Function Of Annexin Research Articles

Genome-Wide Characterization and Expression Analysis of Annexin Genes under High-Temperature Stress in Sorghum bicolor L.

The annexin family is found in animals and plants and responds to a wide range of environmental stressors. In this study, 19 annexin proteins were identified based on their putative annexin repeats in Sorghum bicolor and further grouped on a phylogenic tree. These genes were randomly distributed across seven chromosomes. The amino acid length of sorghum annexins (SbAnn) varied from 241 (SbAnn3) to 370 (SbAnn2). Additionally, selective pressure analysis suggests the role of purification selection in conserving the annexin gene family in sorghum. Most SbAnns were located in the nucleus, cytoplasm, chloroplast, and mitochondrion. Also, higher syntenic relationships were observed between S. bicolor and monocots (S. italica, Z. mays, and O. sativa) than in A. thaliana. Cis-acting elements in the SbAnns gene promoters were grouped into response to light, hormone and environmental stress, and development-related elements. A number of miRNAs related to abiotic stress response targeted eleven SbAnns. Furthermore, five (SbAnn 4, 5, 14, 15, and 18) of the seven SbAnn genes utilized had relatively higher fold change under high-temperature treatment. This research provides insights into the characterization and functions of annexins under high temperatures.

Annexin V: the membrane-binding protein with diverse functions

Annexin V is an eukaryotic protein from the annexin family which is able to reversibly bind to phospholipid membranes in a Ca2+-dependent manner. It possesses a complex mechanism of the membrane binding which includes the two-dimensional lattice formation from annexin V trimers and significant variation of the membrane structure. The precise functions of annexin V are largely unknown, however, its participation in the blood coagulation, membrane repair process and the Ca2+ channel activity is suggested. The usage of annexin V as a marker of phosphatidylserine-positive cells in in vitro and in vivo studies makes the understanding of the protein role in cellular processes critically important.The current review is focused on the structure of annexin V and the mechanism and kinetics of its membrane binding. The lipid specificity and the multimerization process will be described. Finally, some of the proposed annexin V functions including inhibition of the blood coagulation and the Ca2+ transport activity will be discussed.

Role of annexin A family in tumorigenesis and chemoresistance of gastric cancer.

Gastric cancer (GC) is one of the most common cancer types and the fourth leading cause of cancer-related mortality among all malignant tumors worldwide. Due to insidious onset and lack of reliable early diagnostic markers, most GC patients are at an advanced stage at the time of diagnosis. Annexin is an evolutionally-conserved Ca2+-dependent phospholipid-binding protein superfamily, including five members (A, B, C, D, and E). Annexins in the cells of vertebrates comprised the annexin A family, consisting of 12 members in humans. The biological functions of annexin A are Ca2+-signal transduction, vesicle transport, cell proliferation, cell division, cell apoptosis, signal transduction, anti-inflammatory, proangiogenesis, and anticoagulation, most of which overlap with the basic characteristics of tumors. Accumulating evidence indicated that members of the annexin A family are correlated with tumorigenesis and chemoresistance and can be used as potential tumor prognostic factors and targets for biological therapy. Thus, the current review focused on the role and relative mechanisms of the annexin A family in GC.

OsANN4 modulates ROS production and mediates Ca2+ influx in response to ABA

BackgroundPlant annexins are calcium- and lipid-binding proteins that have multiple functions, and a significant amount of research on plant annexins has been reported in recent years. However, the functions of annexins in diverse biological processes in rice are largely unclear.ResultsHerein, we report that OsANN4, a calcium-binding rice annexin protein, was induced by abscisic acid (ABA). Under ABA treatment, the plants in which OsANN4 was knocked down by RNA interference showed some visible phenotypic changes compared to the wild type, such as a lower rooting rate and shorter shoot and root lengths. Moreover, the superoxide dismutase (SOD) and catalase (CAT) activities of the RNAi lines were significantly lower and further resulted in higher accumulation of O2.- and H2O2 than those of the wild-type. A Non-invasive Micro-test Technology (NMT) assay showed that ABA-induced net Ca2+ influx was inhibited in OsANN4 knockdown plants. Interestingly, the phenotypic differences caused by ABA were eliminated in the presence of LaCl3 (Ca2+ channel inhibitor). Apart from this, we demonstrated that OsCDPK24 interacted with and phosphorylated OsANN4. When the phosphorylated serine residue of OsANN4 was substituted by alanine, the interaction between OsANN4 and OsCDPK24 was still observed, however, both the conformation of OsANN4 and its binding activity with Ca2+ might be changed.ConclusionsOsANN4 plays a crucial role in the ABA response, partially by modulating ROS production, mediating Ca2+ influx or interacting with OsCDPK24.

ANNEXIN 8 negatively regulates RPW8.1‐mediated cell death and disease resistance in Arabidopsis

Study on the regulation of broad-spectrum resistance is an active area in plant biology. RESISTANCE TO POWDERY MILDEW 8.1 (RPW8.1) is one of a few broad-spectrum resistance genes triggering the hypersensitive response (HR) to restrict multiple pathogenic infections. To address the question how RPW8.1 signaling is regulated, we performed a genetic screen and tried to identify mutations enhancing RPW8.1-mediated HR. Here, we provided evidence to connect an annexin protein with RPW8.1-mediated resistance in Arabidopsis against powdery mildew. We isolated and characterized Arabidopsis b7-6 mutant. A point mutation in b7-6 at the At5g12380 locus resulted in an amino acid substitution in ANNEXIN 8 (AtANN8). Loss-of-function or RNA-silencing of AtANN8 led to enhanced expression of RPW8.1, RPW8.1-dependent necrotic lesions in leaves, and defense against powdery mildew. Conversely, over-expression of AtANN8 compromised RPW8.1-mediated disease resistance and cell death. Interestingly, the mutation in AtANN8 enhanced RPW8.1-triggered H2 O2 . In addition, mutation in AtANN8 led to hypersensitivity to salt stress. Together, our data indicate that AtANN8 is involved in multiple stress signaling pathways and negatively regulates RPW8.1-mediated resistance against powdery mildew and cell death, thus linking ANNEXIN's function with plant immunity.

Molecular Mechanisms by Which S100A4 Regulates the Migration and Invasion of PGCCs With Their Daughter Cells in Human Colorectal Cancer.

Recently, an increasing number of evidences have shown that polyploid giant cancer cells (PGCCs) could generate daughter cells with a strong migration and invasion ability, which have been implicated in cancer recurrence and metastasis. However, the underlying molecular mechanisms of PGCCs with their daughter cells remain largely unclear. In vitro and in vivo experiments combined with 222 cases of human colorectal cancer (CRC) samples were used to identify the molecular mechanisms of S100A4-related proteins regulating the invasion and metastasis of PGCCs with their daughter cells. PGCCs with their daughter cells had high migration, invasion, and proliferation abilities compared to control cells; these were significantly inhibited after S100A4 knockdown. The high expression of cathepsin B, cyclin B1, TRIM21, and Annexin A2 were significantly downregulated after S100A4 knockdown, while the overexpression of S100A4, cathepsin B, cyclin B1, and S100A10 were significantly downregulated after TRIM21 knockdown in PGCCs with their daughter cells. The tumorigenic and metastatic ability of PGCCs with their daughter cells in vivo was significantly stronger compared to the untreated cells, which was significantly decreased after S100A4 knockdown. Moreover, the expression of S100A4-related proteins was positively correlated with the malignancy degree of human CRC, and maintained a high level in lymph node metastasis. S100A4 and TRIM21 may regulate each other to affect the expression and subcellular localization of cyclin B1, and participate in regulating the structure and function of Annexin A2/S100A10 complex, affecting downstream cathepsin B, resulting in the invasion and metastasis of PGCCs with their daughter cells. Besides, 14-3-3 ζ/δ and Ezrin may be involved in the motility and invasion of PGCCs with their daughter cells via cytoskeletal constructions with S100A4.

Annexins in Adipose Tissue: Novel Players in Obesity.

Obesity and the associated comorbidities are a growing health threat worldwide. Adipose tissue dysfunction, impaired adipokine activity, and inflammation are central to metabolic diseases related to obesity. In particular, the excess storage of lipids in adipose tissues disturbs cellular homeostasis. Amongst others, organelle function and cell signaling, often related to the altered composition of specialized membrane microdomains (lipid rafts), are affected. Within this context, the conserved family of annexins are well known to associate with membranes in a calcium (Ca2+)- and phospholipid-dependent manner in order to regulate membrane-related events, such as trafficking in endo- and exocytosis and membrane microdomain organization. These multiple activities of annexins are facilitated through their diverse interactions with a plethora of lipids and proteins, often in different cellular locations and with consequences for the activity of receptors, transporters, metabolic enzymes, and signaling complexes. While increasing evidence points at the function of annexins in lipid homeostasis and cell metabolism in various cells and organs, their role in adipose tissue, obesity and related metabolic diseases is still not well understood. Annexin A1 (AnxA1) is a potent pro-resolving mediator affecting the regulation of body weight and metabolic health. Relevant for glucose metabolism and fatty acid uptake in adipose tissue, several studies suggest AnxA2 to contribute to coordinate glucose transporter type 4 (GLUT4) translocation and to associate with the fatty acid transporter CD36. On the other hand, AnxA6 has been linked to the control of adipocyte lipolysis and adiponectin release. In addition, several other annexins are expressed in fat tissues, yet their roles in adipocytes are less well examined. The current review article summarizes studies on the expression of annexins in adipocytes and in obesity. Research efforts investigating the potential role of annexins in fat tissue relevant to health and metabolic disease are discussed.

An αIIbβ3- and phosphatidylserine (PS)-binding recombinant fusion protein promotes PS-dependent anticoagulation and integrin-dependent antithrombosis

Blood platelets are required for normal wound healing, but they are also involved in thrombotic diseases, which are usually managed with anticoagulant drugs. Here, using genetic engineering, we coupled the disintegrin protein echistatin, which specifically binds to the platelet integrin αIIbβ3 receptor, to annexin V, which binds platelet membrane-associated phosphatidylserine (PS), to create the bifunctional antithrombotic molecule recombinant echistatin-annexin V fusion protein (r-EchAV). Lipid binding and plasma coagulation studies revealed that r-EchAV dose-dependently binds PS and delays plasma clotting time. Moreover, r-EchAV inhibited ADP-induced platelet aggregation in a dose-dependent manner and exhibited potent antiplatelet aggregation effects. r-EchAV significantly prolonged activated partial thromboplastin time, suggesting that it primarily affects the in vivo coagulation pathway. Flow cytometry results indicated that r-EchAV could effectively bind to the platelet αIIbβ3 receptor, indicating that r-EchAV retains echistatin's receptor-recognition region. In vivo experiments in mice disclosed that r-EchAV significantly prolongs bleeding time, indicating a significant anticoagulant effect in vivo resulting from the joint binding of r-EchAV to both PS and the αIIbβ3 receptor. We also report optimization of the r-EchAV production steps and its purification for high purity and yield. Our findings indicate that r-EchAV retains the active structural regions of echistatin and annexin V and that the whole molecule exhibits multitarget-binding ability arising from the dual functions of echistatin and annexin V. Therefore, r-EchAV represents a new class of anticoagulant that specifically targets the anionic membrane-associated coagulation enzyme complexes at thrombogenesis sites and may be a potentially useful antithrombotic agent.

Molecular Characterization of Annexin B2, B3 and B12 in Taenia multiceps.

Coenurus cerebralis, the metacestode of Taenia multiceps, causes coenurosis, a disease severely affecting goat, sheep, cattle and yak farming and resulting in huge economic losses annually. Annexins bind calcium ions and play an important role in flatworm parasite development. To explore potential functions of annexins in T. multiceps, three homologous genes, namely, TmAnxB2, TmAnxB3 and TmAnxB12, were screened from the transcriptome dataset, amplified from C. cerebralis cDNA and subjected to bioinformatics analysis. Then, polyclonal antibodies recognizing the recombinant TmAnxB2 (rTmAnxB2) and rTmAnxB3 were prepared for localization of TmAnxB2 and TmAnxB3 in different tissues and developmental stages by immunofluorescence. The transcription of all three genes was also measured by relative fluorescent quantitative PCR. The sizes of rTmAnxB2, rTmAnxB3 and rTmAnxB12 were 58.00, 53.06 and 53.51 kDa, respectively, and rTmAnxB12 was unstable. Both rTmAnxB2 and rTmAnxB3 were recognized by goat-positive T. multiceps sera in Western blots. Immunofluorescence revealed that TmAnxB2 and TmAnxB3 were localized in the protoscolex and cyst wall and TmAnxB3 was also detected in adult cortex. TmAnxB2 and TmAnxB12 mRNA levels were determined to be highest in oncospheres and protoscolex, whereas transcription of TmAnxB3 was highest in scolex and immature segments. Taken together, these findings indicate that TmAnxB2 and TmAnxB12 may play critical roles in T. multiceps larvae, while TmAnxB3 may have important functions in adults. These results will lay the foundation for functional research of annexins in T. multiceps.

ANN1 and ANN2 Function in Post-Phloem Sugar Transport in Root Tips to Affect Primary Root Growth.

Annexins are a multigene family of calcium-dependent membrane-binding proteins that play important roles in plant cell signaling. Annexins are multifunctional proteins, and their function in plants is not comprehensively understood. Arabidopsis (Arabidopsis thaliana) annexins ANN1 and ANN2 are 64% identical in their primary structure, and both are highly expressed in seedlings. Here, we showed that ann-mutant seedlings grown in the absence of sugar show decreased primary root growth and altered columella cells in root caps; however, these mutant defects are rescued by Suc, Glc, or Fru. In seedlings grown without sugar, significant up-regulation of photosynthetic gene expression and chlorophyll accumulation was found in ann-mutant cotyledons compared to that in wild type, which indicates potential sugar starvation in the roots of ann-mutant seedlings. Unexpectedly, the overall sugar content of ann-mutant primary roots was significantly higher than that of wild-type roots when grown without sugar. To examine the diffusion of sugar along the entire root to the root tip, we examined the unloading pattern of carboxyfluorescein dye and found that post-phloem sugar transport was impaired in ann-mutant root tips compared to that in wild type. Increased levels of ROS and callose were detected in the root tips of ann-mutant seedlings grown without Suc, the latter of which would restrict plasmodesmal sugar transport to root tips. Our results indicate that ANN1 and ANN2 play an important role in post-phloem sugar transport to the root tip, which in turn indirectly influences photosynthetic rates in cotyledons. This study expands our understanding of the function of annexins in plants.

Annexins - insights from knockout mice.

Annexins are a highly conserved protein family that bind to phospholipids in a calcium (Ca2+) - dependent manner. Studies with purified annexins, as well as overexpression and knockdown approaches identified multiple functions predominantly linked to their dynamic and reversible membrane binding behavior. However, most annexins are found at multiple locations and interact with numerous proteins. Furthermore, similar membrane binding characteristics, overlapping localizations and shared interaction partners have complicated identification of their precise functions. To gain insight into annexin function in vivo, mouse models deficient of annexin A1 (AnxA1), A2, A4, A5, A6 and A7 have been generated. Interestingly, with the exception of one study, all mice strains lacking one or even two annexins are viable and develop normally. This suggested redundancy within annexins, but examining these knockout (KO) strains under stress conditions revealed striking phenotypes, identifying underlying mechanisms specific for individual annexins, often supporting Ca2+ homeostasis and membrane transport as central for annexin biology. Conversely, mice lacking AnxA1 or A2 show extracellular functions relevant in health and disease that appear independent of membrane trafficking or Ca2+ signaling. This review will summarize the mechanistic insights gained from studies utilizing mouse models lacking members of the annexin family.

The Tolerogenic Function of Annexins on Apoptotic Cells Is Mediated by the Annexin Core Domain

Immunological tolerance is constantly being maintained in the periphery by dendritic cells processing material from apoptotic cells (ACs) in the steady-state. Although research has focused on the uptake of ACs by phagocytes, tolerogenic signals exposed by the ACs are much less well defined. In this article, we show that the annexin (Anx) family members AnxA5 and AnxA13 translocate to the surface of ACs to function as redundant tolerogenic signals in vitro and in vivo. Exposure of bone marrow-derived dendritic cells to AnxA5 or AnxA13 in vitro resulted in the inhibition of both proinflammatory cytokine secretion and the upregulation of costimulatory molecules upon TLR stimulation. The highly conserved Anx core domain was sufficient to mediate these effects, whereas recognition by N-formyl peptide receptor family members was dispensable. In vivo, coinjection of OVA-expressing and Anx-expressing ACs prevented induction of Ag-specific CD8(+) T cells. Moreover, mice immunized with Anx-expressing ACs became refractory to an antigenic challenge. These results suggest that several Anxs contribute to AC-induced suppression of dendritic cell activation. Therefore, manipulating Anx-mediated immunosuppression may prove beneficial for patients with cancer or autoimmune diseases and chronic inflammatory disorders.

Annexin A4 is a novel direct regulator of adenylyl cyclase type 5.

Annexin A4 (AnxA4), a Ca(2+)- and phospholipid-binding protein, is up-regulated in the human failing heart. In this study, we examined the impact of AnxA4 on β-adrenoceptor (β-AR)/cAMP-dependent signal transduction. Expression of murine AnxA4 in human embryonic kidney (HEK)293 cells dose-dependently inhibited cAMP levels after direct stimulation of adenylyl cyclases (ACs) with forskolin (FSK), as determined with an exchange protein activated by cAMP-Förster resonance energy transfer (EPAC-FRET) sensor and an ELISA (control vs. +AnxA4: 1956 ± 162 vs. 1304 ± 185 fmol/µg protein; n = 8). Disruption of the anxA4 gene led to a consistent increase in intracellular cAMP levels in isolated adult mouse cardiomyocytes, with heart-directed expression of the EPAC-FRET sensor, stimulated with FSK, and as determined by ELISA, also in mouse cardiomyocytes stimulated with the β-AR agonist isoproterenol (ISO) (anxA4a(+/+) vs. anxA4a(-/-): 5.1 ± 0.3 vs. 6.7 ± 0.6 fmol/µg protein) or FSK (anxA4a(+/+) vs. anxA4a(-/-): 1891 ± 238 vs. 2796 ± 343 fmol/µg protein; n = 9-10). Coimmunoprecipitation experiments in HEK293 cells revealed a direct interaction of murine AnxA4 with human membrane-bound AC type 5 (AC5). As a functional consequence of AnxA4-mediated AC inhibition, AnxA4 inhibited the FSK-induced transcriptional activation mediated by the cAMP response element (CRE) in reporter gene studies (10-fold vs. control; n = 4 transfections) and reduced the FSK-induced phosphorylation of the CRE-binding protein (CREB) measured on Western blots (control vs. +AnxA4: 150 ± 17% vs. 105 ± 10%; n = 6) and by the use of the indicator of CREB activation caused by phosphorylation (ICAP)-FRET sensor, indicating CREB phosphorylation. Inactivation of AnxA4 in anxA4a(-/-) mice was associated with an increased cardiac response to β-AR stimulation. Together, these results suggest that AnxA4 is a novel direct negative regulator of AC5, adding a new facet to the functions of annexins.

Expression of theTpanxb1Gene fromTaenia pisiformisand Its Potential Diagnostic Value by Dot-ELISA

Cysticercosis, caused by the larvae of Taenia pisiformis, is a common disease in rabbits that results in economic losses. To date, there has been limited information available on the early detection of infection by this parasite. This study describes a dot-ELISA method based on an autologous antigen annexin B1 (Tpanxb1). Its potential for serodiagnosis of rabbit cysticercosis was also evaluated. Western blot analysis revealed that the recombinant Tpanxb1 (rTpanxb1) protein could be specifically recognized by rabbit anti-sera. In serum trials, the antibodies could be detected by dot-ELISA using rTpanxb1 at 14 days post-infection. The positive response was present for up to 49 days post-infection. Based on the necropsy results of 169 rabbit samples, the relative sensitivity and specificity of the dot-ELISA were 94.55% and 92.86%, respectively. This study provides a foundation for studying the immunological function of annexin and its application to control Taenia cestodes.

Annexin-Phospholipid Interactions. Functional Implications

Annexins constitute an evolutionary conserved multigene protein superfamily characterized by their ability to interact with biological membranes in a calcium dependent manner. They are expressed by all living organisms with the exception of certain unicellular organisms. The vertebrate annexin core is composed of four (eight in annexin A6) homologous domains of around 70 amino acids, with the overall shape of a slightly bent ring surrounding a central hydrophilic pore. Calcium- and phospholipid-binding sites are located on the convex side while the N-terminus links domains I and IV on the concave side. The N-terminus region shows great variability in length and amino acid sequence and it greatly influences protein stability and specific functions of annexins. These proteins interact mainly with acidic phospholipids, such as phosphatidylserine, but differences are found regarding their affinity for lipids and calcium requirements for the interaction. Annexins are involved in a wide range of intra- and extracellular biological processes in vitro, most of them directly related with the conserved ability to bind to phospholipid bilayers: membrane trafficking, membrane-cytoskeleton anchorage, ion channel activity and regulation, as well as antiinflammatory and anticoagulant activities. However, the in vivo physiological functions of annexins are just beginning to be established.

Expression and function of Annexin II in lung cancer tissue

ObjectiveTo explore the expression of Annexin II and its relationship with the cell differentiation, proliferation in lung cancer. MethodsRT-PCR and Western blot assays were used to detect the expression of Annexin II in lung cancer tissues and cell lines. ResultsAnnexin II was significantly up-regulated in lung cancer tissues, and in lung cancer cell lines, Annexin II had higher mRNA and protein expressions. ConclusionsAnnexin II is up-regulated in lung cancer, suggesting that the Annexin II has a potential value in the human lung cancer.

Insight into plant annexin function

The multifunctionality of plant annexins and their importance for coordinating development and responses to biotic and abiotic environment have been largely reviewed. We recently described a tobacco annexin, named Ntann12, which is mainly localized in the nucleus of root cells when the plant is grown under light conditions. We also found that auxin and polar auxin transport are essential for Ntann12 accumulation in root cells. Under dark condition, Ntann12 is no longer detected in the root system. In the present addendum, light, regulating auxin signaling, is evidenced as an essential determinant for the synchronization of growth and development between the shoot and the root during light/dark cycle. A speculative model for Ntann12 is described and discussed with regards to relevant literature data.

Probing Anticoagulant Fondaparinux for Interference with Prothrombotic B2GPI/Anti-B2GPI Antibody Complexes

Abstract 1209 Background:The presence of autoimmune antibodies directed to beta2-glycoprotein-I (B2GPI) often leads to thrombosis in antiphospholipid syndrome (APS). Heparin, low molecular weight heparin (LMWH) and fondaparinux are commonly used for prophylaxis and treatment of thromboses in APS. These drugs bind and activate antithrombin III to inactivate blood clotting proteases. Fondaparinux is a synthetic pentasaccharide matching a specific sequence within heparin interacting with antithrombin. Aim: We investigated if fondaparinux can bind B2GPI and ameliorate prothrombotic properties of B2GPI/anti-B2GPI antibody complexes. Results: We found that fondaparinux interacts with B2GPI and that the binding is dominated by electrostatic interactions. We measured the binding affinity by monitoring changes in the intrinsic fluorescence of domain V of B2GPI (B2GPI-DV) upon titration with fondaparinux. In the presence of 100 mM NaCl, the binding affinity was about 1.5 uM and stoichiometry of the binding is 1:1. Using solution NMR spectroscopy, we determined that the binding interface of the complex is centered on Lys251 of B2GPI-DV. This observation was confirmed by site-directed mutagenesis. The Lys251/Asp mutant fails to bind B2GPI-DV. Interestingly, the binding site for fondaparinux on B2GPI does not overlap with the major binding site for heparin. Cellular activation by the binding of B2GPI/anti-B2GPI antibody complexes with cell-surface receptors (among them ApoER2, a lipoprotein receptor from the LDLR family) and interference with the protective function of annexin V on anionic phospholipids expressed on the surfaces of activated cells are two potential prothrombotic mechanisms of B2GPI/antibody complexes. We found that fondaparinux does not prevent the association of the ligand-binding modules from ApoER2 with B2GPI-DV. Therefore, fondaparinux does not interfere with the binding of B2GPI/anti-B2GPI antibody complexes with lipoprotein receptors. Neither fondaparinux, nor heparin and LMWH were effective in inhibiting the binding of B2GPI/anti-B2GPI antibody complexes to cardiolipin-coated plates suggesting that these drugs do not prevent the destructive effect of B2GPI/antibody complexes on antithrombotic function of annexin V. Conclusions: At therapeutic concentrations, fondaparinux forms only small number of complexes with B2GPI, given that the binding affinity of the complex is in a micromolar range. When bound to B2GPI, fondaparinux does not interfere with the binding of B2GPI/anti-B2GPI antibody complexes to lipoprotein receptors and anionic phospholipids. Disclosures:No relevant conflicts of interest to declare.

Large-scale Identification of Calcium Oxalate Monohydrate Crystal-binding Proteins on Apical Membrane of Distal Renal Tubular Epithelial Cells

Adhesion of calcium oxalate monohydrate (COM) crystals onto apical surface of renal tubular epithelial cells is a crucial mechanism for crystal retention, leading to kidney stone formation. Various proteins on apical membrane may bind to COM crystals; however, these crystal-binding proteins remained unidentified. The present study therefore aimed to identify COM crystal-binding proteins on apical membrane of distal renal tubular epithelial cells. Madin-Darby Canine Kidney (MDCK) cells were cultivated to be polarized epithelial cells and apical membrane was isolated from these cells using a peeling method established recently. Enrichment and purity of isolated apical membrane were confirmed by Western blot analysis for specific markers of apical (gp135) and basolateral (Na(+)/K(+)-ATPase) membranes. Proteins derived from the isolated apical membrane were then resuspended in artificial urine and incubated with COM crystals. The bound proteins were eluted, resolved by SDS-PAGE, and analyzed by Q-TOF MS and MS/MS, which identified 96 proteins. Among these, expression and localization of annexin II on apical surface of MDCK cells were confirmed by Western blot analysis, immunofluorescence staining, and laser-scanning confocal microscopic examination. Finally, the function of annexin II as the COM crystal-binding protein was successfully validated by COM crystal-binding assay. This large data set offers many opportunities for further investigations of kidney stone disease and may lead to the development of new therapeutic targets.

The Expression Pattern of Annexin A1 in Urinary Bladder Urothelial Carcinoma and Its Clinicopathologic Significance

Annexin A1 (ANXA1) is a 37-kDa protein with calcium and phospholipid binding properties. It is a member of the subfamily A of annexin (ANX), and presents in organisms from molds to mammals. ANX shares a conserved COOH-terminal with repeated domains and an N-terminal region, the variability of which confers specific annexin function. Its anti-inflammatory property is attributed to ANXA1 binding to surface receptors of granulocytes and macrophages, and its ability to inhibit phospholipase A2. It plays various roles in the regulation of apoptosis, intracellular signal transduction, differentiation, and proliferation. ANXA1 is also known to be involved in the carcinogenesis of many tumors. The expression of ANXA1 is increased in hairy cell leukemia, pancreatic cancers, and hepatocellular carcinoma. However, it is decreased in cancers of esophagus, larynx, prostate and B-cell lymphoma. ANX is known to be localized in the cytoplasm and moves to the membrane or the nucleus depending on calcium level and stimulation. Recent studies have shown that specific subcellular localization of ANXA1 is associated with epithelial differentiation and may be a prognostic factor in oral squamous cell carcinoma (SCC). Urothelial carcinoma (UC) of the urinary bladder is the seventh most common cancer worldwide, and comprises approximately 90% of all primary tumors of this organ. Development of UC is associated with chemical exposure, cigarette smoking, schistosomal infections, and genetic alterations. UC is known to progress along two divergent pathways: one is characterized by low grade non-invasive papillary tumors preceded by hyperplasia. The other is that of high grade invasive tumors, which progress from carcinoma in situ (CIS) or arise de novo. While low grade papillary tumors have frequent mutations of HRAS and fibroblast growth factor receptor 3 (FGFR3) genes activating the receptor tyrosine kinase-RAS pathway, high grade invasive tumors harbor defects in the p53 and/or the retinoblastoma protein (RB) pathways. Recently, ANXA1 emerges as a tumorrelated protein in UC, however, the significance of ANXA1 in bladder carcinogenesis is not yet fully understood and study results remain inconsistent. Cui et al. showed that ANXA1 expression was down-regulated in higher histologic grade of UC, but Li et al. reported contradictory outcome with their The Expression Pattern of Annexin A1 in Urinary Bladder Urothelial Carcinoma and Its Clinicopathologic Significance