. However, the exact biological functions of ANXs still remain obscure. Inhibition of phospholipase A(2) (PLA(2)) by ANX-I, a 346-amino acid protein, has been observed in studies with various forms of PLA(2). "Substrate depletion" and "specific interaction" have been proposed for the mechanism of PLA(2) inhibition by ANX-I. Previously, we proposed a specific interaction model for inhibition of a 100-kDa porcine spleen cytosolic form of PLA(2) (cPLA(2)) by ANX-I (Kim, K. M., Kim, D. K., Park, Y. M., and Na, D. S. (1994) FEBS Lett. 343, 251-255). Herein, we present an analysis of the inhibition mechanism of cPLA(2) by ANX-I in detail using ANX-I and its deletion mutants. Deletion mutants were produced in Escherichia coli, and inhibition of cPLA(2) activity was determined. The deletion mutant ANX-I-(1-274), containing the N terminus to amino acid 274, exhibited no cPLA(2) inhibitory activity, whereas the deletion mutant ANX-I-(275-346), containing amino acid 275 to the C terminus, retained full activity. The protein-protein interaction between cPLA(2) and ANX-I was examined using the deletion mutants by immunoprecipitation and mammalian two-hybrid methods. Full-length ANX-I and ANX-I-(275-346) interacted with the calcium-dependent lipid-binding domain of cPLA(2). ANX-I-(1-274) did not interact with cPLA(2). Immunoprecipitation of A549 cell lysate with anti-ANX-I antibody resulted in coprecipitation of cPLA(2). These results are consistent with the specific interaction mechanism rather than the substrate depletion model. ANX-I may function as a negative regulator of cPLA(2) in cellular signal transduction.

Lipocortin 1 binding sites on human T-cells: the population of cells with the binding sites is larger in CD8+ T-lymphocytes than in CD4+ T-lymphocytes.

Lipocortin 1 (LC1) is believed to be a mediator of glucocorticoids in displaying anti-inflammatory and immune suppressive responses. The existence of specific LC1 binding sites (putative receptor) on monocytes and neutrophils has been reported. We have studied the distribution of LC1 binding sites in human peripheral blood leukocytes by flow cytometry. The population of cells with LC1 binding sites was much larger in monocytes than in lymphocytes in both rheumatoid arthritis patients (93.1 +/- 2.3% vs 8.8 +/- 1.0%) and healthy volunteers (97.0 +/- 0.9% vs 9.9 +/- 1.5%). The LC1 binding cell population was larger in CD8+ T-lymphocytes than in CD4+ T-lymphocytes in both healthy volunteers (26.4 +/- 4.5% vs 10.6 +/- 2.0%) and rheumatoid arthritis patients (28.8 +/- 4.7% vs 8.7 +/- 2.1%). No difference in LC1 binding cell populations was found between rheumatoid arthritis patients and healthy controls.

Role of cytosolic phospholipase A(2) as a downstream mediator of Rac in the signaling pathway to JNK stimulation.

Rac is an important regulatory molecule implicated in c-jun N-terminal kinase (JNK) activation in response to stress and cytokines. However, the signaling events that mediate the activation of JNK by Rac are not yet well characterized. To broaden our understanding of downstream mediators that link Rac signals to the JNK pathway, we investigated whether cytosolic phospholipase A(2) (cPLA(2)) is involved in Rac activation of JNK. In this report we demonstrate that either co-transfection with antisense cPLA(2) oligonucleotide or pretreatment with arachidonyltrifluoromethyl ketone (AACOCF3), a potent and specific inhibitor of cPLA(2), inhibits Rac-mediated JNK activation, implying a potential role of cPLA(2) in Rac-signaling to JNK activation. In accordance with this observation, we demonstrate that the addition of exogenous arachidonic acid (AA), a principal product of Rac-activated cPLA(2), or leukotrienes, products of 5-lipoxygenase (5-LO) of AA, caused a specific stimulation of JNK. Together, our findings suggest that cPLA(2) mediates, at least partly, the signaling cascade by which Rac stimulates JNK.

Identification of multiple forms of membrane-associated neutral sphingomyelinase in bovine brain.

Many different stimuli such as bioactive agents and environmental stresses are known to cause the activation of sphingomyelinase (SMase), which hydrolyzes sphingomyelin to generate ceramide as a second messenger playing a key role in differentiation and apoptosis in various cell types. Here we identified multiple forms of the membrane-associated neutral SMase (N-mSMase) activity in bovine brain. They could be classified into two groups according to extracting agents: group T-mSMase, extracted with 0.2% Triton X-100, and group S-mSMase, extracted with 0.5 M (NH(4))(2)SO(4). Group T-mSMase: alpha, beta, gamma, and delta, which were extensively purified from 40,000-g pellets of bovine brain homogenates by 3,150-, 5,275-, 1,665-, and 2,556-fold over the membrane extracts, respectively, by sequential use of several column chromatographies. On the other hand, S-mSMase was eluted as two active peaks of S-mSMase epsilon and zeta in a phenyl-5PW hydrophobic HPLC column and further purified by 1,119- and 976-fold over 40,000-g pellets of the homogenates, respectively. These highly purified N-mSMase enzyme preparations migrated as several bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and showed many different features in biochemical properties such as pH dependence, Mg(2+) requirements, and effects of detergents. Taken together, our data strongly suggest that mammalian brain N-mSMase may exist as multiple forms different in both its chromatographic profiles and biochemical properties.

Rac and p38 kinase mediate 5-lipoxygenase translocation and cell death.

5-Lipoxygenase (5-LO) is a key enzyme involved in the synthesis of leukotrienes from arachidonic acid, and its activation is usually followed by translocation to the nuclear envelope. The details of mechanisms involved in the translocation of 5-LO are not well understood, though Ca(2+) is known to be essential. Here we show that ionomycin, a Ca(2+) ionophore, induces 5-LO translocation and necrotic cell death in Rat-2 fibroblasts, suggesting a potential relationship between activation of 5-LO and cell death. These effects were markedly attenuated in Rat2-Rac(N17) cells expressing a dominant negative Rac1 mutant. Pretreatment with SB203580, a specific inhibitor of p38 MAP kinase, or EGTA, a Ca(2+) chelator, likewise diminished ionomycin-induced 5-LO translocation and cell death, but PD98059, a MEK inhibitor, did not. Thus, Rac and p38 MAP kinase appear to be components in a Ca(2+)-dependent pathway leading to 5-LO translocation and necrotic cell death in Rat-2 fibroblasts.

IkappaBbeta interacts with the retinoid X receptor and inhibits retinoid-dependent transactivation in lipopolysaccharide-treated cells.

To elucidate the molecular action of the NFkappaB inhibitor IkappaBbeta, we isolated a number of IkappaBbeta interactors using the yeast two-hybrid system. These include the retinoid X receptor (RXR), whose interaction with IkappaBbeta is significantly stimulated by the RXR ligand 9-cis-retinoic acid, as shown in the yeast system as well as the glutathione S-transferase pull down assays. RXR is a nuclear protein, whereas IkappaBbeta accumulates in the nucleus only in cells stimulated with lipopolysaccharide or other inducers that result in prolonged activation of NFkappaB. Consistent with this, cotransfection with IkappaBbeta specifically repressed the 9-cis-RA-induced transcriptional activities of RXR in an lipopolysaccharide-dependent manner. These results suggest a novel IkappaBbeta-mediated antagonism between the signaling pathways of NFkappaB and RXR.