Carbohydrate-binding proteins in bovine kidney have consensus amino acid sequences of annexin family proteins.

Ca(2+)-dependent carbohydrate-binding proteins were purified from bovine kidney extracts. Upon SDS-polyacrylamide gel electrophoresis under nonreducing conditions, the purified fraction gave doublet protein bands corresponding to 33 kDa (p33) and 41 kDa (p41). Under reducing conditions, a single protein band (p33) was observed. p33 and p41 were submitted to proteolytic digestion with endoproteinase Lys-C, the peptides produced were separated by reversed-phase high performance liquid chromatography, and their amino acid sequences were determined by an automated gas-phase protein sequenator. Most of the resulting partial amino acid sequences of these proteins were strikingly homologous to annexin IV, an annexin family protein, i.e. Ca2+/phospholipid-binding proteins, especially in the consensus sequences. In the presence of Ca2+, both proteins bound to vesicles composed of phosphatidylserine and phosphatidylethanolamine, but not phosphatidylcholine. These results indicated that p33 and p41 are members of annexin family proteins.     

小鼠膜联蛋白Ⅱ基因剔除重组子的构建及小鼠无膜联蛋白Ⅱ细胞系的建立

为了便于对膜联蛋白II(annexinII)的进一步研究以及今后进一步发展膜联蛋白II-/-动物模型,构建了膜联蛋白II基因封闭重组子(pPNT/annexinII/LacZ),筛选了细胞(L5178Y)克隆,并获得了膜联蛋白II-/-稳定细胞克隆(D4,E2)。所获膜联蛋白II-/-L5178Y克隆有待于以PCR做进一步鉴定。     

Annexin VI regulation of cardiac function

Annexins are a family of membrane binding proteins that are characterized by a hypervariable amino terminus followed by a series of highly conserved Ca2+-phospholipid binding domains. Annexins function by binding to anionic phospholipid surfaces in a Ca2+-dependent manner. They self-associate to form trimers which further assemble into sheets that cover the membrane surface and alter properties such as fluidity and permeability. This submembranous skeleton alters integral protein functions such as ion transport properties and shields the surface from phospholipid binding proteins such as phospholipases and protein kinase C. Transgenic mouse hearts overexpressing wild type annexin VI (AnxVI673), a dominant-negative truncated annexin VI (residues 1-129, Anx129) and an annexin VI-null mouse (AnxVI-/-) have implicated the protein as a regulator of intracellular Ca2+ homeostasis which affects cardiac function.     

Ca(2+)-dependent phospholipid and arachidonic acid binding by the placental annexins VI and IV.

Using an assay system in which phospholipids were immobilised on phenyl-Sepharose, we examined the affinities of the placental annexins VI and IV for binding to specific phosphatidylserine, phosphatidylethanolamine and phosphatidylinositol at Ca2+ concentrations of 0.6, 0.4 and 3.5 microM, respectively, compared to values of 4.5, 4.5 and 20 microM Ca2+, respectively for purified annexin IV. These values did not change significantly in the presence of other proteins from the family. Neither annexin VI or IV bound to phosphatidylinositol bisphosphate and phosphatidylcholine, even at millimolar concentrations of Ca2+. However, both proteins bound to arachidonic acid, oleic acid and palmitic acid in a Ca(2+)-dependent manner, using the same assay system. The level of binding for both proteins was significantly increased when mixtures of phosphatidylcholine and arachidonic acid were examined. A dose-dependent inhibition of phospholipase A2 by both annexins VI and IV, at millimolar concentrations of Ca2+ was observed when phosphatidylcholine liposomes were used as a substrate. These results raise questions about the interpretation of experiments in which the release of arachidonic acid is used as a measure of lipase activity, and of the validity of the substrate-depletion model for the inhibition of phospholipases by the annexins.     

Binding of annexin V/placental anticoagulant protein I to platelets. Evidence for phosphatidylserine exposure in the procoagulant response of activated platelets

Proteins of the annexin/lipocortin family act as in vitro anticoagulants by binding to anionic phospholipid vesicles. In this study, we investigated whether annexin V (placental anticoagulant protein I) would bind to human platelets. Annexin V bound to unstimulated platelets in a reversible, calcium-dependent reaction with an apparent Kd of 7 nM and 5000-8000 sites/platelet. Additional binding sites could be induced by several platelet agonists in the following order of effectiveness: A23187 greater than collagen + thrombin greater than collagen greater than thrombin. However, neither ADP nor epinephrine induced additional binding sites. Three other proteins of the annexin family (annexins II, III, and IV) competed for annexin V platelets binding sites with the same relative potencies previously observed for binding to phospholipid vesicles. Phospholipid vesicles containing phosphatidylserine completely inhibited binding of annexin V to platelets. Annexin V completely blocked binding of 125I-factor Xa to thrombin-stimulated platelets. These results support the hypothesis that phosphatidylserine exposure occurs during platelet activation and may be necessary for assembly of the prothrombinase complex on platelet membranes.     

Annexin A5 is not essential for skeletal development

Annexins are highly conserved proteins that are characterized by their ability to interact with phospholipids in a calcium-dependent manner. Although diverse functions have been ascribed to annexins based on in vitro analyses, their in vivo functions still remain unclear. The intensively studied annexin A5 has been identified by its effects on blood coagulation, and subsequently, its function as a calcium-specific ion channel was described. In vitro experiments and expression studies suggested a potential role of annexin A5 during calcification processes in vivo, especially in endochondral ossification. To gain insights into the relevance of annexin A5 in this process, we generated an annexin A5-deficient mouse mutant. Mice lacking annexin A5 are viable, are fertile, and reveal no significant alterations in the biochemical parameters characteristic for metabolic or functional defects. Neither the development of skeletal elements nor the in vitro calcification properties of isolated chondrocytes is significantly impaired by the absence of annexin A5. Therefore, annexin A5 is dispensable for the formation and maintenance of skeletal elements in the mouse and may possibly be pointing to a compensatory effect of other members from the annexin family due to their high functional and structural similarity.     

Differential expression of annexins I, II and IV in human tissues: an immunohistochemical study

 Annexins constitute a family of Ca²⁺- and phospholipid-binding proteins. Although their functions are still not clearly defined, several members of the annexin family have been implicated in membrane-related events along exocytotic and endocytotic pathways. To elucidate a possible correlation of those functional proposals with the tissue distribution of annexins, we analysed immunohistochemically the expression of annexins I, II and IV in a broad variety of human tissues. Annexins I and II were chosen for this study since their functionally relevant N-terminal domains are structurally closely related, whilst annexin IV is structurally less related to the former two proteins. The study revealed distinct expression patterns of annexins I, II and IV throughout the body. Annexin I was found in leucocytes of peripheral blood, tissue macrophages and T-lymphocytes and in certain epithelial cells (respiratory and urinary system, superficial cells of non-keratinised squamous epithelium), annexin II in endothelial cells, myoepithelial cells and certain epithelial cells (mainly respiratory and urinary system), whereas annexin IV was almost exclusively found in epithelial cells. Epithelia of the upper respiratory system, Bowman’s capsule, urothelial cells, mesothelial cells, peripheral nerves, the choroid plexus, ependymal cells and pia mater and arachnoid of meninges generally strongly expressed all three annexins investigated. The characteristic expression in different tissues and the intracellular distribution indicates that the three annexins investigated are involved in aspects of differentiation and/or physiological functions specific to these tissues. Accepted: 15 January 1998     

Chromosomal mapping of the human annexin IV (ANX4) gene.

Annexin IV (placental anticoagulant protein II) is a member of the annexin or lipocortin family of calcium-dependent phospholipid-binding proteins. A cDNA for human annexin IV was isolated from a placental library that is 675 bases longer in the 3' untranslated region than previously reported, indicating the existence of alternative mRNA processing for this gene. Genomic Southern blotting with a cDNA probe indicated a gene size of 18-56 kb. Primers developed for polymerase chain reaction (PCR) allowed amplification of a 1.6-kb portion of the ANX4 gene. DNA sequence analysis showed that this PCR product contained a single intron with exon-intron boundaries in exactly the same position as in the mouse annexin I and annexin II genes. PCR analysis of a somatic cell hybrid panel mapped the ANX4 gene to chromosome 2, and in situ hybridization with a cDNA probe showed a unique locus for ANX4 at 2p13. This study provides further evidence that genes for the annexins are dispersed throughout the genome but are similar in size and exon-intron organization.     

Structure of soluble and membrane-bound human annexin V.

Annexins are a family of water-soluble proteins that bind to membranes in a calcium-dependent manner. Some members have been shown to exhibit voltage-dependent calcium channel activity, a property characteristic of integral membrane proteins. The structures of human annexin V in crystals obtained from aqueous solution and in two-dimensional crystals when bound to phospholipid layers have been determined by X-ray and electron crystallography, respectively. They are compared here. Both structures show close correspondence, suggesting that annexins attach to phospholipid membranes without substantial structural change. These observations, together with biochemical data, lead to the conclusion that annexin V interacts with phospholipid membranes with its convex face. We propose that binding is mediated by direct interaction between the phosphoryl headgroups and the calcium bound to polypeptide loops protruding from the convex face. The membrane area covered by annexin may thus become disordered and permeable allowing calcium flux through the membrane and the central channel-like structure found in annexin molecules.     

Structural determinant of the vesicle aggregation activity of annexin I.

Some annexins, including annexins I, II, IV, and VII, can promote membrane aggregation. To identify amino acids involved in annexin I-mediated membrane aggregation, we generated truncated mutants of human annexin I lacking various parts of the amino terminus. The in vitro vesicle binding and aggregation activities of these mutants indicated that both the amino-terminal region of annexin I spanning residues 26-29 and the carboxy-terminal core are involved in membrane aggregation. This notion was further supported by the finding that a chimera composed of residues 24-35 of annexin I and the core of annexin V has vesicle aggregation activity that is significantly higher than that of annexin V but lower than that of annexin I. Further site-specific mutations in the amino-terminal region of annexin I indicated that Lys-26 and Lys-29 are essential for its membrane aggregation activity. The comparison of tryptic digest patterns of free and vesicle-bound wild type and K29E mutant suggests that a primary role of Lys-26 and Lys-29 is to induce and stabilize an active conformation of annexin I for vesicle aggregation.     

Annexins as intracellular calcium sensors

The annexins are a multigene family of Ca(2+)- and charged phospholipid-binding proteins. Although they have been ascribed with diverse functions, there is no consensus about the role played by this family as a whole. We have mapped the Ca(2+)-induced translocations of four members of the annexin family and of two truncated annexins in live cells, and demonstrated that these proteins interact with the plasma membrane as well as with internal membrane systems in a highly coordinated manner. Annexin 2 was the most Ca(2+) sensitive of the studied proteins, followed by annexins 6, 4 and 1. The calcium sensitivity of annexin 2 increased further following co-expression with S100A10. Upon elevation of [Ca(2+)](i), annexins 2 and 6 translocated to the plasma membrane, whereas annexins 4 and 1 also became associated with intracellular membranes and the nuclear envelope. The NH(2)-terminus had a modulatory effect on plasma membrane binding: its truncation increased the Ca(2+) sensitivity of annexin 1, and decreased that of annexin 2. Given the fact that several annexins are present within any one cell, it is likely that they form a sophisticated [Ca(2+)] sensing system, with a regulatory influence on other signaling pathways.