The self peptide annexin II (208–223) presented by dendritic cells sensitizes autologous CD4+ T lymphocytes to recognize melanoma cells

Annexin II is known to be over-expressed in different types of tumours. We show here that annexin II protein is expressed by melanoma cell lines in various amounts, consistent with previous findings that an annexin II (208-223) peptide could be eluted from isolated HLA-DR molecules of a constitutively MHC class II-positive melanoma line. T cells sensitized to annexin II (208-223) in vitro using peptide-pulsed autologous dendritic cells responded only to the lines which overexpressed annexin II, in a peptide-specific, HLA-DR-restricted fashion. These CD4+ T cells proliferated strongly and secreted large amounts of type 1 cytokines in response to annexin II (208-223) peptide or annexin II protein-positive melanoma cell lines. These results demonstrate that the annexin II (208-223) peptide, corresponding to a non-mutated sequence of a normal protein, induces antigen-specific T cells which can respond to melanoma cells over-expressing the annexin II molecule. This peptide may therefore be useful in immunotherapy for recruiting CD4+ type 1 helper cells active locally in the tumour environment.     

STARD3NL inhibits the osteogenic differentiation by inactivating the Wnt/β‐catenin pathway via binding to Annexin A2 in osteoporosis

Osteoporosis is one of the leading forms of systemic diseases related to bone metabolism in the world. STARD3 N‐terminal like (STARD3NL) showed robust association with osteoporosis‐related traits. Yet, the molecular functional mechanisms of STARD3NL in osteoblasts is still obscure. In this study, we demonstrated a high level of STARD3NL expression in the bone tissues from the patients with low bone mass and ovariectomized (OVX)‐induced osteoporotic mice. We identified Stard3nl as a potent factor that negatively and positively regulates osteoblast differentiation and cell proliferation, respectively. Furthermore, inhibition of Stard3nl induced β‐catenin gene expression and the nuclear translocation of β‐catenin, as well as Wnt signalling activities, contributing to the activation of Wnt/β‐catenin signalling. Mechanistic studies revealed that Stard3nl bound with Annexin A2 (Anxa2) to suppress β‐catenin expression, resulting into the suppression of Wnt signalling and downstream osteogenic differentiation. Moreover, adeno‐associated virus 9 (AAV9)‐mediated silencing of Stard3nl reversed bone loss in OVX‐induced osteoporotic mice by the injection into the knee joints. Collectively, our study revealed that Stard3nl suppressed osteogenesis via binding with Anxa2, resulting into the inactivation of Wnt signalling. It also highlights the preventive and therapeutic potential of STARD3NL as a specific and novel target for osteoporotic patients.     

Enrichment of non–apoptotic human spermatozoa after cryopreservation by immunomagnetic cell sorting

Cryopreservation increases the rate of apoptotic spermatozoa withdecreased capability to fertilise oocytes. In order to optimise thefertilisation rates, especially in assisted reproduction the use of apoptoticsperms should be avoided. Early events of apoptosis in cryopreservedspermatozoaare not detectable by conventional methods. However, the surface of apoptoticspermatozoa is characterised by externalisation of phosphatidylserine (PS),which has a high affinity to Annexin V. Therefore, colloid paramagneticAnnexin-V-conjugated microbeads (AN-MB) were tested fortheir ability to eliminate apoptotic spermatozoa from a total of 40 fresh andinTEST yolk buffer cryopreserved semen samples which were provided by 15 healthyvolunteers. By passing through a magnetic field (MiniMACS, Miltenyi Biotec) thesperm suspensions were divided into 2 sperm fractions depending on boundmagnetic Annexin V-microbeads (AN-MB) to spermatozoa. Asadditional markers of apoptosis CD95 (Fas, APO-1) on the sperm surfaceand activated caspases in the cytosol were detected in both fractions.Supplementary investigations comprised eosin-supravital staining andcomputer assisted sperm motion analysis. The separation was supervised by flowcytometric analysis of spermatozoa labelled with FITC-conjugated antiAnnexin V-antibodies.Analyses of the magnetic inactive sperm fraction (AN-MB-negative)showed CD95 on 0.6 ± 0.3% (X ± SEM) of spermatozoa andonly3.2 ± 0.5% were stainable with eosin, whereas, 40.6 ±6.7% of the remaining cells in the column appeared to be CD95 positiveand 99.8 ± 0.1% stainable with eosin after cryopreservation.Indeed the overall amount of CD95 positive spermatozoa did not significantlyincrease after cryopreservation (2.5 ± 0.5% vs. 4.3 ±1.2%; p > 0.05). Activated caspases were found in 21.8 ±2.6% of the spermatozoa in fresh and in 47.7 ± 5.8% ofcryopreserved semen samples (p < 0.01). The separation procedure of thecryopreserved spermatozoa reduced significantly the quantity of thosecontainingactivated caspases to 9.3 ± 2.2% within theAN-MB-negative fraction. In contrast 89.1 ± 2.3% ofAN-MB-positive sperms showed activation of these proteolyticenzymes. Flow cytometric analyses using FITC-conjugated anti AnnexinV-antibodies for monitoring of AN-MB-binding to spermatozoashowed 5.2 ± 1.0% labelled spermatozoa in the AN-MBnegative fraction and 72.6 ± 2.7% labelled spermatozoa in theAN-MB positive one. There was no significant influence of the separationcolumn and the magnetic field on the sperm functions. The passage through thecolumn led to a sperm loss of 0.8 ± 1.2%.Conclusion: The binding of paramagnetic AnnexinV-conjugated microbeads is an excellent method to eliminate spermatozoaat early apoptotic stages from cryopreserved semen samples. A deleteriousinfluence of the separation column and the magnetic field on the spermatozoawasnot observed.     

S100 and annexin proteins identify cell membrane damage as the Achilles heel of metastatic cancer cells

Mechanical activity of cells and the stress imposed on them by extracellular environment is a constant source of injury to the plasma membrane (PM). In invasive tumor cells, increased motility together with the harsh environment of the tumor stroma further increases the risk of PM injury. The impact of these stresses on tumor cell plasma membrane and mechanism by which tumor cells repair the PM damage are poorly understood. Ca²⁺ entry through the injured PM initiates repair of the PM. Depending on the cell type, different organelles and proteins respond to this Ca²⁺ entry and facilitate repair of the damaged plasma membrane. We recently identified that proteins expressed in various metastatic cancers including Ca²⁺-binding EF hand protein S100A11 and its binding partner annexin A2 are used by tumor cells for plasma membrane repair (PMR). Here we will discuss the involvement of S100, annexin proteins and their regulation of actin cytoskeleton, leading to PMR. Additionally, we will show that another S100 member – S100A4 accumulates at the injured PM. These findings reveal a new role for the S100 and annexin protein up regulation in metastatic cancers and identify these proteins and PMR as targets for treating metastatic cancers.     

Functional identification of alpha 1-giardin as an annexin of Giardia lamblia

A protein with a relative molecular mass of 31 kDa was specifically extracted by EGTA from a detergent-insoluble fraction of Giardia lamblia. N-terminal sequencing showed this protein to be identical to alpha 1-giardin, a component of the ventral disc which, based on its predicted amino acid sequence, has been classified as annexin XIX. Purified alpha 1-giardin associated with multilamellar phosphatidyl serine-containing vesicles in a Ca(2+)-dependent manner, confirming that it is a functional annexin. Molecular modelling of the amino acid sequence of the giardial annexin into the X-ray structure of annexin V suggests that the Ca(2+)-binding sites, which, as in other annexins, are all located on the convex surface of the molecule, are of the low-affinity type III.     

Identification of Annexin A1 interacting proteins in chronic myeloid leukemia KCL22 cells

In the present study, we used a functional proteomic approach to identify Annexin A1 (Anxa1) interacting proteins in the Philadelphia‐positive KCL22 cell line. We focused on Anxa1 because it is one of the major proteins upregulated in imatinib‐sensitive KCL22S cells versus imatinib‐resistant KCL22R. Our proteomic strategy revealed 21 interactors. Bioinformatic analysis showed that most of these proteins are involved in cell death processes. Among the proteins identified, we studied the interaction of Anxa1 with two phosphatases, Shp1 and Shp2, which were recently identified as biomarkers of imatinib sensitivity in patients affected by chronic myeloid leukemia. Our data open new perspectives in the search for annexin‐mediated signaling pathways and may shed light on mechanisms of resistance to imatinib that are unrelated to Bcr‐Abl activity. All mass spectrometry data have been deposited in the ProteomeXchange with identifier PXD000030.     

Lipid Segregation and Membrane Budding Induced by the Peripheral Membrane Binding Protein Annexin A2

The formation of dynamic membrane microdomains is an important phenomenon in many signal transduction and membrane trafficking events. It is driven by intrinsic properties of membrane lipids and integral as well as membrane-associated proteins. Here we analyzed the ability of one peripherally associated membrane protein, annexin A2 (AnxA2), to induce the formation of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂)-rich domains in giant unilamellar vesicles (GUVs) of complex lipid composition. AnxA2 is a cytosolic protein that can bind PI(4,5)P₂ and other acidic phospholipids in a Ca²⁺-dependent manner and that has been implicated in cellular membrane dynamics in endocytosis and exocytosis. We show that AnxA2 binding to GUVs induces lipid phase separation and the recruitment of PI(4,5)P₂, cholesterol and glycosphingolipids into larger clusters. This property is observed for the full-length monomeric protein, a mutant derivative comprising the C-terminal protein core domain and for AnxA2 residing in a heterotetrameric complex with its intracellular binding partner S100A10. All AnxA2 derivatives inducing PI(4,5)P₂ clustering are also capable of forming interconnections between PI(4,5)P₂-rich microdomains of adjacent GUVs. Furthermore, they can induce membrane indentations rich in PI(4,5)P₂ and inward budding of these membrane domains into the lumen of GUVs. This inward vesiculation is specific for AnxA2 and not shared with other PI(4,5)P₂-binding proteins such as the pleckstrin homology (PH) domain of phospholipase Cδ1. Together our results indicate that annexins such as AnxA2 can efficiently induce membrane deformations after lipid segregation, a mechanism possibly underlying annexin functions in membrane trafficking.     

Use of SILAC for exploring sheddase and matrix degradation of fibroblasts in culture by the PIII SVMP atrolysin A: identification of two novel substrates with functional relevance

Snake venom metalloproteinases (SVMPs) in Viperid venoms primarily function to give rise to local and systemic hemorrhage following snake envenomation. Years of research on these toxins, both in vitro and in vivo, indicate that they function by disrupting capillary basement membranes, stromal matrix and cell-cell and cell-matrix contacts to allow escape of capillary contents under pressure. However, most of these studies used either defined substrates in vitro or were limited by relevant antibodies for detection of sites of action in vivo. In this investigation we use stable isotope-labeled amino acids in culture (SILAC) to determine novel proteolytic activities for exogenously added atrolysin A, a hemorrhagic PIII SVMP isolated from Crotalus atrox venom. When comparing the solubilized products of SILAC-labeled cultured human fibroblasts treated with atrolysin A to that of untreated fibroblasts using LC/MS/MS, several proteins were identified as being released into the culture media specifically due to atrolysin A proteolytic activity. These included collagen VI, fibronectin, fibulin 2 and annexin V. Of particular interest was the observation of collagen VI and annexin V in that the release of these substrates could play a role in altering hemostasis and promote hemorrhage caused by the more typical actions of atrolysin A. In summary, this study demonstrates the utility of SILAC for exploring sheddase activity with cells in culture and suggests the presence of two novel substrates for SVMPs that may play a pathological role in altering host hemostasis during envenomation.     

Involvement of Annexin A2 in p53 induced apoptosis in lung cancer

Tumor suppressor p53 plays important roles in cell cycle regulation, apoptosis and DNA repair in different cell types including lung cancer. There are different p53 apoptotic pathways in high and low metastatic ability lung cancer cells. However, the exactly mechanism in the pathway is still unclear. Here we found that Annexin A2, a Ca²⁺-dependent phospholipid-binding protein, is involved in p53-mediated apoptosis. First, by using mRNA differential display technique, down-regulated Annexin A2 expression was found in all cell lines transfected of Ad-p53 (adenoviral expression construct encoding wild type p53 gene) especially in highly metastatic Anip973 lung cancer cells. Then, decreased expression of Annexin A2 was further confirmed by Northern blot and Western blot analysis. At last, knock down of Annexin A2 by siRNA inhibited cellular proliferation in BE1 cell line with highly metastatic ability. Taken together, our results suggested that Annexin A2 may play roles in p53 induced apoptosis and it is also involved in regulation of cell proliferation. The authors Yun Huang, Yan Jin and Cheng-hui Yan contributed equally to this work.     

Annexin A1 restores Aβ1‐42‐induced blood–brain barrier disruption through the inhibition of RhoA‐ROCK signaling pathway

The blood–brain barrier (BBB) is composed of brain capillary endothelial cells and has an important role in maintaining homeostasis of the brain separating the blood from the parenchyma of the central nervous system (CNS). It is widely known that disruption of the BBB occurs in various neurodegenerative diseases, including Alzheimer's disease (AD). Annexin A1 (ANXA1), an anti‐inflammatory messenger, is expressed in brain endothelial cells and regulates the BBB integrity. However, its role and mechanism for protecting BBB in AD have not been identified. We found that β‐Amyloid 1‐42 (Aβ42)‐induced BBB disruption was rescued by human recombinant ANXA1 (hrANXA1) in the murine brain endothelial cell line bEnd.3. Also, ANXA1 was decreased in the bEnd.3 cells, the capillaries of 5XFAD mice, and the human serum of patients with AD. To find out the mechanism by which ANXA1 recovers the BBB integrity in AD, the RhoA‐ROCK signaling pathway was examined in both Aβ42‐treated bEnd.3 cells and the capillaries of 5XFAD mice as RhoA was activated in both cases. RhoA inhibitors alleviated Aβ42‐induced BBB disruption and constitutively overexpressed RhoA‐GTP (active form of RhoA) attenuated the protective effect of ANXA1. When pericytes were cocultured with bEnd.3 cells, Aβ42‐induced RhoA activation of bEnd.3 cells was inhibited by the secretion of ANXA1 from pericytes. Taken together, our results suggest that ANXA1 restores Aβ42‐induced BBB disruption through inhibition of RhoA‐ROCK signaling pathway and we propose ANXA1 as a therapeutic reagent, protecting against the breakdown of the BBB in AD.