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Background Finding effective and safe therapeutic drugs for atrial fibrillation (AF) is an important concern for clinicians. Proteome-wide Mendelian randomization analysis provides new ideas for finding potential drug targets. Methods and Results Using a proteome-wide Mendelian randomization approach, we assessed the genetic predictive causality between thousands of proteins and AF risk and found that genetically predicted plasma levels of phosphomevalonate kinase, tumor necrosis factor ligand superfamily member 12, sulfhydryl oxidase 2, interleukin-6 receptor subunit alpha, and low-affinity immunoglobulin gamma Fc region receptor II-b might decrease AF risk, while genetically predicted plasma levels of beta-mannosidase, collagen alpha-1(XV) chain, ANXA4 (annexin A4), COF2 (cofilin-2), and RAB1A (Ras-related protein Rab-1A) might increase AF risk ( P <3.4×10 -5 ). By using different Mendelian randomization methods and instrumental variable selection thresholds, we performed sensitivity analyses in 30 scenarios to test the robustness of positive findings. Replication analyses were also performed in independent samples to further avoid false-positive findings. Drugs targeting tumor necrosis factor ligand superfamily member 12, interleukin-6 receptor subunit alpha, low-affinity immunoglobulin gamma Fc region receptor II-b, and annexin A4 are approved or in development. The results of the phenome-wide Mendelian randomization analysis showed that changing the plasma levels of phosphomevalonate kinase, cofilin-2, annexin A4, Ras-related protein Rab-1A, sulfhydryl oxidase 2, and collagen alpha-1(XV) chain did not increase the risk of other diseases while decreasing the risk of AF. Conclusions We found a significant causal association between genetically predicted levels of 10 plasma proteins and AF risk. Four of these proteins have drugs targeting them that are approved or in development, and our results suggest the potential for these drugs to treat AF or cause AF. Sulfhydryl oxidase 2, low-affinity immunoglobulin gamma Fc region receptor II-b, and beta-mannosidase have not been suggested by previous laboratory or epidemiological studies to be associated with AF and may reveal new pathophysiological pathways as well as therapeutic targets for AF.

Objective: In the present study, we aimed to explore lncRNA HOXD cluster antisense RNA 1 (HOXD-AS1) expression in oral squamous cell carcinoma (OSCC) tissues, its biological roles, and the underlying potential mechanisms in OSCC progression., Materials and Methods: HOXD-AS1 expression in paired OSCC and non-tumor tissues from 60 OSCC patients was determined by RT-qPCR. The effects of HOXD-AS1 and miR-203a-5p overexpression on expression of Annexin A4, a validated target of miR-203a-5p, were analyzed by RT-qPCR and Western blot. The roles of HOXD-AS1, miR-203a-5p, and Annexin A4 in the invasion and migration of OSCC cells were analyzed by Transwell assays., Results: HOXD-AS1 overexpression in OSCC predicted poor survival. HOXD-AS1 was predicted to interact with miR-203a-5p, but its expression was not significantly correlated with miR-203a-5p. HOXD-AS1 overexpression increased Annexin A4 expression, while miR-203a-5p overexpression decreased Annexin A4 expression in OSCC cells. Transwell assays showed that invasion and migration of OSCC cells were enhanced by HOXD-AS1 and Annexin A4 overexpression but were reduced by miR-203a-5p overexpression. In addition, miR-203a-5p overexpression suppressed the role of HOXD-AS1 in cell movement and Annexin A4 expression., Conclusions: HOXD-AS1 may upregulate Annexin A4 by sponging miR-203a-5p in OSCC to promote cancer cell invasion and migration., (© 2022 The Authors. Oral Diseases published by Wiley Periodicals LLC.)

Annexin A4 (encoded by the ANXA4 gene) is a calcium ion (Ca 2+ )- and phospholipid-binding protein of the Annexin family. In this study, we checked the expression profile of ANXA4 in basal-like breast cancer (BLBC) and its association with survival outcomes using pan-cancer data from The Cancer Genome Atlas (TCGA) and the Genotype-Tissue Expression (GTEx) project. Then, using MDA-MB-231 and MDA-MB-468 cells, we explored the functional role of ANXA4 in regulating a cancer-related signaling pathway and identified potential partners of ANXA4. The results showed that expression of total ANXA4 and the two dominant ANXA4 protein-coding transcripts (ENST00000409920.5 and ENST00000394295.4) was consistently upregulated in tumor tissues compared with normal breast tissues. BLBC patients with high ANXA4 expression had significantly worse overall survival, progression-free survival, and disease-free survival than those with low ANXA4 expression. ANXA4 could positively modulate cyclin D1 expression and G1/S progression in the two cell lines. An in vivo tumor model showed that ANXA4 inhibition significantly slowed the growth of tumors derived from the two BLBC cell lines. ANXA4 could increase JAK1 expression and STAT3 phosphorylation (Y705). ANXA4 colocalized with ANXA1 in some MDA-MB-231 cells. A co-immunoprecipitation assay confirmed direct binding between ANXA4 and ANXA1. Knockdown of ANXA1 reduced JAK1 expression and STAT3 phosphorylation and impaired ANXA4-induced upregulation of JAK1 and p-STAT3. In conclusion, this study revealed that aberrant ANXA4 upregulation is associated with poor survival in BLBC. ANXA4 could activate JAK-STAT3 signaling by elevating the expression of JAK1 and p-STAT3, which was mediated by direct interaction with ANXA1.

Many studies have shown that downregulated miR-203 level is in a variety of cancers including gastric cancer (GC). However, the precise molecule mechanisms of miR-203 in GC have not been well clarified. In the current study, we investigated the biological functions and molecular mechanisms of miR-203 in GC cell lines. We found that miR-203 is downregulated in GC tissues and cell lines. Moreover, the low level of miR-203 was associated with increased expression of annexin A4 in GC tissues and cell lines. The invasion and EMT of GC cells were suppressed by overexpression of miR-203. However, downregulation of miR-203 promoted invasion and EMT of GC cells. Bioinformatics analysis predicted that annexin A4 was a potential target gene of miR-203. Next, luciferase reporter assay confirmed that miR-203 could directly target annexin A4. Consistent with the effect of miR-203, downregulation of annexin A4 by siRNA inhibited the invasion and EMT of GC cells. Introduction of annexin A4 in GC cells partially blocked the effects of miR-203 mimic. Introduction of miR-203 directly targeted annexin A4 to inhibit the invasion and EMT of GC cells. Overall, reactivation of the miR-203/annexin A4 axis may represent a new strategy for overcoming metastasis of GC.

The present study aimed to investigate the association between Lewis(y) antigen and chemoresistance in ovarian cancer and to elucidate the underlying molecular mechanisms. Lewis(y) expression in chemoresistant ovarian cancer tissues and cells was detected by immunohistochemistry. α1,2‑fucosyltransferase (FUT1) expression in different ovarian cancer chemotherapy-resistant cells was analyzed by reverse transcription-quantitative PCR (RT-qPCR). Genes differentially expressed in the chemoresistant and sensitive groups were screened using a gene chip followed by validation using RT-qPCR and western blot analysis. We found that Lewis(y) and FUT1 expression in ovarian cancer cells was significantly increased following the induction of drug resistance. The positive expression rate and intensity of Lewis(y) in ovarian cancer chemoresistant tissues were also significantly higher than those in the sensitive group. Compared with the non-resistant cell lines, the differentially expressed genes were mainly enriched in the terms related to the transmembrane receptor protein tyrosine kinase signaling pathway and positive regulation of cell proliferation. Interaction network analysis predicted genes participating in the regulation of apoptotic processes. The highly differential expression of Annexin A4 (ANXA4), BCL2 interacting killer (BIK), transmembrane 4 L six family member 4 (TM4SF4) and pleckstrin homology-like domain family A member 1 (PHLDA1) was validated using RT-qPCR in ovarian cancer cell lines. Finally, ANXA4 expression was increased at both the mRNA and protein level in the drug‑resistant cells, and in addition, ANXA4 contained a Lewis(y) structure. The expression of Bcl-2 and other anti-apoptotic proteins increased with the increase of Lewis(y) expression. After blocking Lewis(y) using an antibody, the expression of the involved signaling pathway and apoptosis-related proteins decreased significantly. These findings provide strong evidence that Lewis(y) is a component of the structure of the ANXA4 membrane protein. Its overexpression can abnormally activate signaling pathways and regulate the expression of a number of factors, forming a positive feedback loop to induce the chemoresistance of ovarian cancer cells, and ultimately promoting the progression of ovarian cancer.

Genetic studies have shown that FGF10/FGFR2 signaling is required for airway branching morphogenesis and FGF10 functions as a chemoattractant factor for distal epithelial cells during lung development. However, the detail downstream cellular and molecular mechanisms have not been fully characterized. Using live imaging of ex vivo cultured lungs, we found that tip airway epithelial progenitor cells migrate faster than cleft cells during airway bud formation and this migration process is controlled by FGFR2-mediated ERK1/2 signaling. Additionally, we found that airway progenitor cells that migrate faster tend to become distal airway progenitor cells. We identified that Anxa4 is a downstream target of ERK1/2 signaling. Anxa4 -/- airway epithelial cells exhibit a "lag-behind" behavior and tend to stay at the stalk airways. Moreover, we found that Anxa4-overexpressing cells tend to migrate to the bud tips. Finally, we demonstrated that Anxa4 functions redundantly with Anxa1 and Anxa6 in regulating endoderm budding process. Our study demonstrates that ERK1/2/Anxa4 signaling plays a role in promoting the migration of airway epithelial progenitor cells to distal airway tips and ensuring their distal cell fate.

Efficient cell membrane repair mechanisms are essential for maintaining membrane integrity and thus for cell life. Here we show that the Ca 2+ - and phospholipid-binding proteins annexin A4 and A6 are involved in plasma membrane repair and needed for rapid closure of micron-size holes. We demonstrate that annexin A4 binds to artificial membranes and generates curvature force initiated from free edges, whereas annexin A6 induces constriction force. In cells, plasma membrane injury and Ca 2+ influx recruit annexin A4 to the vicinity of membrane wound edges where its homo-trimerization leads to membrane curvature near the edges. We propose that curvature force is utilized together with annexin A6-mediated constriction force to pull the wound edges together for eventual fusion. We show that annexin A4 can counteract various plasma membrane disruptions including holes of several micrometers indicating that induction of curvature force around wound edges is an early key event in cell membrane repair.

Aim: To explore disease-associated molecules in rheumatoid arthritis (RA), we comprehensively analyzed phosphoproteins purified from RA synoviocytes., Method: Synoviocytes were obtained from three patients with RA and three patients with osteoarthritis (OA). Profiles of phosphoproteins purified from the synoviocytes were compared by two-dimensional differential gel electrophoresis (2D-DIGE) between the RA and OA groups. Protein spots with significantly different phosphorylation levels were identified by mass spectrometry. Recombinant protein of annexin A4 (ANXA4), one of the identified phosphoproteins, was transfected into synoviocytes from an OA patient to mimic RA synoviocytes and humoral factor secretion was compared between rANXA4-transfected and non-transfected synoviocytes under a tumor necrosis factor-α (TNFα)-stimulated condition., Results: In 2D-DIGE, 318 phosphoprotein spots were detected, of which 94 spots showed significantly different intensities between the two groups (P < 0.05). Among the 94 spots, 22 spots showed two-fold or higher intensity and one spot showed less than 1/2-fold intensity in the RA group compared to the OA group. From the 22 spots, 11 phosphoproteins were identified, which included kinases, carrier and chaperone proteins, cytoskeletal proteins, proteases and calcium-binding proteins. One of the identified calcium-binding proteins was ANXA4, an exocytosis-regulating protein. The transfected rANXA4 was found to be phosphorylated intracellularly, and secretion of chemokine (C-X-C motif) ligand 1 and interleukin-8 induced by TNFα stimulation was significantly suppressed by the transfection (P < 0.01)., Conclusion: The phosphoprotein profile of RA synoviocytes was different from that of OA synoviocytes. This difference would reflect the different pathophysiologies of the diseases. ANXA4 may be one of therapeutic targets in RA., (© 2017 Asia Pacific League of Associations for Rheumatology and John Wiley & Sons Australia, Ltd.)

The prognosis of hepatocellular carcinoma (HCC) after surgical resection remains unsatisfactory for the majority of HCC patients who developed early recurrence or metastasis. There is still a lack of reliable biomarkers that can be used to predict the possibility of recurrence/metastasis in HCC patients after operation. In the current study, annexin A4, a calcium-dependent phospholipid-binding protein, has been found to be significantly elevated in HCC patients with early recurrence/metastasis, and had a strong correlation with portal vein tumor thrombosis (p = 0.03) and advanced BCLC stage (p = 0.002). Cox proportional hazards regression analysis revealed that annexin A4 was an independent prognostic predictor for both early recurrence/metastasis (HR = 1.519, p = 0.032) and overall survival (HR = 1.827, p = 0.009) after surgical resection. Meanwhile, Kaplan-Meier analysis showed that Patients with high-expression levels of annexin A4 had higher recurrence rate and shorter overall survival than those with low expression (log-rank test, p < 0.001). Furthermore, in vitro studies have demonstrated that overexpression of annexin A4 facilitated HCC cell migration and invasion via regulating epithelial-mesenchymal transition (EMT). In conclusion, annexin A4 has played important roles in the progression of HCC, and might act as a potential prognostic biomarker for HCC.

Annexin A4 (ANXA4) is a member of the annexin family that binds to both calcium ions and phospholipids. Studies indicate that ANXA4 modulates membrane permeability and membrane trafficking, participates in cellular growth and apoptosis, enhances tumor invasion and promotes anti-tumor drug resistance. The overexpression of ANXA4 has been identified in various clinical epithelial tumors including: lung, gastric, colorectal, pancreatic, gallbladder, breast, renal, ovarian, laryngeal, and prostate cancers. In addition, upregulation and nuclear translocation of ANXA4 have been observed in the progression of colorectal cancer and ovarian serous carcinoma. Knockdown of ANXA4 attenuated migration in ovarian cancer and breast cancer cells. In contrast, knockdown of ANXA4 increased susceptibility to platinum in ovarian cancer and malignant mesothelioma cells. It is conceivable that ANXA4 is an indicator for tumor development, invasion, chemo-resistance, poor outcomes of cancer patients, and may be a potential target for therapeutic intervention.

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., (© FASEB.)

Large protein complexes form in the cytosol of prokaryotes and eukaryotes as assemblies of functional enzymes or aggregates of misfolded proteins. Their roles in the cell range from critical components of metabolism to disease-causing agents. We have observed a novel structure in the cells of transgenic Arabidopsis thaliana that appears to be a form of inclusion body. These long, spindle-shaped structures form when Arabidopsis are transformed to express high levels of the protein Annexin4 fused to a fluorescent protein. These structures, previously named darts, are visible in all cells of the plant throughout development. Darts take on a variety of morphologies including rings and figure-eights. These structures are not associated with the endomembrane system and are not membrane bounded. Darts appear to be insoluble aggregates of protein analogous to bacterial inclusion bodies and eukaryotic aggresomes. Similar structures have not been observed in untransformed plants, suggesting darts are artifacts of transgenic overexpression., (Copyright © 2015 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Epithelial ovarian cancer (EOC) consists of four major subtypes: clear cell carcinoma (CCC), endometrioid adenocarcinoma (EA), mucinous adenocarcinoma (MA) and serous adenocarcinoma (SA). Relative to the other subtypes, the prognosis of CCC is poor due to a high recurrence rate and chemotherapy resistance, but CCC-specific biomarkers have yet to be identified. With the aim of identifying diagnostic and treatment biomarkers for CCC, we analyzed 96 cases of EOC (32 CCC, 13 EA, 19 MA, 32 SA) using liquid chromatography/mass spectrometry (LC/MS) followed by immunohistochemistry (IHC) and quantitative reverse transcription PCR (RT-qPCR). Semi-quantification of protein differences between subtypes showed upregulation of 150 proteins and downregulation of 30 proteins in CCC relative to the other subtypes. Based on hierarchical clustering that revealed a marked distinction in the expression levels of cystatin B (CYTB) and Annexin A4 (ANXA4) in CCC relative to the other subtypes, we focused the study on CYTB and ANXA4 expression in EOCs by IHC, RT-qPCR and western blot analyses using tissue specimens and cultured cells. As a result, compared to the other subtypes, CCC showed significantly high expression levels of CYTB and ANXA4 in the analyses. To examine the possibility of CYTB and ANXA4 as serum diagnostic biomarkers of CCC, we checked the protein levels in conditioned media and cell lysates using culture cells. Compared with the other subtypes, CCC cell lines showed a significantly higher level of expression of CYTB in both conditioned media and cell lysates, while ANXA4 showed a higher level of expression in cell lysates only. Our results demonstrate that CYTB and ANXA4 overexpression may be related to carcinogenesis and histopathological differentiation of CCC. CYTB may be a secreted protein, and may serve as a potential serum diagnostic biomarker of CCC, while ANXA4 may be useful as an intracellular marker.

Annexin A4 (Anx4) is a cytosolic calcium-binding protein with four repeat domains, each containing one calcium-binding site (CBS). The protein interacts with the phospholipid membrane through the CBS-coordinated calcium ion, although the role of each CBS in the calcium-dependent association is unclear. To determine the role of each CBS, 15 CBS-abolished variants were produced in various combinations by substitution of a calcium-liganding residue on each CBS by Ala. Various mutant combinations produced different influences on calcium-dependent membrane-binding behavior and on the sodium-dependent dissociation of membrane-bound Anx4. Our data suggest the interaction of Anx4 with the lipid membrane consists of strong and weak interactions. CBSs I and IV mediate formation of strong interactions, while CBSs II and III are important for weak interactions. We also suggest Anx4 binds the lipid membrane through CBSs I and IV in the cytoplasmic fluids.

To gain insight into liver and pancreas development, we investigated the target of 2F11, a monoclonal antibody of unknown antigen, widely used in zebrafish studies for labeling hepatopancreatic ducts. Utilizing mass spectrometry and in vivo assays, we determined the molecular target of 2F11 to be Annexin A4 (Anxa4), a calcium binding protein. We further found that in both zebrafish and mouse endoderm, Anxa4 is broadly expressed in the developing liver and pancreas, and later becomes more restricted to the hepatopancreatic ducts and pancreatic islets, including the insulin producing ß-cells. Although Anxa4 is a known target of several monogenic diabetes genes and its elevated expression is associated with chemoresistance in malignancy, its in vivo role is largely unexplored. Knockdown of Anxa4 in zebrafish leads to elevated expression of caspase 8 and Δ113p53, and liver bud specific activation of Caspase 3 and apoptosis. Mosaic knockdown reveal that Anxa4 is required cell-autonomously in the liver bud for cell survival. This finding is further corroborated with mosaic anxa4 knockout studies using the CRISPR/Cas9 system. Collectively, we identify Anxa4 as a new, evolutionarily conserved hepatopancreatic factor that is required in zebrafish for liver progenitor viability, through inhibition of the extrinsic apoptotic pathway. A role for Anxa4 in cell survival may have implications for the mechanism of diabetic ß-cell apoptosis and cancer cell chemoresistance., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Ovarian clear cell adenocarcinoma (CCC) is the second most common subtype of ovarian cancer after high-grade serous adenocarcinomas. CCC tends to develop resistance to the standard platinum-based chemotherapy, and has a poor prognosis when diagnosed in advanced stages. The ANXA4 gene, along with its product, a Ca(++)-binding annexin A4 (ANXA4) protein, has been identified as the CCC signature gene. We reported two subtypes of ANXA4 with different isoelectric points (IEPs) that are upregulated in CCC cell lines. Although several in vitro investigations have shown ANXA4 to be involved in cancer cell proliferation, chemoresistance, and migration, these studies were generally based on its overexpression in cells other than CCC. To elucidate the function of the ANXA4 in CCC cells, we established CCC cell lines whose ANXA4 expressions are stably knocked down. Two parental cells were used: OVTOKO contains almost exclusively an acidic subtype of ANXA4, and OVISE contains predominantly a basic subtype but also a detectable acidic subtype. ANXA4 knockdown (KO) resulted in significant growth retardation and greater sensitivity to carboplatin in OVTOKO cells. ANXA4-KO caused significant loss of migration and invasion capability in OVISE cells, but this effect was not seen in OVTOKO cells. We failed to find the cause of the different IEPs of ANXA4, but confirmed that the two subtypes are found in clinical CCC samples in ratios that vary by patient. Further investigation to clarify the mechanism that produces the subtypes is needed to clarify the function of ANXA4 in CCC, and might allow stratification and improved treatment strategies for patients with CCC.

Fhit protein is lost or reduced in a large fraction of human tumors, and its restoration triggers apoptosis and suppresses tumor formation or progression in preclinical models. Here, we describe the identification of candidate Fhit-interacting proteins with cytosolic and plasma membrane localization. Among these, Annexin 4 (ANXA4) was validated by co-immunoprecipitation and confocal microscopy as a partner of this novel Fhit protein complex. Here we report that overexpression of Fhit prevents Annexin A4 translocation from cytosol to plasma membrane in A549 lung cancer cells treated with paclitaxel. Moreover, paclitaxel administration in combination with AdFHIT acts synergistically to increase the apoptotic rate of tumor cells both in vitro and in vivo experiments.

Aspirin-exacerbated respiratory disease (AERD) is a clinical syndrome characterized by bronchoconstriction after ingestion of nonsteroidal anti-inflammatory drugs including aspirin. The Ca concentration in bronchial epithelial cells is an important factor for bronchoconstriction. Human annexin A4 (ANXA4) is predominantly expressed in the secretory epithelia in the lung, stomach, intestine, and kidney. Furthermore, translocation and induction of ANXA4 have been observed in human Ca-depleted neutrophils. To investigate the association between annexin A4 polymorphisms and the risk of AERD, we have genotyped 21 variants from 102 AERD subjects and 429 aspirin-tolerant asthma (ATA) controls. Logistic analyses controlling for sex, smoking status, and atopy as covariates were performed to estimate the association between the annexin A4 polymorphisms and AERD. Among these variants, 8 polymorphisms (rs2168116, rs4853017, rs6546547, rs13428251, rs7577864, rs7559354, rs7588022, and rs3816491) and 2 haplotypes (ANXA4-ht3 and ANXA4-ht5) were significantly associated with the risk of AERD. One common polymorphism in intron 11, rs3816491, showed the strongest association signal with susceptibility to aspirin-AERD even after multiple testing corrections (OR=0.57; 95% confidence interval 0.40-0.83; P=0.003; P=0.045 in the codominant model). Although further functional evaluations of replication studies in larger cohorts are required, our findings suggest that the annexin A4 could have susceptibility for AERD.

Genetically encoded FRET (Foerster resonance energy transfer) sensors are exciting tools in modern cell biology. Changes in the conformation of a sensor lead to an altered emission ratio and provide the means to determine both temporal and spatial changes in target molecules, as well as the activity of enzymes. FRET sensors are widely used to follow phosphorylation events and to monitor the effects of elevated calcium levels. Here, we report for the first time, to our knowledge, on the analysis of the conformational changes involved in sensor function at low resolution using a combination of in vitro and in cellulo FRET measurements and small-angle scattering of x rays (SAXS). The large and dynamic structural rearrangements involved in the modification of the calcium- and phosphorylation-sensitive probe CYNEX4 are comprehensively characterized. It is demonstrated that the synergistic use of SAXS and FRET methods allows one to resolve the ambiguities arising due to the rotation of the sensor molecules and the flexibility of the probe., (Copyright © 2012 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Mesothelioma is a highly malignant tumor with a poor prognosis and limited treatment options. Although cisplatin (CDDP) is an effective anticancer drug, its response rate is only 20%. Therefore, discovery of biomarkers is desirable to distinguish the CDDP-susceptible versus resistant cases. To this end, differential proteome analysis was performed to distinguish between mesothelioma cells of different CDDP susceptibilities, and this revealed that expression of annexin A4 (ANXA4) protein was higher in CDDP-resistant cells than in CDDP-susceptible cells. Furthermore, ANXA4 expression levels were higher in human clinical malignant mesothelioma tissues than in benign mesothelioma and normal mesothelial tissues. Finally, increased susceptibility was observed following gene knockdown of ANXA4 in mesothelioma cells, whereas the opposite effect was observed following transfection of an ANXA4 plasmid. These results suggest that ANXA4 has a regulatory function related to the cisplatin susceptibility of mesothelioma cells and that it could be a biomarker for CDDP susceptibility in pathological diagnoses., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Annexin A1 (ANXA1) is a well-known anti-inflammatory protein that is expressed on the surface of apoptotic cells. Annexin A4 (ANXA4) is also recruited from the nucleus to the cytoplasm in apoptotic cells, although it is not known whether or not ANXA4 is expressed on the surface of apoptotic cells. In this study, we obtained rabbit anti-human ANXA1 and ANXA4 antibodies, and then examined whether or not ANXA1 and ANXA4 are expressed on the surface of early and late human apoptotic cells. ANXA1 and, to a lesser extent, ANXA4 were detected on late but not early apoptotic HeLa cells, whereas ANXA1 and a small amount of ANXA4 were detected on both early and late apoptotic human neutrophils. We then examined the effects of the anti-human ANXA1 and ANXA4 antibodies on the mouse or human macrophage response to human apoptotic cells. Upon coculturing of mouse or human macrophages with late apoptotic human neutrophils, anti-human ANXA1 antibodies and, to a lesser extent, anti-human ANXA4 antibodies increased MIP-2 or IL-8 production significantly, suggesting that ANXA1 and ANXA4 suppress MIP-2 or IL-8 production by macrophages in response to late apoptotic human neutrophils., (Copyright © 2011 Elsevier B.V. All rights reserved.)

NME/NDPK family proteins are involved in the control of intracellular nucleotide homeostasis as well as in both physiological and pathological cellular processes, such as proliferation, differentiation, development, apoptosis, and metastasis dissemination, through mechanisms still largely unknown. One family member, NME1/NDPK-A, is a metastasis suppressor, yet the primary physiological functions of this protein are still missing. The purpose of this study was to identify new NME1/NDPK-A-dependent biological functions and pathways regulated by this gene in the liver. We analyzed the proteomes of wild-type and transgenic NME1-null mouse livers by combining two-dimensional gel electrophoresis and mass spectrometry (matrix-assisted laser desorption/ionization time of flight and liquid chromatography-tandem mass spectrometry). We found that the levels of three proteins, namely, phenylalanine hydroxylase, annexin IV, and elongation factor 1 Bα (EF-1Bα), were strongly reduced in the cytosolic fraction of NME1(-/-) mouse livers when compared to the wild type. This was confirmed by immunoblotting analysis. No concomitant reduction in the corresponding messenger RNAs or of total protein level was observed, however, suggesting that NME1 controls annexin IV and EF-1Bα amounts by post-translational mechanisms. NME1 deletion induced a change in the subcellular location of annexin IV in hepatocytes resulting in enrichment of this protein at the plasma membrane. We also observed a redistribution of EF-1Bα in NME1(-/-) hepatocytes to an intracytoplasmic compartment that colocalized with a marker of the reticulum endoplasmic. Finally, we found reduced expression of annexin IV coincident with decreased NME1 expression in a panel of different carcinoma cell lines. Taken together, our data suggest for the first time that NME1 might regulate the subcellular trafficking of annexin IV and EF-1Bα. The potential role of these proteins in metastatic dissemination is discussed.

The protein annexin IV (ANX4) is elevated specifically and characteristically in ovarian clear cell adenocarcinoma (CCA), a highly malignant histological subtype of epithelial ovarian cancer. On the basis of the hypothesis that the expression of ANX4 in CCA is regulated by a unique transcription mechanism, we explored the cis-elements involved in CCA-specific ANX4 expression using a luciferase reporter. We compared the transcriptional activities of the region from -1534 to +1010 relative to the ANX4 transcription start site in CCA and non-CCA-type cell lines, and found that two repeated binding motifs for the tumor suppressor protein, p53, in the first intron of ANX4 were involved in CCA-specific transcriptional activity. Furthermore, chromatin immunoprecipitation showed that endogenous p53 bound to this site in CCA cell lines. Moreover, the use of short interference RNA to silence the p53 gene decreased the transcriptional activity and mRNA expression of ANX4 in CCA cell lines. Thus, the ANX4 gene is, at least in part, regulated by p53 in CCA cells. Mutations in the p53 gene were absent and levels of p53 target genes were higher in several CCA-derived cell lines. Although the expression of ANX4 is typically low in these non-CCA cell lines, ANX4 levels were elevated more than three-fold by the overexpression of wild-type but not mutant p53. Therefore, we conclude that the ANX4 gene is a direct transcriptional target of p53, and its expression is enhanced by wild-type p53 in CCA cells., (© 2011 The Authors Journal compilation © 2011 FEBS.)

Background: Annexins (ANXAs) belong to a superfamily of closely related calcium and membrane-binding proteins and are overexpressed in some carcinomas. The overexpression of ANXAs presumably induces an autoantibody response, but this has not been demonstrated for sera from colorectal cancer (CRC) patients., Study: We examined serum samples from 220 CRC patients and 216 healthy volunteers to evaluate the ANXA autoantibody response in patients by using an enzyme-linked immunosorbent assay with recombinant ANXA A4 as the antigen., Results: The sensitivity of the anti-ANXA response from CRC patient sera was about 85% and the specificity was about 61.6%. When a cut-off value of 5.0 ng/mL was chosen for carcinoembryonic antigen (CEA) in the same sera samples, the sensitivity and specificity values were 43.2% and 85.2%, respectively. Combined detection using ANXA autoantibodies and CEA produced better sensitivity (71.8%) and specificity (79.2%) compared with CEA sensitivity (43.2%) and anti-ANXA specificity (61.6%). The area under a receiver operating characteristic curve was 0.794 for ANXA autoantibodies, 0.666 for CEA, and 0.84 for both markers together. Importantly, in comparison to CEA (27.5% seropositivity), ANXA autoantibody showed a remarkable change (81.3% seropositivity) at the early stage of CRC., Conclusions: Measurement of ANXA autoantibody levels may provide an alternative detection indicator for CRC, particularly among early-stage patients.

Previously, we identified annexin A4 (ANXA4) as a candidate substrate of caspase-3. Proteomic studies were performed to identify interacting proteins with a view to determining the roles of ANXA4. ANXA4 was found to interact with the p105. Subsequent studies revealed that ANXA4 interacts with NF-kappaB through the Rel homology domain of p50. Furthermore, the interaction is markedly increased by elevated Ca(2+) levels. NF-kappaB transcriptional activity assays demonstrated that ANXA4 suppresses NF-kappaB transcriptional activity in the resting state. Following treatment with TNF-alpha or PMA, ANXA4 also suppressed NF-kappaB transcriptional activity, which was upregulated significantly early after etoposide treatment. This difference may be due to the intracellular Ca(2+) level. Additionally, ANXA4 translocates to the nucleus together with p50, and imparts greater resistance to apoptotic stimulation by etoposide. Our results collectively indicate that ANXA4 differentially modulates the NF-kappaB signaling pathway, depending on its interactions with p50 and the intracellular Ca(2+) ion level.

Objective: To investigate the genes that were differentially expressed between clear cell carcinoma (CCC) and serous carcinoma (SAC) of the ovary with complementary DNA microarray., Methods: Complementary DNA microarray was carried out in 8 CCCs and 8 SACs of the ovary. Differentially expressed genes were identified and verified by real-time polymerase chain reaction. The expression of the protein was also verified with immunohistochemistry and Western blot in cells and tissues of ovarian CCC., Results: Comparison of the gene expression profiling identified 21 genes with more than 2-fold different expression between CCC and SAC of the ovary. The up-regulated and down-regulated genes were 9 and 12, respectively. The verification of Annexin IV in the cell line and tissues was in accordance with the result of the microarray., Conclusions: The complementary DNA microarray technique is a feasible way to explore the difference of the gene expression profiling between the 2 types of ovarian carcinoma. The overexpression of Annexin IV may be an ovarian CCC-specific molecular marker.

Tiam1 (T-cell lymphoma invasion and metastasis-inducing protein 1), a guanine nucleotide exchange factor that activates Rac, is a colorectal cancer metastasis-related gene. In this study, we aimed to better understand the mechanism underlying Tiam1-mediated metastasis. We applied gene microarray and proteome analysis and compared expression of genes and proteins in a stable Tiam1-silencing colorectal cancer cell line and in a control cell line. Our analysis identified three genes, high-mobility group box1 (HMGB1), annexin IV (ANXA4) and phosphoglycerate mutase 1 (PGAM1) that were associated with Tiam1. Analysis of these proteins, which may be directly or indirectly regulated by Tiam1, may provide insight into the role and mechanism of Tiam1 in colorectal cancer metastasis.

Phospholipase A(2) (PLA(2))-activating protein (PLAA) is a novel signaling molecule that regulates the production of prostaglandins (PGE(2)) and tumor necrosis factor (TNF)-alpha. To characterize the function of native PLAA in situ, we generated HeLa (Tet-off) cells overexpressing plaa (plaa(high)) and control (plaa(low)) cells, with the plaa gene in opposite orientation in the latter construct. The plaa(high) cells produced significantly more PGE(2) and interleukin (IL)-6 compared to plaa(low) cells in response to TNF-alpha. There was an increased activation and/or expression of cytosolic PLA(2), cyclooxgenase-2, and NF-kappaB after induction of plaa(high) cells with TNF-alpha compared to the respective plaa(low) cells. Microarray analysis of plaa(high) cells followed by functional assays revealed increased production of proinflammatory cytokine IL-32 and a decrease in the production of annexin A4 and clusterin compared to plaa(low) cells. We demonstrated the role of annexin A4 as an inhibitor of PLA(2) and showed that addition of exogeneous clusterin limited the production of PGE(2) from plaa(high) cells. To understand regulation of plaa gene expression, we used a luciferase reporter system in HeLa cells and identified one stimulatory element, with Sp1 binding sites, and one inhibitory element, in exon 1 of the plaa gene. By using decoy DNA oligonucleotides to Sp1 and competitive binding assays, we showed that Sp1 maintains basal expression of the plaa gene and binds to the above-mentioned stimulatory element. We demonstrated for the first time that the induction of native PLAA by TNF-alpha can perpetuate inflammation by enhancing activation of PLA(2) and NF-kappaB.

Annexin A4 (anxA4) is a member of the Ca(2+)-dependent membrane-binding family of proteins implicated in the regulation of ion conductances, Ca(2+) homeostasis, and membrane trafficking. We demonstrate, in mice, that annexins 1-6 are present in whole bladder and exhibit differential expression in the urothelium. An anxA4a-knockout (anxA4a(-/-)) mouse model shows no protein in the urothelium by immunofluorescence and immunoblotting. In wild-type bladders, anxA4a in umbrella cells showed uniform cytoplasmic staining and some association with the nuclear membrane. Application of a hydrostatic pressure to bladders mounted in Ussing chambers resulted in redistribution of anxA4a from cytoplasm to cellular boundaries in the basal and intermediate cells but not in superficial umbrella cells. We hypothesized that anxA4a might be important for barrier function or for stretch-activated membrane trafficking. To test these hypotheses, we conducted a series of functional and morphological analyses on bladders from control and anxA4a(-/-) animals. The transepithelial resistances, water permeabilities, and urea permeabilities of anxA4a(-/-) bladders were not different from controls, indicating that barrier function was intact. Membrane trafficking in response to hydrostatic pressure as measured by capacitance increases was also normal for anxA4a(-/-) bladders. Cystometrograms performed on live animals showed that voiding frequency and intrabladder pressures were also not different. There were no differences in bladder surface morphology or cellular architecture examined by scanning and transmission electron microscopy, respectively. We conclude that loss of anxA4 from the urothelium does not affect barrier function, membrane trafficking, or normal bladder-voiding behavior.

Many cell types, including neurons and epithelial cells, express a variety of annexins. Although the overall function has only been partially unravelled, a dominant feature is the formation of two-dimensional assemblies under the plasma membrane in a calcium-dependent manner. Here we show that fluorescently tagged annexins A1, A2, A4, A5, and A6 translocate and assemble at the plasma membrane and the nuclear envelope, except annexin A2, which only attaches to the plasma membrane. All annexins have different response times to elevated calcium levels as was shown by the translocation of co-expressed proteins. Fluorescence recovery after photobleaching revealed the static nature of all annexin assemblies. Analysis of the assemblies by Foerster resonance energy transfer (FRET) using acceptor bleaching demonstrated mostly annexin-specific self-assembly. Heterogeneous assembly formation was shown between annexins A5 and A1, and A5 and A2. The formation of homo- and heterogeneous annexin assemblies may play an important role when high increases in calcium occur, such as after disruption of the plasma membrane.

Annexin IV (ANXA4) belongs to a ubiquitous family of Ca(2+)-dependent phospholipid-binding proteins. ANXA4 has been shown to be involved in a range of physiological functions including ion channel regulation, exocytosis and Ca(2+)-dependent signal transduction. The aims of this study were to fully characterize ANXA4 mRNA and protein in human endometrium during the menstrual cycle and to investigate the hormonal regulation of ANXA4. ANXA4 mRNA expression was quantified by real-time PCR in fresh endometrial tissue from cycling women, and protein expression was analysed by immunohistochemistry and western blotting. Hormonal regulation of ANXA4 transcription and translation was investigated using an endometrial explant system. ANXA4 mRNA was significantly up-regulated during mid-secretory (MS) and late-secretory (LS) phases compared with proliferative phases during the menstrual cycle. ANXA4 protein was localized to glandular and luminal epithelium and was present in high levels throughout the menstrual cycle except during early-secretory (ES) phase, when it was significantly reduced. Our data also show that, in proliferative explants, progesterone significantly increased the ANXA4 mRNA and protein after 48h in culture. Estrogen did not have any significant effects. This is the first study to show that ANXA4 transcription and translation are regulated by progesterone and suggests that ANXA4 may be important in regulating ion and water transport across the endometrial epithelium.

Compared to glutathione S -transferase (GST), tagging with hexahistidine residues (His) has several merits: low levels of toxicity and immunogenicity, a smaller size and no electric charge. We have constructed a novel expression vector, designated as pHisJM (EMBL/GenBank/DDJB accession no. AB116367), for producing recombinant His-fusion proteins. This vector was constructed by replacing GST and multiple cloning site (MCS) cassettes in pGEX-5X-3 with those of hexahistidine and MCS derived from pRSET C vector. Human annexin IV (Anx IV) was used as target protein. His-Anx IV fusion protein was expressed using pHisJM and gave a 40 kDa band when immuno-stained with anti-His mAb or anti-Anx IV mAb as predicted. To compare expression efficiency, a Anx IV cDNA inserted-pHisJM or pGEX-5X-3 was transformed into Escherichia coli DH5alpha, JM109, BL21 and BL21(DE3). Using pHisJM, Anx IV protein was highly expressed in all cell strains. In addition to the merits of using His-tag, pHisJM has several advantages: 1) it has high expression efficiency; 2) it can be used in any Escherichia coli strain; and 3) it can be used in a single strain of Escherichia coli in all steps from plasmid construction to the expression of the target gene.

Fetuin-A is a major constituent of the fetal bovine serum used extensively in cell culture media. We hereby present data demonstrating that breast carcinoma cells can adhere to immobilized fetuin-A in a calcium-dependent fashion. Interestingly, the cells can also divide and attain confluency under these conditions. Using a proteomic approach, we have identified annexin-II and -VI as the putative cell surface receptors for fetuin-A in the presence of Ca2+ ions. Biotinylation of cell surface proteins followed by immunoprecipitation revealed that annexin-VI was expressed on the extracytoplasmic surface of the cell membranes. Finally, to demonstrate that annexin-II and -VI were the adhesive receptors for fetuin-A, siRNA knockdown of expression of the annexins significantly reduced the calcium-mediated adhesion. Interestingly, we demonstrated that the tumor cells could also adhere to immobilized fetuin-A in the presence of magnesium ions, and that this adhesion was most likely mediated by integrins because neutralizing antibodies against beta1 integrins substantially reduced the adhesion. Our studies suggest that the expression of annexin-II and -VI and possibly other members of the family mediate novel adhesion and signaling mechanisms in tumor cells.

Characterizing the alterations of protein expression in cancer cells can be very useful in providing insight into the changes in the functional pathways and thus the fundamental mechanisms of cancer development at the molecular level. In this study, we profiled protein expressions in eleven pairs of primary cell cultures derived from renal-cell carcinoma (RCC) tissues and patient-matched normal kidney tissues utilizing two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). Together with the immunoblot analysis of proteins from the RCC tissues, the study also demonstrated that the alterations of protein expression observed in RCC primary cell cultures reflected those observed in the original RCC tissues. We analyzed the expression profiles and identified proteins differentially expressed in RCC primary cell cultures by 2-D PAGE and mass spectrometry (MS). We found sixteen proteins were overexpressed and seven proteins underexpressed in RCC. The deregulated expressions of proteins include those involved in metabolism, cellular morphology, heat shock response, cell growth, etc. Overexpression of three proteins, alphabeta-crystallin, manganese superoxide dismutase (MnSOD), and annexin IV, most commonly observed in primary RCC cell cultures, were also observed by immunoblot analysis of proteins from the RCC tissues from which the primary cell cultures were derived. Semi-quantitative reverse transcription (RT)-polymerase chain reaction (PCR) analysis revealed the direct correlation between deregulated gene expression and the corresponding protein abundance in two of the three most commonly upregulated proteins we found in RCC., (Copyright 2004 Wiley-Liss, Inc.)

Annexin IV (AIV), a Ca2+-dependent membrane-binding protein, is expressed in many epithelia. Annexin IV modifies membrane bilayers by increasing rigidity, reducing water and H+ permeability, promoting vesicle aggregation, and regulating ion conductances, all in a Ca2+-dependent manner. We have characterized a mouse in which a gene trap has been inserted into the first intron of annexin IV. Processing of the primary transcript is disrupted. Northern blot and immunoblot data indicated that annexin IV expression was eliminated in many but not all tissues. Immunohistochemical analysis, however, demonstrated that annexin IV expression was eliminated in some cell types, but was unaltered in others. 5'-Rapid amplification of cDNA ends analysis of intestinal and kidney RNA revealed three transcripts, AIVa, AIVb, and AIVc. AIVa is widely distributed. AIVb is expressed only in the digestive tract. AIVc expression is very restricted. A selected number of epithelial cells of unique morphology demonstrate high concentrations. All three transcripts produce an identical annexin IV protein. The different tissue and cell-specific expression profiles of the three transcripts suggest that regulation of both the annexin IV gene expression and the cellular role of the protein are complex. The AIVa-/- mouse may become a valuable model to further study transcription and the physiological role of annexin IV.

The annexins are a family of highly homologous Ca(2+) and phospholipid binding proteins. The expressive amounts of several annexins have been shown to alter in certain pathological states such as brain ischemia and Alzheimer's disease. It has been demonstrated that ethanol induces cytotoxicity, which results in brain damage. In this study, we examined the relationship between ethanol-induced cytotoxicity and the intrinsic amount of annexins using cell lines (rat glioma C6 cells and human adenocarcinoma A549 cells). A decrease in the mitochondrial enzyme (dehydrogenase) activity, which is widely used to measure cytotoxicity, was observed with a high concentration of ethanol (200 mM or more) after a 24-h exposure in both C6 and A549 cells. Western blot analysis revealed that the amount of annexin IV was augmented in both cells by ethanol, whereas levels of annexins I and V were unchanged. The amount of annexin IV was augmented with increasing concentration of ethanol. The overexpression of annexin IV in C6 cells by transfection with annexin IV-DNA enhanced ethanol-induced cell lesion and was accompanied by NFkappaB activation. Thus, it might be indicated that the amount of annexin IV is selectively augmented and this augmentation facilitates the development of cell lesion by ethanol.

We studied mRNA structure of 31 kDa annexin of zebra fish Brachydanio rerio using previously obtained 3'-terminal incomplete cDNA. The size of this protein mRNA was determined by Northern hybridization. PCR screening of cDNA library of zebra fish gastrula allowed us to obtain cDNA of the 5'-terminal regions of the mRNA. The primary structure of the protein deduced from the mRNA sequence allowed us to identify it as an annexin IV with threonine in position 6--a phosphorylation target for protein kinase C.

ZAP36, a zymogen granule membrane associated protein with 36 kDa, was cloned from both canine and rat pancreas. ZAP36 is found to be a novel member of annexin IV, and showed an apical localization in exocrine pancreas and an ubiquitous expression in epithelial tissues. ZAP36 may be involved in exocytosis in apical regions of polarized cells.

Vertebrate kidney organogenesis is characterised by the successive formation of the pronephros, the mesonephros and the metanephros. The pronephros is the first to form and is the functional embryonic kidney of lower vertebrates; although it is vestigial in higher vertebrates, it is a necessary precursor for the other kidney types. The Xenopus pronephros is a simple paired organ; each nephron consists of a single large glomus, one set of tubules and a single duct. The simple organisation of the pronephros and the amenability of Xenopus laevis embryos to manipulation make the Xenopus pronephros an attractive system in which to study organogenesis. It has been shown that pronephric tubules can be induced to form in presumptive ectodermal tissue by treatment with RA and activin. We have used this system in a subtractive hybridisation screen that resulted in the cloning of Xenopus laevis annexin IV (Xanx-4). Xanx-4 transcripts are specifically located to the developing pronephric tubules, and the protein to the luminal surface of these tubules. Temporal expression shows zygotic transcription is upregulated at the time of pronephric tubule specification and persists throughout pronephric development. The temporal and spatial expression pattern of Xanx-4 suggests it may have a role in pronephric tubule development. Overexpression of Xanx-4 yields no apparent phenotype, but Xanx-4 depletion, using morpholinos, produces a shortened, enlarged tubule phenotype. The phenotype observed can be rescued by co-injection of Xanx-4 mRNA. Although the function of annexins is not yet clear, studies have suggested a role for annexins in a number of cellular processes. Annexin IV has been shown to have an inhibitory role in the regulation of epithelial calcium-activated chloride ion conductance. The enlarged pronephric tubule phenotype observed may be attributed to incorrect modulation of exocytosis, membrane plasticity or ion channels and/or water homeostasis. In this study, we demonstrate an in vivo role for annexin IV in the development of the pronephric tubules in Xenopus laevis.

Site-directed mutagenesis, electron microscopy, and X-ray crystallography were used to probe the structural basis of annexin IV-induced membrane aggregation and the inhibition of this property by protein kinase C phosphorylation. Site-directed mutants that either mimic (Thr6Asp, T6D) or prevent (Thr6Ala, T6A) phosphorylation of threonine 6 were produced for these studies and compared with wild-type annexin IV. In vitro assays showed that unmodified wild-type annexin IV and the T6A mutant, but not PKC-phosphorylated wild-type or the T6D mutant, promote vesicle aggregation. Electron crystallographic data of wild-type and T6D annexin IV revealed that, similar to annexin V, the annexin IV proteins form 2D trimer-based ordered arrays on phospholipid monolayers. Cryo-electron microscopic images of junctions formed between lipid vesicles in the presence of wild-type annexin IV indicated a separation distance corresponding to the thickness of two layers of membrane-bound annexin IV. In this orientation, a single layer of WT annexin IV, attached to the outer leaflet of one vesicle, would undergo face-to-face self-association with the annexin layer of a second vesicle. The 2.0-A resolution crystal structure of the T6D mutant showed that the mutation causes release of the N-terminal tail from the protein core. This change would preclude the face-to-face annexin self-association required to aggregate vesicles. The data suggest that reversible complex formation through phosphorylation and dephosphorylation could occur in vivo and play a role in the regulation of vesicle trafficking following changes in physiological states.

The roles of the four domains of annexin IV in binding to phospholipids and glycolipids were assessed by analyzing the binding of a group of mutant annexins IV in which one or more of the four domains was inactivated by replacing a critical amino residue(s) (Asp or Glu) with the neutral residue Ala. The data reveal that individual annexin domains may have characteristic affinities for different lipids. In particular, inactivation of the fourth domain inhibits the binding to phosphatidylserine (PS) and phosphatidylinositol (PI) but not to phosphatidylglycerol (PG), suggesting that this domain specifically can accommodate the larger head groups of PS and PI whereas the other three domains may form more restricted binding pockets. In order to block binding to PG, domain 1, or both domains 2 and 3 must be inactivated in addition to domain 4, suggesting that all four domains may be able to accommodate the headgroup of PG to some extent. Binding to acidic glycolipids (sulfatides) was also sensitive to inactivation of domain 4. However, in the case of sulfatides the nature of the binding reaction is fundamentally different compared with the binding to phospholipids since the interaction with sulfatides was highly sensitive to an increase in ionic strength. The binding to sulfatides may depend therefore on charge-charge interactions whereas the binding to phospholipid may involve a more specific interaction between the lipid headgroup and the protein surface, and/or interaction of the protein with the hydrophobic portion of a lipid bilayer.

A recurring problem with cancer therapies is the development of drug resistance. While investigating the protein profile of cells resistant to a novel antimitotic compound (A204197), we discovered an increase in annexin IV expression. When we examined the annexin IV protein expression level in a paclitaxel-resistant cell line (H460/T800), we found that annexin IV was also overexpressed. Interestingly a closely related protein, annexin II, was not overexpressed in H460/T800 cells. Immunostaining with either annexin II or IV antibody revealed that annexin IV was primarily located in the nucleus of paclitaxel-resistant H460/T800 cells. Short-term treatment of H460 cells with 10 nM paclitaxel for up to 4 days resulted in induction of annexin IV, but not annexin II expression. In addition, there was an increase in annexin IV staining in the nucleus starting at day 1. Furthermore, cells pretreated with 10 nM paclitaxel for 4 days resulted in cells becoming approximately fivefold more resistant to paclitaxel. Transfection of annexin IV cDNA into 293T cells revealed that there was a threefold increase in paclitaxel resistance. Thus our results indicate that annexin IV plays a role in paclitaxel resistance in this cell line and it is among one of the earliest proteins that is induced in cells in response to cytotoxic stress such as antimitotic drug treatment.

We have shown previously that surfactant protein A (SP-A) binds to annexin IV in a Ca2+-dependent manner [Sohma, Matsushima, Watanabe, Hattori, Kuroki and Akino (1995) Biochem. J. 312, 175-181]. Annexin IV is a member of the annexin family having four consensus repeats of about 70 amino acids and a unique N-terminal tail. In the present study, the functional site of both annexin IV and SP-A for the Ca2+-dependent binding was investigated using mutant proteins. SP-A bound in a Ca2+-dependent manner to an annexin-IV truncation mutant consisting of the N-terminal domain and the first three domains (T(N-1-2-3)). SP-A also bound to T3-4, but this interaction was not Ca2+-dependent. SP-A bound weakly to the other truncation mutants (T(N-1-2), T(2-3) and T(2-3-4)). Each consensus repeat of annexin IV possesses a conserved acidic amino acid residue (Glu70, Asp142, Glu226 and Asp301) that putatively ligates Ca2+. Using annexin-IV DE mutants in which one, two or three residues out of the four Asp/Glu were altered to Ala by site-directed mutagenesis [Nelson and Creutz (1995) Biochemistry 34, 3121-3132], it was revealed that Ca2+ binding in the third domain is more important than in the other Ca2+-binding sites. SP-A is a member of the animal lectin group homologous with mannose-binding protein A. The substitution of Arg197 of rat SP-A with Asp or Asn eliminated binding to annexin IV, whereas the substitution of Glu195 with Gln was silent. These results suggest that the Ca2+ binding to domain 3 of annexin IV is required for the Ca2+-dependent binding by SP-A and that Arg197 of SP-A is important in this binding.

Annexin IV was cloned and sequenced from a mouse bone marrow-derived macrophage cDNA library, and was found to exist as three different alternatively spliced transcripts. One transcript contained an additional 688 base pairs inserted within the coding region of the gene including an in-frame stop codon. Translation of this transcript in vitro confirmed the premature arrest of translation which resulted in a truncated annexin IV protein of approximately 22 kDa. Like other members of the annexin family, the product of the wild-type annexin IV transcript bound in a calcium-dependent manner to both phenyl-sepharose and phospholipid vesicles. In contrast, the truncated annexin IV product bound to these substrates in a Ca2+-independent fashion. The existence of a novel form of annexin IV in mouse macrophages may aid in further defining the role of members of the annexin family., (Copyright 1999 Academic Press.)

Annexin IV was found to be highly expressed in various human adenocarcinoma cell lines, but not in an erythroleukemia cell line, K562. We investigated the effects of transfection of human annexin IV cDNA into K562 cells on cell surface lectin receptors. cDNA transfectants were found to be more sensitive to cytotoxic lectins such as Ricinus communis agglutinin and wheat germ agglutinin than mock transfectants. The results of flow cytometric analyses with various lectins showed that the transfectants expressed more sugar chains which bind to Ulex europaeus agglutinin I and Maackia amurensis mitogen than mock transfectants. These results suggest that transfection of annexin IV cDNA increases the expression of alpha-2,3-sialylated and/or fucosylated sugar chains on the surface.

Bovine annexin IV, a Ca(2+)-dependent, membrane-binding protein, was expressed in E. coli using four different prokaryotic expression vector systems. An annexin IV cDNA was mutated in the 5' noncoding region to introduce an NcoI restriction site at the translation initiation site. The coding sequence was then excised and ligated into the expression vectors: pKK233-2 (which uses a hybrid trc promoter), pFOG405 (which uses the alkaline phosphatase promoter and generates a fusion protein with the alkaline phosphatase signal sequence that targets the protein for secretion), pOTSNco12 (which provides temperature-sensitive expression from the lambda phage promoter), and pET11d (which uses the T7lac promoter and a protease-deficient host). Expression of wild type and mutant annexin IV in the various systems was compared. Differences in level of expression, formation of inclusion bodies, and yield of purified protein were observed. The pET11d system was found to be the most effective expression system for annexin IV and various annexin IV mutant constructs, providing the highest yield of functional protein from the soluble fraction of cell lysates. Bovine chromaffin granule binding and aggregating activities of recombinant annexin IV were found to be virtually indistinguishable from those of bovine annexin IV isolated from liver tissue. Truncation constructs containing one, two, or three of the four conserved 70-amino-acid domains of native annexin IV were successfully created and expressed in E. coli, but the recombinant proteins were generally insoluble. pET11d annexin constructs containing point mutations in residues involved in binding calcium produced soluble protein at levels comparable to those of constructs expressing wild type protein.

This study addresses the roles of individual annexin IV domains in calcium-dependent membrane binding and aggregation through an analysis of the activities of mutant annexin IV proteins in which critical residues in one or more domains have been altered. The consensus sequence for high-affinity Ca(2+)-binding pockets obtained from the annexin V crystal structure is GXGT-38 residues-D/E [Huber, R., et al. (1992) J. Mol. Biol. 223, 683-704]. Site-directed mutagenesis was used to change the conserved acidic residues (D/E) in these sequences to alanine residues in each of the four domains of bovine annexin IV, singly or in combinations. Fourteen mutants with one, two, three, or four mutated domains were constructed and expressed in Escherichia coli. Purified recombinant product was evaluated for Ca(2+)-dependent binding to and aggregation of bovine chromaffin granules. Increases in the number of mutated domains resulted in increased Ca2+ requirements for both granule binding and aggregation. Further analysis revealed that mutations in individual domains had preferential effects on the binding or aggregating activities of the protein. For example, mutation of the first or fourth domains had a greater effect on membrane binding than aggregation, while conversely, mutation of the second domain had a more dramatic effect on membrane aggregation. Mutation of the third domain was largely silent in these assays. An additional mutation was made in the fourth domain to substitute a serine for a highly conserved arginine residue (Arg274) present at the C-terminus of the fourth endonexin fold. This mutation increased the calcium requirement for membrane binding 2-fold and for membrane aggregation 3-fold. This mutant protein was found to be an effective inhibitor of membrane aggregation by native annexin IV at intermediate levels of calcium.

The mouse genes encoding early growth response 4 (Egr4), annexin IV (Anx4), and transforming growth factor alpha (Tgfa) have been mapped to a linkage group on mouse chromosome 6 that is conserved on human chromosome 2p11-p13. The genes are closely linked, with 0/215 recombinants between Anx4 and Tgfa and 1/215 recombinants between these genes and Egr4. The genes are located approximately 2 cM distal to mnd2, a mouse mutation causing neuromuscular disease. The results demonstrate that mnd2 is located at an internal position within this conserved linkage group.