Structure, Function And Evolution Of Novel Members Of Annexin Family

Annexins are Ca²⁺ and phosphol ipid 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 Annex in.Anx B2 and AnxB3 isolated using the degeneracy of the code to design PCR primers from multiplr-sequence alignments, are two novel members of the annexin family of Ca²⁺ and phospholipid binding proteins. Studies on Anx B2 and AnxB3 can help to make clear what the role they play in the infection, loading and other activities of Cysticercus cellulosae, which may provide some new ways to prevent and control this disease. Additionally, Anx B2 and AnxB3 can also act as a molecular model for studying the evolutionary divergence of Annexins. Fusion expression vectors of Anx B2 and AnxB3 were constructed and the expressed product were purified by affinity or ion exchange chromatography; Homology modeling and sequence-deleted mutagenesis; prothrombin time (PT), Activated partial thromboplastin time (aPTT) assay and platelet aggregation inhibition were performed to explore the biological activities of Anx B2 and AnxB3. The main results of our research are as follows:1. Anx B2 and AnxB3 isolated using the degeneracy of the code to design PCR primers from multiplr-sequence alignments are two novel members of the annexin family of Ca2+ and phospholipid binding proteins. Anx B2 comprises 1285bp with an open reading frame of 1065bp encoding a protein of 355 amino acids with a calculated molecular mass of 38682. 05 Dalton, Anx B3 comprises 1126bp with an open reading frame of 910bp encoding a protein of 310 amino acids with a calculated molecular mass of 35874. 19 Dalton。2. A BLAST search with the amino acid sequence of Anx B2 and Anx B3 led to the alignmentof 100 Annexin in different species with high match scores. Further searching in protein structural database showed that Anx B2 and Anx B3 contain four typical Annexin repeats, each repeat harboring the type II Ca2* binding site. The insilico secondary structure prediction showed the protein was made up of mainly alpha-helices without any secretory signal peptide, suggesting it is a cytosolic protein coincided with other Annexins. The cDNA of Anx B2 and Anx B3 respectively were inserted into the prokaryotic expression vector pGEX⁵T. These evidences confirmed that Anx B2 and Anx B3 belong to Annexin family and are two novel members of this family since the N-terminal is distinct from other Annexins. So they are respectively designated as Anx B2 and Anx B3 according to the new nomenclature of Annexins.3. Based on the X-ray crystal structures of other Annexin family members, the 3-D structures of Anx B2 and Anx B3 respectively were generated by homology modeling. Thepredicted Anx B2 and Anx B3 structure frames are very similar with other members of Annexin family. Every model contains four homologous internal-domains and the Ca trace of domain I, II and IV exhibits high similarity.4. In order to explore the functional of Anx B2 and Anx B3, we assayed their immunogenicity and anticoagulant activity according to Anx B2, Anx B3 and M_Anx B2protein structure stigmata by Bioinformatics analysis. As Anx B2 and Anx B3 were found from homologous Cysticercus cellulose, studies on Anx B2, Anx B3 and their relationship with Annexin Bl(Anx Bl) in the structure and the function can help to make clear what the role they play in the infection, invasion and other activities of Cysticercus cellulosae, which may provide some new ways to prevent and control this disease. Additionally, Anx B family can also act as a molecular model for studying the evolutionary divergence of annexins.5. To investigate the correlation of Anx B2 and Anx B3 with other Annexins and their biological function. 80 amino acid sequences from Annexins were selected by speciesclassification from Swiss^prot and NCBI-Structure database. The structure, function and evolution of Anx B2 and Anx B3 were analyzed with bioinformatics. Anx B2 and Anx B3 contain respectively 7 and 5 Ca2*binding sites. Anx B2 contains specific amino acid residues of...