Cloning And Structure-function Study Of A Novel Member Of Annexin Family

Annexins are Ca2+ and phospholipid binding proteins forming an evolutionary conserved multigene family widely distributed in eukaryotes. Proteins of this family have been implicated in a widely range of important biological processes, such as membrane-cytoskeleton interaction, cell growth control, ion channel regulation and signal transduction. Each member of this protein family contains a conserved protein core characterized by high alpha-helices content that includes the calcium- and phospholipid-binding sites, and a variable N-terminal domain that is specific in sequence and length for each annexin.Annexin Bl is a new gene isolated from the cDNA library of Cysticercus cellulosae by immunological screening. Bioinformatics analysis revealed that it is indeed a novel member of annexin family and has common structural features of the family. As the first annexin was found in platyhelminth, studies on annexin Bl can help to make clear what the role it plays hi the infection, loading and other activities of Cysticercus cellulosae, which may provide some new ways to prevent and control this disease. Additionally, annexin Bl can also act as a molecular model for studying the evolutionary divergence of annexins. Fusion and non-fusion expression vectors of annexin Bl were constructed and the expressed product was purified by affinity or ion exchange chromatography; Northern blot and immunohistochemistry were used to investigate the stage-specific expression and histological localization of annexin Bl; Homology modeling, .site-directed and sequence-deleted mutagenesis, protein crystallization were taken to study the relationship between structure and function of annexin Bl; Liposome-pelleting, activated platelets and apoptotic cells binding assay were performed and animal thrombus models were established to explore the biological activities of annexin Bl. The main results of our research are as follows:1 . Cysticercus cellulosae cDNA library was constructed with gtl 1 expression vector and screened by antiserum of humans and pigs infected with Cysticercus cellulosae. One clone, designated as cCl was identified respectively by sera from humans and pigs. cC1comprises 1299bp with an open reading frame of 1044bp, encoding a protein of 347 amino acids with a calculated molecular mass of 37958.19 Dalton.2. A BLAST search with the amino acid sequence of cCl led to the alignment of 100 annexin in different species with high match scores. Further searching in protein structural database showed that cCl contains four typical annexin repeats* each repeat harboring the type II Ca2+ binding site. The in-silico secondary structure prediction showed the protein was made up of mainly alpha-helices without any secretory signal peptide, suggesting it isa cytosolic protein coincided with other annexins. The cDNA of cCl was inserted into the prokaryotic expression vector pGEX-5T and the recombinant protein was purified with affinity chromatography. By liposome-pelleting assay , cCl protein could bind phosphatidylserin with the presence of calcium ion. These evidences confirmed that cCl belongs to annexin family and is a novel member of this family since the N-terminal is distinct from other annexins. So it is designated as annexin Bl according to the new nomenclature of annexins.3. mRNA was extracted from Cysticercus cellulosae and different proglottids of adult worm, and was hybridized with annexin Bl cDNA probe pre-labeled with 32P. The result suggested that annexin Bl is a stage-specific expression gene. Anti-annexin Bl polyclonal antibody was prepared and annexin Bl localization was examined by immunohistochemistry. The result showed the annexin Bl expresses mainly in Cysticercus cellulosae cyst wall.4. Based on the X-ray crystal structures of other annexin family members, the 3-D structure of annexin Bl was generated by homology modeling. The predicted annexin Bl structure frame is very similar with other members of annexin family. This model contains four homologous internal-domains an...