流感嗜血杆菌ATCC49247来源的IgA酶理化性质及其对低糖基化IgA1的分解作用
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摘要
【背景】近年来,能够特异性切割人IgA1分子的IgA蛋白酶(IgA酶)被认为是治疗IgA肾病(IgAN)的潜在药物,但各种物理化学因素均可能影响其生物学活性。【目的】研究流感嗜血杆菌(Haemophilus influenzae) ATCC49247 IgA酶的理化性质,确定IgA酶最适的作用条件后观察IgA酶对低糖基化IgA1的作用。【方法】从细菌培养液中分离纯化IgA酶,SDS-PAGE电泳后银染法分别检测多种理化条件下IgA酶的水解活性及其对低糖基化IgA1的消化作用。【结果】H. influenzae ATCC49247IgA酶可耐受的反应温度较广,最适温度为50°C;在高于60°C的环境中,IgA酶便不可逆地丧失了稳定性;IgA酶在pH 6.0-9.0的环境下能够保持完整催化活性;1 mmol/L的PMSF和高于10 mmol/L的SDS能够强烈地抑制IgA酶的活性,DTT和EDTA对IgA酶活性无明显影响;所有浓度的Al~(3+)、Fe~(3+)和高浓度的Cu~(2+)、Zn~(2+)、Fe~(2+)对IgA酶均表现出强烈的抑制作用,同时Co~(2+)、Mn~(2+)、Ca~(2+)、Ni~(2+)和Mg~(2+)都对IgA酶活性没有明显影响。选择了最适宜的IgA酶作用条件,发现IgA酶能够有效降解低糖基化IgA1底物。【结论】确定IgA酶最适宜的作用条件能保持良好的酶活性,更好地发挥了IgA酶对低糖基化IgA1的降解作用,为IgA酶的临床研究和药用价值的进一步开发提供一定的依据。
[Background] Recently, IgA proteases that specifically cleave human IgA1 molecules have been considered as potential drugs for the treatment of IgA nephropathy(IgAN), but its biological activity may be affected by various physical and chemical factors. [Objective] To explore the physicochemical properties of Haemophilus influenzae ATCC49247 IgA protease for determining the optimal conditions and then to observe its decomposition of low glycosylated IgA1. [Methods] IgA protease was isolated and purified from the bacterial culture solution and the hydrolysis activity of IgA protease and its decomposition of low glycosylated IgA1 were detected by SDS-PAGE electrophoresis under various physicochemical conditions. [Results] H. influenzae ATCC49247 IgA protease tolerated a wide range of temperatures and the optimum temperature is 50 °C. IgA protease irreversibly lost stability above 60 °C. IgA protease maintained full catalytic activity between pH 6.0 and 9.0. PMSF of 1 mmol/L and SDS of above 10 mmol/L strongly inhibited the activity of IgA protease, whereas DTT and EDTA had no significant effect on its activity. IgA protease was obviously inhibited by all concentrations of Al~(3+), Fe~(3+) and high concentration of Cu~(2+), Zn~(2+) and Fe~(2+), whereas Co~(2+), Mn~(2+), Ca~(2+), Ni~(2+) and Mg~(2+) had no significant effect on its activity. By selecting the most suitable conditions for IgA protease, we found that most of the low-glycosylated IgA1 substrate could be degraded by IgA protease. [Conclusion] IgA protease can maintain good enzyme activity under the optimal conditions, when exerting the degradation of low glycosylated IgA1 and the result lays the fundation for the clinical research and the further development of the medicinal value for IgA protease.
[Background] Recently, IgA proteases that specifically cleave human IgA1 molecules have been considered as potential drugs for the treatment of IgA nephropathy(IgAN), but its biological activity may be affected by various physical and chemical factors. [Objective] To explore the physicochemical properties of Haemophilus influenzae ATCC49247 IgA protease for determining the optimal conditions and then to observe its decomposition of low glycosylated IgA1. [Methods] IgA protease was isolated and purified from the bacterial culture solution and the hydrolysis activity of IgA protease and its decomposition of low glycosylated IgA1 were detected by SDS-PAGE electrophoresis under various physicochemical conditions. [Results] H. influenzae ATCC49247 IgA protease tolerated a wide range of temperatures and the optimum temperature is 50 °C. IgA protease irreversibly lost stability above 60 °C. IgA protease maintained full catalytic activity between pH 6.0 and 9.0. PMSF of 1 mmol/L and SDS of above 10 mmol/L strongly inhibited the activity of IgA protease, whereas DTT and EDTA had no significant effect on its activity. IgA protease was obviously inhibited by all concentrations of Al~(3+), Fe~(3+) and high concentration of Cu~(2+), Zn~(2+) and Fe~(2+), whereas Co~(2+), Mn~(2+), Ca~(2+), Ni~(2+) and Mg~(2+) had no significant effect on its activity. By selecting the most suitable conditions for IgA protease, we found that most of the low-glycosylated IgA1 substrate could be degraded by IgA protease. [Conclusion] IgA protease can maintain good enzyme activity under the optimal conditions, when exerting the degradation of low glycosylated IgA1 and the result lays the fundation for the clinical research and the further development of the medicinal value for IgA protease.
引文
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[4]Wang L,Wang HL,Shen HC,et al.Decomposition of low glycosylated IgA1 and its immune complexes by bacterial-derived IgA protease[A]//The 11th National Congress of Basic Theory of Integrated Medicine[C].Yinchuan:Chinese Association of Integrative Medicine,2015:2(in Chinese)王丽,王洪连,沈宏春,等.细菌源性IgA蛋白酶对低糖基化IgA1及其免疫复合物的分解作用[A]//第十一届全国中西医结合基础理论学术研讨会论文集[C].银川:中国中西医结合学会,2015:2
[5]Li XY.IgA protease degradate the deposited IgA immune complex in IgAN in vitro and vivo[D].Luzhou:Master’s Thesis of Sichuan Medical University,2015(in Chinese)李雪英.IgA蛋白酶清除IgA肾病免疫复合物的体内外研究[D].泸州:四川医科大学硕士学位论文,2015
[6]Wang L,Li XY,Shen HC,et al.Bacterial IgA protease-mediated degradation of agIgA1 and agIgA1 immune complexes as a potential therapy for IgA nephropathy[J].Scientific Reports,2016,6:30964
[7]Wang HL,Zhong X,Li JC,et al.Cloning and expression of H.influenzae 49247 IgA protease in E.coli[J].Molecular Biotechnology,2018,60(2):134-140
[8]Walker SG,Carnu OI,Tüter G,et al.The immunoglobulin A1proteinase from Streptococcus pneumoniae is inhibited by tetracycline compounds[J].FEMS Immunology&Medical Microbiology,2006,48(2):218-222
[9]Simpson DA,Hausinger RP,Mulks MH.Purification,characterization,and comparison of the immunoglobulin A1proteases of Neisseria gonorrhoeae[J].Journal of Bacteriology,1988,170(4):1866-1873
[10]Bachovchin WW,Plaut AG,Flentke GR,et al.Inhibition of IgA1proteinases from Neisseria gonorrhoeae and Hemophilus influenzae by peptide prolyl boronic acids[J].The Journal of Biological Chemistry,1990,265(7):3738-3743
[11]Wyatt RJ,Julian BA.IgA nephropathy[J].New England Journal of Medicine,2013,368(25):2402-2414
[2]Lamm ME,Emancipator SN,Robinson JK,et al.Microbial IgAprotease removes IgA immune complexes from mouse glomeruli in vivo:potential therapy for IgA nephropathy[J].The American Journal of Pathology,2008,172(1):31-36
[3]Lomholt H,Poulsen K,Kilian M.Comparative characterization of the IgA gene encoding IgA1 protease in Neisseria meningitidis,Neisseria gonorrhoeae and Haemophilus influenzae[J].Molecular Microbiology,1995,15(3):495-506
[4]Wang L,Wang HL,Shen HC,et al.Decomposition of low glycosylated IgA1 and its immune complexes by bacterial-derived IgA protease[A]//The 11th National Congress of Basic Theory of Integrated Medicine[C].Yinchuan:Chinese Association of Integrative Medicine,2015:2(in Chinese)王丽,王洪连,沈宏春,等.细菌源性IgA蛋白酶对低糖基化IgA1及其免疫复合物的分解作用[A]//第十一届全国中西医结合基础理论学术研讨会论文集[C].银川:中国中西医结合学会,2015:2
[5]Li XY.IgA protease degradate the deposited IgA immune complex in IgAN in vitro and vivo[D].Luzhou:Master’s Thesis of Sichuan Medical University,2015(in Chinese)李雪英.IgA蛋白酶清除IgA肾病免疫复合物的体内外研究[D].泸州:四川医科大学硕士学位论文,2015
[6]Wang L,Li XY,Shen HC,et al.Bacterial IgA protease-mediated degradation of agIgA1 and agIgA1 immune complexes as a potential therapy for IgA nephropathy[J].Scientific Reports,2016,6:30964
[7]Wang HL,Zhong X,Li JC,et al.Cloning and expression of H.influenzae 49247 IgA protease in E.coli[J].Molecular Biotechnology,2018,60(2):134-140
[8]Walker SG,Carnu OI,Tüter G,et al.The immunoglobulin A1proteinase from Streptococcus pneumoniae is inhibited by tetracycline compounds[J].FEMS Immunology&Medical Microbiology,2006,48(2):218-222
[9]Simpson DA,Hausinger RP,Mulks MH.Purification,characterization,and comparison of the immunoglobulin A1proteases of Neisseria gonorrhoeae[J].Journal of Bacteriology,1988,170(4):1866-1873
[10]Bachovchin WW,Plaut AG,Flentke GR,et al.Inhibition of IgA1proteinases from Neisseria gonorrhoeae and Hemophilus influenzae by peptide prolyl boronic acids[J].The Journal of Biological Chemistry,1990,265(7):3738-3743
[11]Wyatt RJ,Julian BA.IgA nephropathy[J].New England Journal of Medicine,2013,368(25):2402-2414