ABSTRACT
Annexin is a protein of evolutionarily conserved polygene family that binds to cell membrane phosphatidylserine (PS). PS is closely related to many diseases with a potential as a new drug target. Annexin has a good value in drug discovery and new drug development. Annexin A4 is a member of the annexins family. Annexin A4 involves in a number of cellular functions, such as exocytosis and coagulation. These functions are related to binding of annexin to acidic phospholipids. However, the detail function(s) of annexin A4 has not been fully uncovered. Production of annexin A4 in large quantity is prerequisite for indepth investigation of the structure-function relationship of annexin A4. Human annexin A4 was originally purified from the natural resource at a low yield due to the complex procedure. In the present study, annexin A4 was expressed in a prokaryotic system with a high yield of soluble protein. The plasmid pET28a-annexin A4-EGFP was constructed for the expression. Recombinant annexin A4-EGFP was purified using two methods. Affinity chromatography approach gave a protein yield at purity of 80%. While, the membrane absorption method produced the protein with the purity over 90%. Flow cytometric analysis showed that the annexin A4-EGFP fusion protein could recognize and bind to the apoptotic cells with an affinity PS at 79.58±11.68 nmol·L-1, which is at the same order of magnitude as A5-EGFP. We successfully achieved the efficient expression of annexin A4-EGFP in prokaryotic system, and provided an easy and convenient method for purifying a large amount of annexin A4-EGFP with a high purity. This study has laid a solid foundation for our study of the function of annexin A4 in the future.
ABSTRACT
ObjectiveTo identify the differential expressed proteins,and to investigate the relationship between altered expression of annexin A4 during window of implantation [ WOI ( at day-6 after ovulatory day )] in infertile patients with endometriosis and endometrial receptivity.MethodsTwo-dimensional fluorescence differential in-gel electrophoresis (2D-DIGE) and matrix-assist laser desorption ionization-time of flight-mass spectrometry (MALDI-TOF-MS) were used to detect protein expression in endometrial WOI in 10 infertile cases with endometriosis as endometriosis group and 10 infertile cases with tubal factors as control group.The semi-quantitative validation of annexin A4 in the eutopic endometrial tissue during WOI was analyzed by western blot.Results By comparing protein profiles,there were 7 meaningful differential proteins during WOI in infertile patients with endometriosis.One protein with an isoelectric point of 5.84 and relative molecular weight of 36 100 were down regulated 348％ in samples of endometriosis group.It was identified as annexin A4 by mass spectrometry.By western blot,relative intensity of annexin A4 in endometriosis group was 7.2 ±0.9,which was lower than 17.8 ± 2.6 in control group significantly (t =7.654,P =0.002 ).ConclusionLower expresssion of annexin A4 during WOI in infertile patients with endometriosis might be associated with the decrease of endometrial receptivity.
ABSTRACT
Objective To investigate the differential expression of annexin A4 (ANXA4)protein in different phenotype of human hepatocellular carcinoma (HCC) tissues, and discuss the correlative clinico-pathologic significance. Methods The mRNA levels of ANXA4 in different liver tissues were validated by using semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR), and the expressions of ANXA4 protein in different liver tissues specimens were observed by immunohistochemistry assay. The relative analysis between the results of immunohistochemistry assay and the clinicopathologic parameters were studied. Results In mRNA level, the results from RT-PCR were significantly increased of ANXA4 in HCC tissues than that in cirrhosis tissues and normal liver tissues. Immunohistochemistry assay of different type liver tissues also confirmed that ANXA4 protein overexpressed in HCC tissues than that in the cirrhosis tissues post hepatitis B and normal liver tissues (49.4% vs 28.0% vs 24.0%), and the higher positive rate could be observed in different phenotype HCC tissues (53.8% in small HCC, 64.5% in large HCC, 28.6% in multiple HCC, respectively),according the positive rate of para-tumorous tissues was 53.8%, 64.5% and 67.9%, respectively. The results of statistics analysis showed that the expression of ANXA4 up-regulated gradually going with the hepatocellular malignant metaplasia and tumorous progression, but the expression of ANXA4 down-regulated when invasion were emerging in HCC. Furthermore, expression of ANXA4 had a negative correlation with the tumor size and maybe responsible for carcinogenesis and progression in human hepatocellular carcinoma. Conclusions The mRNA level of ANXA4 is significantly increased in HCC and cirrhosis tissues compared to the normal liver tissues by semi-quantitative RT-PCR. The same trend can be observed in immunohistochemistry assay. ANXA4 might be concerned with the carcinogenesis of HCC. Unbalance of expression of ANXA4 could be observed in different phenotype HCC tissues. ANXA4 might take part in the process of progression and invasion of HCC.