Identification Of A Novel Annexin Subfamily-AnxB1 And Its Anticoagulation Function

Using immunoscreen method, Sun S.H. et al isolated an antigen gene cCl from the cDNA library of Cysticercus cellulosae. Lots of findings have indicated that cCl not only means a promising protective antigen, but also might be involved in the transduction of mitogenic signal and cell's proliferation and differentiation. In order to make a deep study on its physiological role, bioinformatics analysis was performed on the nucleic acid sequence and amino acid sequence of cCl, including similar sequence search, secondary and crystal structure prediction, biochemical and biophysical character prediction. The results are encouraging: cCl have 40 ~60% amino acid sequence similarity to other annexin genes. It has been believed that if two amino acid sequences have 30% similarity or more, the two genes might belong to the same family. This means that cCl maybe also a homologue of annexins. By searching the domain databases such as PROSTTE, BLOCKS we found that cCl contains four typical "Annexins repeats". Each repeat was composed of 70 ~ 100 amino acid residues and containing a motif of "M-K/R-G-X-G-T-(38 residues)-D/E" ,which was suggested to be the type II Ca2+ binding site in annexins. Prediction of the secondary structure revealed that 57.8% of the total amino acid residues formed 18-20 a-helixes, no p-fold could be predicted. The crystal structure of cCl was established by homology modeling, interestingly, it shows a similar overall topology to the structure of annexinAS. All above data indicted cC 1 has the typical structure of annexins.Annexins are a large family of calcium binding proteins widely distributed in eukaryotes. Proteins of this family have the common biochemical character: binding phospholipid membrane in the present of calcium. To identify whether cCl also have the property, full-length cDNA was inserted into the plasmid pGEX-5T and transformed the E.coli strain k802, then the recombinant expressed under the tac promoter and induced by IPTG. In the present of 2 mM Ca2+, the recombinant cCl could be co-deposited with phosphatidylserine-contained vesicles. When added 4 mM EGTA to the pellets, cCl can be separated from the vesicles. This experiment identified that cCl also has the common biochemical property of annexins.Since cCl have the two typical annexins features, it can be concluded that cCl is also be a member of annexins. In addition, the amino acid similarity between the conserved tetrad in cCl and other annexins is in the range of 40 ~ 60%, and between N-termini in cCl and other annexins is no more than 20%, this means that cCl is not belong to any of the extant subfamilies, but behaves a novel subfamily. According to the new nomenclature of annexins, it was designed as Annexin Bl (abbreviation AnxBl) .AnxBl is the first annexin isolated from platyhelminth. Studies on itcan help to make clear what the physiological role it plays in Cysticercus cellulosae's infection, loading and other processes, which may provides some new methods to prevent and control this disease. Nevertheless, as one of the few annexins from parasites, AnxB 1 can act as a model to study the evolution profile of annexins. The next step we studied the expression of AnxBl in different stages ofTaenia solium by RT-PCR analysis. Strikingly, Annexin Bl is a stage-specific expression gene. It only expresses in Cysticercus cellulosae, but not in different proglottids of adult worm. What the role AnxBl plays in Cysticercus cellulosae's growth and transformation is not clear.Another property of AnxBl is its anticoagulation activity. The recombinant GST-AnxBl could prolong the kaolin partial thromboplastin time (KPTT) significantly in a dose-dependent way, whereas GST alone lacks this properity. Previous studies have proposed that the anticoagulant properties of annexins are associated with its calcium-dependent phospholipid binding activity. Indeed, some steps in the coagulation process (e.g., activation of factor X by Villa and Ixa) require phosphatidylserine (PS) as cofactor. In vivo, this phospholipid is found in th...