Calcium-dependent Phospholipid-binding Protein Research Articles

Annexin V disruption impairs mechanically induced calcium signaling in osteoblastic cells

The mechanical environment of the skeleton plays an important role in the establishment and maintenance of structurally competent bone. Biophysical signals induced by mechanical loading elicit a variety of cellular responses in bone cells, however, little is known about the underlying mechanotransduction mechanism. We hypothesized that bone cells detect and transduce biophysical signals into biological responses via a mechanism requiring annexin V (AnxV). AnxV, a calcium-dependent phospholipid binding protein, has several attributes, which suggest it is ideally suited for a role as a mechanosensor, possibly a mechanosensitive ion channel. These include the ability to function as a Ca 2+ selective ion channel, and the ability to interact with both extracellular matrix proteins and cytoskeletal elements. To test the hypothesis that AnxV has a role in mechanosensing, we studied the response of osteoblastic cells to oscillating fluid flow, a physiologically relevant physical signal in bone, in the presence and absence of AnxV inhibitors. In addition, we investigated the effects of oscillating flow on the cellular location of AnxV. Oscillating fluid flow increased both [Ca 2+] i levels and c-fos protein levels in osteoblasts. Disruption of AnxV with blocking antibodies or a pharmacological inhibitor, K201 (JTV-519), significantly inhibited both responses. Additionally, our data show that the cellular location of AnxV was modulated by oscillating fluid flow. Exposure to oscillating fluid flow resulted in a significant increase in AnxV at both the cell and nuclear membranes. In summary, our data suggest that AnxV mediates flow-induced Ca 2+ signaling in osteoblastic cells. These data support the idea of AnxV as a Ca 2+ channel, or a component of the signaling pathway, in the mechanism by which mechanical signals are transduced into cellular responses in the osteoblast. Furthermore, the presence of a highly mobile pool of AnxV may provide cells with a powerful mechanism by which cellular responses to mechanical loading might be amplified and regulated.

Nucleocytoplasmic lectins.

This review summarizes studies on lectins that have been documented to be in the cytoplasm and nucleus of cells. Of these intracellular lectins, the most extensively studied are members of the galectin family. Galectin-1 and galectin-3 have been identified as pre-mRNA splicing factors in the nucleus, in conjunction with their interacting ligand, Gemin4. Galectin-3, -7, and -12 regulate growth, cell cycle progression, and apoptosis. Bcl-2 and synexin have been identified as interacting ligands of galectin-3, involved in its anti-apoptotic activity in the cytoplasm. Although the annexins have been studied mostly as calcium-dependent phospholipid-binding proteins mediating membrane-membrane and membrane-cytoskeleton interactions, annexins A4, A5 and A6 also bind to carbohydrate structures. Like the galectins, certain members of the annexin family can be found both inside and outside cells. In particular, annexins A1, A2, A4, A5, and A11 can be found in the nucleus. This localization is consistent with the findings that annexin A1 possesses unwinding and annealing activities of a helicase and that annexin A2 is associated with a primer recognition complex that enhances the activity of DNA polymerase alpha. Despite these efforts and accomplishments, however, there is little evidence or information on an endogenous carbohydrate ligand for these lectins that show nuclear and/or cytoplasmic localization. Thus, the significance of the carbohydrate-binding activity of any particular intracellular lectin remains as a challenge for future investigations.

Human annexin V binds to sulfatide: contribution to regulation of blood coagulation.

Annexin V is a calcium-dependent phospholipid-binding protein that exhibits anticoagulant activity on binding to phosphatidylserine exposed on the activated surfaces of endothelial cells and platelets, inhibiting activation of factor X and prothrombin in the blood coagulation cascade. Sulfatide (galactosylceramide I(3)-sulfate), one of the glycosphingolipids of the platelet cell membrane, is thought to be involved in blood coagulation systems via activation of factor XII. In this study, we examined whether or not annexin V binds to sulfatide and affects the coagulant activity of sulfatide. Solid phase assaying of annexin V revealed that it binds specifically to sulfatide, i.e. not to galactosylceramide or gangliosides, in the presence of calcium ions. Affinity analysis by means of surface plasmon resonance showed that the K(D) of the interaction between annexin V and sulfatide is 1.2 micro M. Kinetic turbidometric assaying of plasma coagulation initiated by CaCl(2) revealed that the coagulation rate in the presence of sulfatide or phosphatidylserine was decreased by annexin V. These results suggest that annexin V regulates coagulability in the blood stream by binding not only to phosphatidylserine but also to sulfatide.

Nucleocytoplasmic lectins

This review summarizes studies on lectins that have been documented to be in the cytoplasm and nucleus of cells. Of these intracellular lectins, the most extensively studied are members of the galectin family. Galectin-1 and galectin-3 have been identified as pre-mRNA splicing factors in the nucleus, in conjunction with their interacting ligand, Gemin4. Galectin-3, -7, and -12 regulate growth, cell cycle progression, and apoptosis. Bcl-2 and synexin have been identified as interacting ligands of galectin-3, involved in its anti-apoptotic activity in the cytoplasm. Although the annexins have been studied mostly as calcium-dependent phospholipid-binding proteins mediating membrane-membrane and membrane-cytoskeleton interactions, annexins A4, A5 and A6 also bind to carbohydrate structures. Like the galectins, certain members of the annexin family can be found both inside and outside cells. In particular, annexins A1, A2, A4, A5, and A11 can be found in the nucleus. This localization is consistent with the findings that annexin A1 possesses unwinding and annealing activities of a helicase and that annexin A2 is associated with a primer recognition complex that enhances the activity of DNA polymerase α. Despite these efforts and accomplishments, however, there is little evidence or information on an endogenous carbohydrate ligand for these lectins that show nuclear and/or cytoplasmic localization. Thus, the significance of the carbohydrate-binding activity of any particular intracellular lectin remains as a challenge for future investigations.

The annexins.

Annexins are traditionally thought of as calcium-dependent phospholipid-binding proteins, but recent work suggests a more complex set of functions. More than a thousand proteins of the annexin superfamily have been identified in major eukaryotic phyla, but annexins are absent from yeasts and prokaryotes. The unique annexin core domain is made up of four similar repeats approximately 70 amino acids long, each of which usually contains a characteristic 'type 2' motif for binding calcium ions. Animal and fungal annexins also have non-homologous amino-terminal domains of varying length and sequence, which are responsible for the distinct localizations and specialized functions of the proteins through post-translational modification and binding to other proteins. Annexins interact with various cell-membrane components that are involved in the structural organization of the cell, intracellular signaling by enzyme modulation and ion fluxes, growth control, and they can act as atypical calcium channels. Analysis of site-specific conservation in the core domain suggests a role for certain buried residues in the calcium-channel activity of vertebrate annexins and in the structural stability of their core domains. Evolutionarily significant differences between subfamilies are preferentially localized to accessible sites on the protein surface that determine membrane binding and interactions with cytosolic proteins.

Annexin A1 processing is associated with caspase-dependent apoptosis in BZR cells

Annexins are widely distributed and have been described in lung as well as in other cells and tissues. Annexin I (ANX AI) is a member of the calcium-dependent phospholipid binding protein family. Besides its anti-inflammatory function, ANX AI has been involved in several mechanisms such as the Erk repression pathway or apoptosis. To investigate the role of ANX AI on apoptosis in broncho-alveolar cells, we have constructed a plasmid containing the ANX AI full length cDNA. Transfected BZR cells displayed a higher level of both forms of ANX AI (37 and 33 kDa) as well as a decrease in cell viability (two-fold versus cells transfected with an empty vector). In order to analyse the endogenous ANX AI processing during stimulus-induced apoptosis, BZR cells were treated with a commonly used inducer, i.e. C2 ceramides. In these conditions, microscopic analysis revealed chromatin condensation in dying cells and the Bcl-2, Bcl-x L/Bax mRNA balance was altered. Caspase-3 is one of the key executioners of apoptosis, being responsible for the cleavage of many proteins such as the nuclear enzyme poly(ADP-ribose) polymerase (PARP). We demonstrate that caspase-3 was activated after 4 h treatment in the presence of ceramide leading to the cleavage of PARP. Dose–response experiments revealed that cell morphology and viability modifications following ceramide treatment were accompanied by an increase in endogenous ANX AI processing. Interestingly, in both ceramide and transfection experiments, the ANX AI cleaved form was enhanced whereas pre-treatment with the caspase inhibitor Z-VAD-fmk abolished ANX AI cleavage. In conclusion, this study demonstrates a complex regulatory role of caspase-dependent apoptosis where ANX AI is processed at the N-terminal region which could give susceptibility to apoptosis upon ceramide treatment.

Intracellular Localization of Endothelial Cell Annexins Is Differentially Regulated by Oxidative Stress

Annexins are calcium-dependent phospholipid binding proteins that are implicated in the regulation of both intracellular and extracellular thrombostatic mechanisms in the vascular endothelium. Tight control of annexin gene expression and targeting of annexin proteins is therefore of importance in maintaining the health of the endothelium. Because annexins are abundant in vascular endothelial cells and could be either dysregulated by or contribute to anomalies in Ca2+ signaling, we investigated annexin gene expression and subcellular localization in human umbilical vein endothelial cells (HUVEC) in a model of chronic oxidative stress. HUVEC were cultured under mild hyperoxic conditions in a custom-built chamber to induce oxidative stress over a period of 12 days. Although annexin expression levels did not change significantly in response to hyperoxic stress, immunofluorescence analysis revealed striking effects on the subcellular localization of certain annexins, including the redistribution of annexins 5 and 6 from the cytosol to the nucleus. In addition, oxidative stress modulated the responses of certain annexins to stimulation with a range of pharmacological and physiological Ca2+-mobilizing agonists, in a manner that suggested that annexin localization is regulated via the complex integration of both Ca2+ and intracellular signaling pathways. These results show that differential regulation of annexin localization by oxidative stress may have a causative role in the cellular pathophysiology of vascular endothelial cell disease.

Expression of a releasable form of annexin II by human keratinocytes.

Annexin II is a multifunctional calcium-dependent phospholipid binding protein whose presence in epidermis has previously been reported. However, like other members of annexin family, annexin II has been regarded as either an intracellular protein or associated with the cellular membrane. Here, we report the presence of a releasable annexin II and p11, two monomers of annexin II tetramer, in keratinocyte-conditioned medium (KCM). Proteins present in KCM were fractionated on a gel filtration column and following further evaluation, a releasable protein with apparent MW of 36 kDa was identified. Further characterization identified this protein as the p36 monomer of annexin II tetramer. The phospho-tyrosine antibody did not visualize this protein as the phosphorylated form of p36. Several experiments were conducted to examine whether this protein is soluble or associated with keratinocyte cell membranes in the conditioned medium. A centrifugation of conditioned medium was not able to bring this protein down into the pellet. Surprisingly, the results of Western analysis identified p36 and p11, two monomers of the annexin II tetramer, in conditioned medium derived from either keratinocytes cultured alone or keratinocytes co-cultured with fibroblasts. In contrast to the keratinocyte-conditioned medium in which annexin II was easily detectable, both monomers were barely detectable in conditioned medium collected from dermal fibroblasts. This finding was in contrast to the cell lysates in which p36 was detectable in both keratinocytes and fibroblasts. However, the amount of this protein was markedly higher in keratinocyte lysate relative to that of dermal fibroblasts. Conditioned medium derived from keratinocyte established from adult showed a higher level of annexin II compared to that of keratinocytes established from newborn babies. The expression of p11 seems to increase with differentiation of keratinocytes derived from either adult or newborn skin samples. When the site of annexin synthesis in human skin was examined by immunohistochemical staining, the antibody for p36 localized the annexin to the keratinocyte cell members in the basal and suprabasal keratinocytes. In conclusion, Western blot detection of both p36 and p11 in conditioned medium from skin cells revealed that human keratinocytes, but not fibroblasts, express a releasable monomer form of annexin II which is regulated by differentiation status of keratinocytes. This finding is consistent with the localization of annexin II detected by immunohistochemical staining.

Effect of annexin-1 on experimental autoimmune encephalomyelitis (EAE) in the rat.

Annexin-1, a calcium-dependent phospholipid binding protein, has been shown to act as an endogenous central neuroprotectant, notably against cerebral ischaemic damage. In the present study we extend these findings to an animal model of multiple sclerosis, EAE, and report that endogenous annexin-1 is expressed in ED1+ macrophages and resident astrocytes localized within the lesions in the central nervous system (CNS). Intracerebroventricular (i.c.v.) administration of an NH2-terminal fragment spanning amino acids 1-188 of annexin-1 after the onset of the clinical symptoms significantly reduced both the neurological severity as well as weight loss of mild EAE. Immunoneutralization of endogenous brain annexin-1 failed to exacerbate the clinical features of EAE. Thus, although the role of endogenous annexin-1 in the pathogenesis of EAE remains to be determined, our findings suggest that annexin-1 may be of therapeutic benefit to the treatment of multiple sclerosis.

Microarray Analysis of Differential Gene Expression in Lead-Exposed Astrocytes

The toxic metal lead is a widespread environmental health hazard that can adversely affect human health. In an effort to better understand the cellular and molecular consequences of lead exposure, we have employed cDNA microarrays to analyze the effects of acute lead exposure on large-scale gene expression patterns in immortalized rat astrocytes. Our studies identified many genes previously reported to be differentially regulated by lead exposure. Additionally, we have identified novel putative targets of lead-mediated toxicity, including members of the family of calcium/phospholipid binding annexins, the angiogenesis-inducing thrombospondins, collagens, and tRNA synthetases. We demonstrate the ability to distinguish lead-exposed samples from control or sodium samples solely on the basis of large-scale gene expression patterns using two complementary clustering methods. We have confirmed the altered expression of candidate genes and their encoded proteins by RT–PCR and Western blotting, respectively. Finally, we show that the calcium-dependent phospholipid binding protein annexin A5, initially identified as a differentially regulated gene by our microarray analysis, is directly bound and activated by nanomolar concentrations of lead. We conclude that microarray technology is an effective tool for the identification of lead-induced patterns of gene expression and molecular targets of lead.

Annexin II overexpression correlates with stromal tenascin-C overexpression: a prognostic marker in colorectal carcinoma.

Overexpression of annexin II, a calcium-dependent phospholipid-binding protein, has been reported in various carcinomas. One of its ligands is tenascin-C, an extracellular matrix glycoprotein with predominantly antiadhesive qualities that also has been reported to be a prognostic marker for several carcinomas. In the current study, the authors investigated the correlation between the overexpression of annexin II and tenascin-C in colorectal carcinoma. Western blot analysis of annexin II expression was examined in four human colorectal carcinoma cell lines. Using immunohistochemical methods, the authors also examined expression of annexin II and tenascin-C in 105 primary colorectal carcinoma cases. Although annexin II was expressed in human colon carcinoma cell lines, there was no apparent correlation between its expression level and the metastatic potential of these cell lines. The authors observed overexpression of annexin II and tenascin-C proteins in 29.5% and 49.5%, respectively, of colorectal carcinoma cases. Overexpression of annexin II was found to be correlated significantly with histologic type, tumor size, depth of invasion, and pTNM stage, whereas tenascin-C overexpression was noted to be correlated significantly with histologic type, depth of invasion, lymphatic invasion, venous invasion, lymph node metastasis, and pTNM stage. Expression of annexin II was shown to be correlated significantly with that of tenascin-C. Multivariate analysis demonstrated that annexin II and tenascin-C cooverexpression was an independent factor of poor prognosis in patients with colorectal carcinoma. The data from the current study suggest that both annexin II and tenascin-C are overexpressed in advanced colorectal carcinoma and that they may be related to the progression and metastatic spread of colorectal carcinoma.

Evidence for the Involvement of Annexin 6 in the Trafficking between the Endocytic Compartment and Lysosomes

Annexins are a family of calcium-dependent phospholipid-binding proteins, which have been implicated in a variety of biological processes including membrane trafficking. The annexin 6/lgp120 prelysosomal compartment of NRK cells was loaded with low-density lipoprotein (LDL) and then its transport from this endocytic compartment and its degradation in lysosomes were studied. NRK cells were microinjected with the mutated annexin 6 (anx61–175), to assess the possible involvement of annexin 6 in the transport of LDL from the prelysosomal compartment. The results indicated that microinjection of mutated annexin 6, in NRK cells, showed the accumulation of LDL in larger endocytic structures, denoting retention of LDL in the prelysosomal compartment. To confirm the involvement of annexin 6 in the trafficking and the degradation of LDL we used CHO cells transfected with mutated annexin 61–175. Thus, in agreement with NRK cells the results obtained in CHO cells demonstrated a significant inhibition of LDL degradation in CHO cells expressing the mutated form of annexin 6 compared to controls overexpressing wild-type annexin 6. Therefore, we conclude that annexin 6 is involved in the trafficking events leading to LDL degradation.

Functional mapping of surfactant protein A.

Surfactant protein A (SP-A) is a highly ordered, oligomeric glycoprotein that is secreted into the airspaces of the lung by alveolar type II cells and Clara cells of the pulmonary epithelium. Although research has shown that SP-A is both a calcium-dependent phospholipid-binding protein that affects surfactant structure and function and a lectin that opsonizes diverse microbial species, our understanding of the physiologically relevant roles of SP-A in the lung remains incomplete. My review focuses on the putative biological functions of SP-A that are supported by experiments in mammals and on the structural basis of SP-A function.

Isolation and localisation of an annexin gene (gp-nex) from the potato cyst nematode, Globodera pallida

AbstractA full length cDNA clone was isolated by screening a mixed stage Globodera pallida expression library using the monospecific polyclonal antibody IACR-PC320. Sequence comparison indicated that the predicted 332 amino acid protein encoded by the cDNA was a member of the annexin gene family named gp-nex and very similar to annexins found in other nematode species. Annexins are calcium-dependent phospholipid binding proteins characterised by four repeated domains approximately 70 amino acids in length. The predicted amino acid sequence of the protein did not contain an N-terminal secretion signal peptide; however, the protein was shown to be present in excretory/secretory products from G. pallida second stage juveniles treated with the neurotransmitter 5 methoxy-N, N dimethyl tryptamine. Gp-nex encodes a protein of 35 kDa and was immunolocalised in the amphids, genital primordium and in the constraining muscles above and below the metacorpus pump chamber of G. pallida second stage juveniles. It is also present in eggs and adult females of the two species of the potato cyst nematodes.

Construction of recombinant adenoviral vector of annexin II.

Annexin II is a member of the annexin family of calcium-dependent phospholipid binding proteins expressed in vascular endothelium. Recently this molecule was reported to play a role in control of fibrinolysis on the endothelial surface. To examine the role of annexin II in vascular endothelium critically, we developed a recombinant adenoviral vector containing the annexin II cDNA. A full-length annexin II cDNA was inserted into a shuttle vector, pAdRSV4, and co-transfected into 293 cells with a replication-deficient type 5 adenovirus, pJM17. Resulting plaques were isolated and checked for protein expression. The verified clone (AdRSV-ANII) was further analyzed. Characterization of this vector will facilitate the investigation of the mechanism of fibrinolysis on vascular endothelium.

Annexin II increases osteoclast formation by stimulating the proliferation of osteoclast precursors in human marrow cultures.

Annexin II (AXII), a calcium-dependent phospholipid-binding protein, has been recently found to be an osteoclast (OCL) stimulatory factor that is also secreted by OCLs. In vitro studies showed that AXII induced OCL formation and bone resorption. However, the mechanism of action by which AXII acts as a soluble extracellular protein to induce OCL formation is unknown. In this paper, we demonstrate that AXII gene expression is upregulated by 1,25-dihydroxyvitamin D3 [1, 25-(OH)2D3] and that addition of AXII significantly increased OCL-like multinucleated cell formation. Time-course studies suggested that AXII acted on the proliferative stage of OCL precursors and that AXII increased thymidine incorporation in OCL precursors. Moreover, AXII enhanced the growth of CFU-GM, the earliest identifiable OCL precursor, when bone marrow cultures were treated with low concentrations of GM-CSF. This capacity of AXII to induce OCL precursor proliferation was due to induction of GM-CSF expression, because the addition of neutralizing antibodies to GM-CSF blocked the stimulatory effect of AXII on OCL formation. RT-PCR analysis using RNA from highly purified subpopulations of marrow cells demonstrated that T cells, especially CD4(+) T cells, produced GM-CSF in response to AXII. Furthermore, FACS(R) analysis of T-cell subpopulations treated with fluorescein-labeled AXII suggested that the CD4(+), but not CD8(+), subpopulation of T cells express an AXII receptor. Taken together, these data suggest that AXII stimulates OCL formation by activating T cells through a putative receptor to secrete GM-CSF. GM-CSF then expands the OCL precursor pool to enhance OCL formation.

Binding of annexin V to bilayers with various phospholipid compositions using glass beads in a flow cytometer

Annexin V belongs to the family of calcium-dependent phospholipid binding proteins and binds almost solely to phosphatidylserine (PS). When annexin V is used to detect loss of membrane asymmetry in cellular systems, the binding properties under physiological conditions are of importance. Most biochemical studies use optimized binding conditions, conditions that are often far from physiological. For the interpretation of flow cytometric studies that use fluorescent annexin V to probe PS exposure, it is important to know what mixture of lipid species exposed in the outer leaflet of a membrane can evoke a positive annexin V signal. The lipid species is important in this respect as well as the concentration that just evokes a positive signal (detection level). Furthermore, the influence of the composition of the lipid matrix (cholesterol content, other phospholipid species) was investigated, as well as the influence of the calcium concentration on annexin V binding. In this study, we report on the binding of annexin V to phospholipid bilayers (adsorbed to glass beads) as measured by flow cytometry at physiological conditions. Annexin V binding was found to increase rapidly, with increasing PS concentrations up to a certain level (attained at 6 mol% PS). Further increase of the PS concentration resulted only in a slight increase of annexin V binding. Calcium concentrations below 3 mM were found to reduce the sensitivity of the binding assay. Phosphatidylethanolamine incorporated in the phospholipid bilayer resulted in a lower threshold for the binding assay, whereas sphingomyelin had no influence on the binding of annexin V and cholesterol reduces binding of annexin V to lipid bilayers. These data may help in the interpretation of results obtained from binding of annexin V to cell membranes (e.g., involved in apoptosis).

NMR analyses of the interactions of human annexin I with ATP, Ca 2+, and Mg 2+

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 (Δ-annexin I) and its interactions with Ca 2+, Mg 2+, and ATP were studied at the atomic level using nuclear magnetic resonance (NMR) spectroscopy. Since Δ-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 Δ-annexin I with Ca 2+, Mg 2+, and ATP. The 13C NMR study focused on the carbonyl carbon resonances of the histidine residues of Δ-annexin I. We found that ATP binds to Δ-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 Δ-annexin I is 1:1.

Activated protein kinase C α associates with annexin VI from skeletal muscle

We have previously detected a number of protein kinase C (PKC) alpha-binding proteins in skeletal muscle cytosol by blot overlay assay, and now identify the major, 69 kDa binding protein as annexin VI by immunoblotting and overlay assay of hydroxyapatite chromatography fractions. Annexin VI was also detected in immunoprecipitates of PKC alpha. Annexin VI and PKC alpha are both calcium-dependent phospholipid-binding proteins, and detection of the interaction was dependent on the presence of calcium and phosphatidylserine (PS). The association probably involves specific protein-protein interactions rather than mere bridging by lipid molecules: firstly, detection of PKC alpha-annexin VI complexes by overlay assay was not diminished when PS concentrations were increased over a 10-fold range, while that of other PKC alpha-binding protein complexes was reduced or abolished; secondly, the presence in the overlay assay of a PKC pseudosubstrate peptide, analogous to a PKC sequence previously found to be involved in PKC binding activity, reduced complex formation; thirdly, we were also able to detect annexin VI interaction with PKC beta by overlay of skeletal muscle cytosol, but not with PKC theta, the major novel PKC in this tissue, suggesting sequences specific to calcium-dependent PKC isoenzymes are involved. While other annexin isoforms may be PKC substrates or inhibitors, annexin VI phosphorylation by PKC alpha could not be detected after co-purification, while phosphorylation of subsequently-added histone IIIS was readily observed. Annexin VI is a major skeletal muscle protein and our data are consistent with a role for this isoform in the control of calcium-dependent PKC.

Degradation of annexin I in bronchoalveolar lavage fluid from patients with cystic fibrosis.

Annexin I is a 36 kilodalton (kD) calcium-dependent phospholipid-binding protein which may have anti-inflammatory properties. Previous investigations which sampled lower respiratory tract epithelial lining fluid (ELF) via bronchoalveolar lavage (BAL) have demonstrated that annexin I can be degraded in inflammatory lung disease. We analyzed BAL fluid from patients with cystic fibrosis (CF) to determine the effects of lung inflammation on the structure and activity of annexin I. Intact annexin I was absent in 17 out of 20 BAL fluid samples from patients with CF, due largely to degradation to a 33 kD protein. The three CF BAL fluids in which annexin I was detectable had very little or no unopposed neutrophil elastase activity in contrast to the 17 in which no annexin I was detectable. Annexin I was present in all BAL fluid samples from 10 normal volunteer (NV) subjects and 12 patients with interstitial lung disease (ILD). The 33 kD annexin I breakdown product was not detectable in samples from NV, but was detectable only in ILD patients with relatively high percentages of neutrophils on BAL differential cell counts. Annexin I appeared to be cleaved by neutrophil elastase at the N-terminal portion between Val-36 and Ser-37 to yield the 33 kD protein. Cleavage of the N-terminal portion of annexin I was accompanied by a marked change in the annexin I isoelectric point (pI) value (from 6.0 to 8.5-9.0) and greatly diminished annexin I functional activity. Our findings demonstrate that annexin I degradation in epithelial lining fluid is closely related to lung inflammation.