IgA Protease Degradate The Deposited IgA Immune Complex In IgAN In Vitro And Vivo

Objective:IgA nephropathy is the most common glomerulonephritis worldwide which is one of main reasons causing end-stage renal disease.Recently,IgA nephropathy is considered as a kind of autoimmune disease.Interestingly,the previous studies found the Galactose-Deficient IgAl and formation of IgA1-containing immune complex areplay critical role in thepathogenesis.Therefore,removal of mesangial deposited dIgAl and immune complex may be a potential biological therapeutic for IgAN.The previous studies have confirmed that a bacterial IgA protease exhibits substrate specificity for human IgA1 and IgAl-containing immune complexe in a passive mouse model of IgAN.However,these studies did not prove whether the IgA protease could cleared human Galactose-Deficient IgAl and dIgA1-containing immune complexes.The purpose of the present paper is to study the decomposition of dIgA1 and immune complexes which deposited in mice or human vitro specimen by the Neisseria gonorrhoeae IgA protease,and provide trial basis for IgA Protease treatment IgAN.Methods:First,detect the activity of IgA protease in vitro.We firstly deglycosylated the human IgA1 with neuraminidase or P-galactosidase or both of them,and the glycosylation status was confirmed by Helix aspersa(HAA)-based ELISA.After Commercialization IgAl and Galactose-Deficient IgAl were incubated with IgA protease at 37?for one to four hours,respectively,the decomposition activity of IgA1 protease was confirmed by SDS-PAGE electrophoresis and silver stain.Second,Performed the effect of IgA1 protease on IgA1 and dIgA1 in serum and tissue in vitro.Collection the serum,kidney,intestine,tonsil,skin specimens of IgAN and nIgAN patients.Detected and analysis the concentration of IgA1 and dIgA1 in serum and the decomposition effect of IgAl and dIgA1 by IgA protease via ELISA.Compared and analysis the amount of deposited IgA and dIgA1 in kidney tissues of the two group patients.At the same time,we investigated the time and dose effect of the IgA protease by immunofluorescence IgA and fluorescent HAA staining.In addition,we also conducted a study by immunofluorescence IgA staining to assess the decomposition effect of IgA enzyme in tonsils,intestinal,skin,respectively.Third,Confirmed the degradation ability of IgA protease to dIgA1 and dIgA1-containing immune complex in vivo.To study the function of bacterial IgA protease in a pathologically resembling condition,we constructed a passive IgAN mouse model,BALB/c femal mice(8 to 10 weeks old)were intravenously injected with different amount of dIgA-IgG immune complex,respectively,and sacrificed at different time.The same amount of dIgA1-IgG(no special),IgA1,PBS injected into mice as control.Compared the state of complex deposition at different doses and time,choosing the optimal compound dose and deposition time of the animal models,and observe whether the IgA protease could degradation the deposits IgA and IgG in situ of model mice glomerulus by immunofluorescence IgA(Fc,Fab)and IgG staining.To further validate the potential therapeutic usage of purified bacterial IgA protease,the passive IgAN mice were further intravenously administrated with different amount of IgA protease and kidney sections were checked for immune complex by immunofluorescence.Results:First,the neuraminidase or ?-galactosidase or both of them could deglycosylate the human IgA1 by HAA and silver stain.Incubation of IgA 1 or dIgA1 with Neisseria gonorrhoeaed IgA protease(ilution to 1:100)at 37? for one to four hours could crack the hinge region of IgA1 and dIgA1 to Fc and Fab fragments,which suggested the cleavage activity of IgA Protease in vitro.Second,we found that patients with IgAN had more IgA1 and dIgA1 in the sera(both P<0.001)and kidney tissues(both P<0.001)than the nIgAN by ELISA and immunofluorescence methods.Additionally,comparing with the control groups,we also found IgA protease could decreased the IgA1 in serum,tonsil,skin,kidney,intestines and the dIgA1 in serum and kidney by using the optimum concentration(100 ug/ml)and optimum duration time(8 h)(all P<0.001).And the enzyme degradated the IgA1 was more reactive than the dIgA1.Third,to establish a IgAN mouse model,single 900ug dIgA-IgG immune complex was injected in the animals.After 6-hour post-injection,there is massive deposition of immune complex in glomeruli.But,injection of dIgA only or a mixture of dIgA and non-specific goat IgG caused a little deposition of the globin,no immune complex can be detected in saline-injected mice.After IgA Protease(5-30ug per mouse)was injected into IgAN mouse model can cause reduction of Fab fragment immunostaining intensity of both IgA1 and IgG in a dosage-dependent manner compared with the control manipulation(both P<0.001).Treating with 30ug protease,it could caused the most effect of degradation,and in fact it reduced the IgA and IgG deposition more than 50%when compared with control cohort.At the same time,IgA protease had the capacity to degrade the dIgA1 immune complex in situ when it access the glomerulus of the IgAN model mice.Conclusion:First,Neisseria gonorrhoeae IgA protease had cracking activity to commercialization IgA1 and galactose-deficient IgA1.Second,IgA protease can degrade IgA1 and galactose-deficient IgAl deposited in serum or glomeruli of IgAN patient in vitro.Third,IgA protease can clear dIgA-IgG deposited in passive animal model in vivo.