ABSTRACT
Asparaginyl endopeptidases (AEPs) in plants belong to the family of cysteine protease that undergo self-activation in the form of zymogen in acidic vacuole and play important physiological roles in maturation of seed storage proteins, protein degradation, programmed cell death and host defense. Bioprocessing enzymes (peptidyl Asx-specific ligases, PALs) that promote the maturation of cyclotides have recently been isolated and identified from several cyclotide-rich plants. PALs derived from AEPs can site-specifically catalyze the formation of asparagine or aspartate peptide bonds. Due to the advantages of relatively traceless peptide bonds and broad substrate spectrum and high catalytic efficiency, they have been playing important roles in the cyclization and modification of peptides and proteins, and are powerful tools for improving the stability of peptide drugs. This review describes the physiological functions of AEPs in plants and summarizes the discoveries, structural characteristics, catalytic mechanism and protein engineering of PALs, as well as the limitation of their applications and future trends. In addition, the applications of PALs in cyclotides biosynthesis and the development of macrocyclic peptides are highlighted, with the aim of providing a new idea for the biocatalytic synthesis of cyclic peptides.
ABSTRACT
Objectives To establish a model of carotid atherosclerotic (AS)stenosis in rabbits and to preliminarily investigate the expression of asparaginyl endopeptidase. Methods Fourteen New Zealand white rabbits were divided into either an model group (n = 8)or a sham operation group (n = 6)according to the random number table. The carotid intima was injured by operation in the model group. The rabbits in both groups were fed with high fat diets containing magnesium for 10 weeks. The rabbits were weighted and their blood lipids were tested every 2 weeks. At the end of the fifth and tenth weeks after procedure,the plaque and vessel stenosis of the rabbits were observed by MRI. At the end of the tenth week after proce-dure,the specimens were collected and sliced. Hematoxylin and eosin (HE)staining was used to observe the pathological changes. Immunohistochemical staining was used to analyze the expression of asparaginyl endopeptidase (AEP). Results One rabbit in the model group died of carotid artery injury. After being fed with high-fat diets,the body quality and the level of blood lipid were increased in the rabbits of both groups compared with those before procedure (all P < 0. 01). At the end of the fifth and tenth weeks after procedure,MRI revealed that the luminal stenosis rates in the operation group were 16 ± 11% and 53 ± 20% respectively. There was significant difference within the group (t = - 4. 83,P < 0. 01). MRI revealed no luminal stenosis twice in the sham operation group. HE staining showed intimal hyperplasia,AS plaque formation,lipid deposition in plaques,macrophage and smooth muscle cells migration and infiltration forming foam cells in the model group. No AS formation was observed in the sham operation group. The expression of AEP was higher in the rabbit carotid artery tissue in the model group,and it expressed rarely in the sham surgery group. The absorbance values were 0. 072 0 ± 0. 028 0 and 0. 002 0 ± 0. 000 9 respectively. There was significant difference (t = 6. 61,P < 0. 01). Conclusions The methods of injuring carotid intima combined with magnesium containing high-fat diet may exactly,reliably,and quickly establish an AS carotid artery stenosis model. AEP may associat with the occurrence of AS plaques.
ABSTRACT
Objective To express the gene encoding mature protease of Schistosoma japonicum asparaginyl endopeptidase (Sj32) and evaluate the potential of the recombinant protein rSj32 in diagnosis of domestic animal schistosomiasis. Methods The DNA fragment encoding mature protease of Schistosoma japonicum asparaginyl endopeptidase was cloned with PCR from pET-28(a)/Sj32, and a recombinant plasmid was previously constructed in the laboratory, which contained the ORF of the gene encoding the pro-enzyme Sj32. The amplified DNA fragment was subcloned into pET-28a( + ) and the recombinant plasmid was transformed into E. coli BL21 (DE3) to express the mature protease of Sj32. Then the recombinant antigen (rSj32) was used in ELISA assay to diagnose schistosomiasis of mice, rabbits and water buffalo artificially infected. The detection effects of soluble Schistosoma japonicum egg antigen (SEA) , rSj32 and the recombinant 23 KDa membrane protein were compared. Results The recombinant antigen rSj32 with a molecular weight 41 KDa was successfully produced in E. coli BL21 ( DE3) and was purified with His Column with a yield of 25 mg/L E. coli culture. By using rSj32 as coating antigen in ELISA assay to detect the specific antibody in artificially infected mice, rabbits and buffalo, the sensitivities were 88.9% , 85.0% and 71.8% , respectively, the specificities were 100% , 96.7% and 96. 9% , respectively. There were no significant differences among the detection results of rSj32, SEA and rSj23. Conclusion rSj32 is a promising antigen for serological diagnosis of domestic animal schistosomiasis.