Mammalian cells express two isoforms of Cu- and Zn-containing superoxide dismutases（SODs）, CuZn-SOD and extracellular SOD（EC-SOD）, involved in the defense system against reactive oxygen species（ROS）. The two SOD...Mammalian cells express two isoforms of Cu- and Zn-containing superoxide dismutases（SODs）, CuZn-SOD and extracellular SOD（EC-SOD）, involved in the defense system against reactive oxygen species（ROS）. The two SODs have structurally homologous centre domain with distinct N- and C-terminuses, resulting in the different characteristics of the structure and function of the two molecules. We generated a hybrid SOD molecule（namely hy- SOD） via replacing the N- and C-terminuses of hCuZnSOD with the counterparts of hEC-SOD. The hySOD was expressed in host Pichia pastoris and the purified protein was a dimer with a molecular weight of about 34000. A series of activity analyses indicates that the hySOD is similar to hEC-SOD in heat-stability, and has the activity of protecting the host cell against heat shock and oxidative stress. Our results show evidence for the study on the compound activity of multiple SOD molecules, and may be important for understanding the relationship between structure and function of hEC-SOD and hCuZnSOD.展开更多
Extracellular superoxide dismutase(EcSOD)protects tissues from oxidative stress,and thus is considered as a therapeutic agent for many diseases such as atherosclerosis,hypertension,and cancer.However,cost-effective pr...Extracellular superoxide dismutase(EcSOD)protects tissues from oxidative stress,and thus is considered as a therapeutic agent for many diseases such as atherosclerosis,hypertension,and cancer.However,cost-effective production of bioactive recombinant human EcSOD(rhEcSOD)remains a challenge.Herein,we developed an efficient strategy for producing active rhEcSOD by transgenic silkworms.rhEcSOD was successfully synthesized as homodimers and homotetramers in the middle silk gland and spun into the cocoons with a concentration of 9.48±0.21 mg/g.Purification of rhEcSOD from the cocoons could be conveniently achieved with a purity of 99.50%and a yield of 3.5±0.5 mg/g.Additionally,N-glycosylation at the only site of N89 in rhEcSOD with 10 types were identified.The purified rhEcSOD gained the potent enzymatic activity of 4162±293 U/mg after Cu/Zn ions incorporation.More importantly,rhEcSOD was capable of penetrating and accumulating in the nuclei of cells to maintain cell morphology and attenuate ultraviolet B-induced cell apoptosis by eliminating reactive oxygen species and inhibiting the C-Jun N-terminal kinase signaling pathway.These results demonstrated that the transgenic silkworm could successfully produce rhEcSOD with enzymatic and biological activities for biomedical applications.展开更多
Plasma extracellular superoxide dismutase (ecSOD) concentrations are significantly (2 - 3-fold) higher in 129 mice than in the C57 strain. We reported earlier that the 129 strain carries a different allele of this enz...Plasma extracellular superoxide dismutase (ecSOD) concentrations are significantly (2 - 3-fold) higher in 129 mice than in the C57 strain. We reported earlier that the 129 strain carries a different allele of this enzyme, characterized by two amino acid substitutions and a 10 bp deletion in the 3’UTR of the mRNA. One of the substitutions is at the signal peptide cleavage site (position 21) while the other is within the catalytic site. Since the differences in plasma enzyme concentrations persist in congenic mice expressing the different alleles, the differences in plasma concentrations appear to be largely allele-driven and independent of the remainder of the genome. As expected, in vitro translation using rabbit reticulocyte lysate showed no differences in translational efficiency of transcripts and processing of the newly synthesized enzymes. Determinations of the rates of synthesis and secretion of the two isoforms of this enzyme in stably transfected CHO cells clearly demonstrate that the mature 129 variant is synthesized at rates nearly twice those of the wild-type isoform and has an intracellular pool size twice as large. However, both isoforms are secreted at the same fractional rate and have identical intracellular T1/2 as well as similar extent of degradation. These data explain, at least partially, the higher levels of ecSOD in congenic mice expressing the 129 variant, as well as enzyme levels in the 129 strain of mice. In silico calculations support this conclusion and indicate that the aa change at the signal peptide cleavage site (N21D) results in a substantial increase in cleavage probability at this site for the 129 variant. Our results also highlight the importance of signal peptide cleavage in determining steady-state levels of secretory proteins.展开更多
Objective To investigate the correlation between drinking behavior combined with polymorphisms of extracellular superoxide dismutase （EC-SOD） and aldehyde dehydrogenase-2 （ALDH2） genes and pancreatic cancer. Meth...Objective To investigate the correlation between drinking behavior combined with polymorphisms of extracellular superoxide dismutase （EC-SOD） and aldehyde dehydrogenase-2 （ALDH2） genes and pancreatic cancer. Methods The genetic polymorphisms of EC-SOD and ALDH2 were analyzed by polymerase chain reaction restriction fragment length polymorphism in the peripheral blood leukocytes obtained from 680 pancreatic cancer cases and 680 non-cancer controls. Subsequently the frequency of genotype was compared between the pancreatic cancer patients and the healthy controls.The relationship of drinking with pancreatic cancer was analyzed. Results The frequencies of EC-SOD （C/G） and ALDH2 variant genotypes were 37.35% and 68.82% respectively in the pancreatic cancer cases, and were significantly higher than those in the healthy controls （21.03% and 44.56%, all P〈0.01）. People who carried EC-SOD （C/G） （0R=2.24, 95% C1= 1.81-4.03, P〈0.01） or ALDH2 variant genotypes （OR=2.75, 95% CI=1.92-4.47, P〈0.01） had a high risk to develop pancreatic cancer. Those who carried EC-SOD （C/G） genotype combined with ALDH2 variant genotype had a high risk for pancreatic cancer （29.56% vs. 6.76%, 0R=7.69, 95% CI=3.58-10.51, P〈0.01）. The drinking rate of the pancreatic cancer group （64.12%） was significantly higher than that of the control group （40.15%; OR=2.66, 95% CI=1.30-4.42, P〈0.01）. An interaction between drinking and EC-SOD （C/G）/ALDH2 variant genotypes increased the risk of occurrence of pancreatic cancer （OR=25.00, 95% CI= 11.87-35.64, P〈0.01）. Conclusion EC-SOD （C/G）, ALDH2 variant genotypes and drinking might be the risk factors of pancreatic cancer.展开更多
Superoxide dismutases （SODs） are a group of important antioxidant defense enzymes. In this study, a putative extracellular Cu/Zn superoxide dismutase （ecCuZnSOD） complementary DNA was cloned and characterized from...Superoxide dismutases （SODs） are a group of important antioxidant defense enzymes. In this study, a putative extracellular Cu/Zn superoxide dismutase （ecCuZnSOD） complementary DNA was cloned and characterized from the whitefly, Bemisia tabaci. Quantitative polymerase chain reaction analysis showed that the expression level of BtecCuZnSOD was more than 10-fold higher in the invasive Middle East Asia Minor 1 （MEAM1） than in the native Asia II 3 species of the B. tabaci species complex. After exposure to low temperature （4 ℃）, the expression of Bt-ecCuZnSOD gene was significantly up-regulated in MEAM1 but not in Asia II 3. Furthermore, the expression level ofB. tabaci intracellular CuZnSOD （Bt-icCuZnSOD）, Bt-ecCuZnSOD and mitochondrial MnSOD （Bt-mMnSOD） was compared after transferring MEAM1 and Asia II 3 whiteflies from favorable （cotton） to unfavorable host plants （tobacco）. On cotton, both CuZnSOD genes were expressed at a higher level in MEAM1 compared with Asia II 3. Interestingly, after transferring onto tobacco, the expression of Bt-ecCuZnSOD was significantly induced in Asia II 3 but not in MEAM1. On the other hand, while Bt-mMnSOD was expressed equally in both species on cotton, Bt-mMnSOD messenger RNA was up-regulated in MEAM 1 on tobacco. Consistently, enzymatic activity assays of CuZnSOD and MnSOD demonstrated that CuZnSOD might play an important protective role against oxidative stress in Asia II 3, whereas MnSOD activation was critical for MEAM1 whiteflies during host adaptation. Taken together, our results suggest that the successful invasion ofMEAM 1 is correlated with its constitutive high activity of CuZnSOD and inducible expression of MnSOD under stress conditions.展开更多
文摘Mammalian cells express two isoforms of Cu- and Zn-containing superoxide dismutases（SODs）, CuZn-SOD and extracellular SOD（EC-SOD）, involved in the defense system against reactive oxygen species（ROS）. The two SODs have structurally homologous centre domain with distinct N- and C-terminuses, resulting in the different characteristics of the structure and function of the two molecules. We generated a hybrid SOD molecule（namely hy- SOD） via replacing the N- and C-terminuses of hCuZnSOD with the counterparts of hEC-SOD. The hySOD was expressed in host Pichia pastoris and the purified protein was a dimer with a molecular weight of about 34000. A series of activity analyses indicates that the hySOD is similar to hEC-SOD in heat-stability, and has the activity of protecting the host cell against heat shock and oxidative stress. Our results show evidence for the study on the compound activity of multiple SOD molecules, and may be important for understanding the relationship between structure and function of hEC-SOD and hCuZnSOD.
基金supported by the National Key Research and Development Program of China(2022YFD1201600)Chongqing Science and Technology Commission(cstc2020jcyj-cxttX0001,CSTB2022TIAD-KPX0083)Yongchuan District Science and Technology Commission(2022ycyfjg20002).
文摘Extracellular superoxide dismutase(EcSOD)protects tissues from oxidative stress,and thus is considered as a therapeutic agent for many diseases such as atherosclerosis,hypertension,and cancer.However,cost-effective production of bioactive recombinant human EcSOD(rhEcSOD)remains a challenge.Herein,we developed an efficient strategy for producing active rhEcSOD by transgenic silkworms.rhEcSOD was successfully synthesized as homodimers and homotetramers in the middle silk gland and spun into the cocoons with a concentration of 9.48±0.21 mg/g.Purification of rhEcSOD from the cocoons could be conveniently achieved with a purity of 99.50%and a yield of 3.5±0.5 mg/g.Additionally,N-glycosylation at the only site of N89 in rhEcSOD with 10 types were identified.The purified rhEcSOD gained the potent enzymatic activity of 4162±293 U/mg after Cu/Zn ions incorporation.More importantly,rhEcSOD was capable of penetrating and accumulating in the nuclei of cells to maintain cell morphology and attenuate ultraviolet B-induced cell apoptosis by eliminating reactive oxygen species and inhibiting the C-Jun N-terminal kinase signaling pathway.These results demonstrated that the transgenic silkworm could successfully produce rhEcSOD with enzymatic and biological activities for biomedical applications.
文摘Plasma extracellular superoxide dismutase (ecSOD) concentrations are significantly (2 - 3-fold) higher in 129 mice than in the C57 strain. We reported earlier that the 129 strain carries a different allele of this enzyme, characterized by two amino acid substitutions and a 10 bp deletion in the 3’UTR of the mRNA. One of the substitutions is at the signal peptide cleavage site (position 21) while the other is within the catalytic site. Since the differences in plasma enzyme concentrations persist in congenic mice expressing the different alleles, the differences in plasma concentrations appear to be largely allele-driven and independent of the remainder of the genome. As expected, in vitro translation using rabbit reticulocyte lysate showed no differences in translational efficiency of transcripts and processing of the newly synthesized enzymes. Determinations of the rates of synthesis and secretion of the two isoforms of this enzyme in stably transfected CHO cells clearly demonstrate that the mature 129 variant is synthesized at rates nearly twice those of the wild-type isoform and has an intracellular pool size twice as large. However, both isoforms are secreted at the same fractional rate and have identical intracellular T1/2 as well as similar extent of degradation. These data explain, at least partially, the higher levels of ecSOD in congenic mice expressing the 129 variant, as well as enzyme levels in the 129 strain of mice. In silico calculations support this conclusion and indicate that the aa change at the signal peptide cleavage site (N21D) results in a substantial increase in cleavage probability at this site for the 129 variant. Our results also highlight the importance of signal peptide cleavage in determining steady-state levels of secretory proteins.
文摘Objective To investigate the correlation between drinking behavior combined with polymorphisms of extracellular superoxide dismutase （EC-SOD） and aldehyde dehydrogenase-2 （ALDH2） genes and pancreatic cancer. Methods The genetic polymorphisms of EC-SOD and ALDH2 were analyzed by polymerase chain reaction restriction fragment length polymorphism in the peripheral blood leukocytes obtained from 680 pancreatic cancer cases and 680 non-cancer controls. Subsequently the frequency of genotype was compared between the pancreatic cancer patients and the healthy controls.The relationship of drinking with pancreatic cancer was analyzed. Results The frequencies of EC-SOD （C/G） and ALDH2 variant genotypes were 37.35% and 68.82% respectively in the pancreatic cancer cases, and were significantly higher than those in the healthy controls （21.03% and 44.56%, all P〈0.01）. People who carried EC-SOD （C/G） （0R=2.24, 95% C1= 1.81-4.03, P〈0.01） or ALDH2 variant genotypes （OR=2.75, 95% CI=1.92-4.47, P〈0.01） had a high risk to develop pancreatic cancer. Those who carried EC-SOD （C/G） genotype combined with ALDH2 variant genotype had a high risk for pancreatic cancer （29.56% vs. 6.76%, 0R=7.69, 95% CI=3.58-10.51, P〈0.01）. The drinking rate of the pancreatic cancer group （64.12%） was significantly higher than that of the control group （40.15%; OR=2.66, 95% CI=1.30-4.42, P〈0.01）. An interaction between drinking and EC-SOD （C/G）/ALDH2 variant genotypes increased the risk of occurrence of pancreatic cancer （OR=25.00, 95% CI= 11.87-35.64, P〈0.01）. Conclusion EC-SOD （C/G）, ALDH2 variant genotypes and drinking might be the risk factors of pancreatic cancer.
基金Acknowledgments Financial support for this study was provided by the National Natural Science Foundation of China （Projects 31071686, 31171848）, the Program for New Century Excellent Talents in University （NCET-12-0483） and the Specialized Research Fund for the Doctoral Program of Higher Education （Project 20120101110077）.
文摘Superoxide dismutases （SODs） are a group of important antioxidant defense enzymes. In this study, a putative extracellular Cu/Zn superoxide dismutase （ecCuZnSOD） complementary DNA was cloned and characterized from the whitefly, Bemisia tabaci. Quantitative polymerase chain reaction analysis showed that the expression level of BtecCuZnSOD was more than 10-fold higher in the invasive Middle East Asia Minor 1 （MEAM1） than in the native Asia II 3 species of the B. tabaci species complex. After exposure to low temperature （4 ℃）, the expression of Bt-ecCuZnSOD gene was significantly up-regulated in MEAM1 but not in Asia II 3. Furthermore, the expression level ofB. tabaci intracellular CuZnSOD （Bt-icCuZnSOD）, Bt-ecCuZnSOD and mitochondrial MnSOD （Bt-mMnSOD） was compared after transferring MEAM1 and Asia II 3 whiteflies from favorable （cotton） to unfavorable host plants （tobacco）. On cotton, both CuZnSOD genes were expressed at a higher level in MEAM1 compared with Asia II 3. Interestingly, after transferring onto tobacco, the expression of Bt-ecCuZnSOD was significantly induced in Asia II 3 but not in MEAM1. On the other hand, while Bt-mMnSOD was expressed equally in both species on cotton, Bt-mMnSOD messenger RNA was up-regulated in MEAM 1 on tobacco. Consistently, enzymatic activity assays of CuZnSOD and MnSOD demonstrated that CuZnSOD might play an important protective role against oxidative stress in Asia II 3, whereas MnSOD activation was critical for MEAM1 whiteflies during host adaptation. Taken together, our results suggest that the successful invasion ofMEAM 1 is correlated with its constitutive high activity of CuZnSOD and inducible expression of MnSOD under stress conditions.
