The association between production of under-O-glycosylated IgA1 in the patient with IgA nephropathy and the staphylococcal cell membrane antigen

(1)Staphylococcal cell membrane antigen，a possible antigen in post-MRSA infection glomerulonephritis and IgA nephropathy，interacts directly with mesangial cellsBackground：The staphylococcal cell membrane antigen(GenBank accession number：BAB41819.1)is a possible antigen involved in post-MRSA infection GN and IgA nephropathy2.Since Toll-like receptors(TLRs)activate the host inate immresune to conthange to commonange，The clinical findings suggest that the staphylococcal cell membrane antigen interacts directly with intrinsic cells of the kidney viaTLRs.Methods：Mouse mesangial cells(ATCC：CRL-1927)were mixed with purified FLAG-tagged staphylococcal cell membrane antigen under various conditions and stained with fluorescein isothiocyanate(FITC)-conjugated anti-FLAG antibody。Cellular attachment of the FLAG-tagged antigen was analyzed by flow cytometry.The cells were also cultured with or without anti-mouse TLR2 or TLR4 IgG antibody，and…More the growth stimulation effect of the antigen on these cells was determined using ELISA based on bromodeoxyuridine(BrdU)incorporation。Genomic DNA obtained from mesangial cells after incubation with or without staphylococcal cell membrane antigen was analyzed by electrophoresis.Results：Attachment of staphylococcal membrane antigen to cultured mesangial cells occurred in the exponential growth phase，whereas supraconfluent cells did not interact with the antigen.Incubation of the mesangial cells with the antigen at 37℃for 15minutes resulted in higher fluorescence intensity in flow cytometry，when compared with incubation at 4℃.Treatment with a low dose of recombinant staphylococcal cell membrane antigen in the absence of anti-TLR antibodies did not accelerate incorporation of BrdU by the mesangial cells，whereas treatment with anti-TLR antibodies attenuated BrdU incorporation。A moderate to high dose of antigen without anti-TLR antibodies also attenuated BrdU incorporation in a dose-dependent manner。Genomic DNA from mesangial cells incubated with the antigen showed fragmentation.Conclusion：These results suggest that staphylococcal cell membrane antigen interacts with mesangial cells directly and transmits weak growth acceleration signals via TLRs and inhibitory signals via an apoptotic pathway.(2)A novel analysis of under-O-glycosylated IgA1 in IgA nephropatpathays Aberrant glycosylation of IgA1hyberrant glycosylation of IgA(IgA)。This abnormality is manifested by a deficiency of galactose in the hinge-region O-linked glycans of IgA1.When compared with the healthy control patients，a decrease in beta1，3-galactosyltransferase activity and an increase in N-acetylgalactosamine-specific alpha2，6-sialyltransferase activity were observed in IgA nephropathy patients。This abnormally O-glycosylated IgA1is likely to be involved in the pathogenesis of IgA nephropathy.However，correlation between the clinilcal parameters，(proteinuria，hematuria，pathological findings and so on)，clinical therapeutic efficacy(steroid therapy，tonsillectomy etc.)And this aberrant glycosylated IgA1 are still unclear.The purpose of this study is to determine how this aberrant glycosylation is effected by the treatment of IgA nephropathy.Fcα/µreceptor(Fcα/µR)is costitutively expressed on the majority of B lymphocytes and macrophages and its human homolog，which bind both IgA and IgM with high affinity.We established a method to capture serum IgA from whole serum by mouse Fcα/µR transfected cells。The immunoglobulin domain of Fcα/µR which can fix IgA and IgM has marked homology for the domain of polymeric immunoglobulin receptor(poly-IgR)。We also hypothesized that this system can evaluate the underglycosylation of serum polymeric IgA mainly observed in the patients‘glomeruli.BW5147(Parent)or moFcα/µR·BW5147 transfectant was treated with normal serum.Captured IgA was detected by FITC conjugated anti-human IgA in flow cytometry.We also measured lectin binding(Helix aspersa-phycoerythrin；HA-PE)by this flow cytometry analysis.The transfectant did not bind to the monomeric IgA but bound to polymeric IgA.Observation by a confocal laser microscope revealed IgA binding of Fcα/µR grouped on the cell surface.In the HAA-PE binding assay between patients with IgA nephropathy(n=33)and healthy controls(n=22)，the patients showed significantly higher HAA-binding，indicating underglycosylation of IgA，when compared with controls。Less：Less

(1)Staphylococcal cell membrane antigen, a possible antigen in post-MRSA infection glomerulonephritis and IgA nephropathy, interacts directly with mesangial cellsBackground : The staphylococcal cell membrane antigen (GenBank accession number : BAB41819.1) is a possible antigen involved in post-MRSA infection GN and IgA nephropathy 2. Since Toll-like receptors (TLRs) activate the host innate immune response to recognize components common to a range of bacteria, the clinical findings suggest that the staphylococcal cell membrane antigen interacts directly with intrinsic cells of the kidney viaTLRs.Methods : Mouse mesangial cells (ATCC : CRL-1927) were mixed with purified FLAG-tagged staphylococcal cell membrane antigen under various conditions and stained with fluorescein isothiocyanate (FITC)-conjugated anti-FLAG antibody. Cellular attachment of the FLAG-tagged antigen was analyzed by flow cytometry. The cells were also cultured with or without anti-mouse TLR2 or TLR4 IgG antibody, and … More the growth stimulation effect of the antigen on these cells was determined using ELISA based on bromodeoxyuridine (BrdU) incorporation. Genomic DNA obtained from mesangial cells after incubation with or without staphylococcal cell membrane antigen was analyzed by electrophoresis.Results : Attachment of staphylococcal cell membrane antigen to cultured mesangial cells occurred in the exponential growth phase, whereas supraconfluent cells did not interact with the antigen. Incubation of the mesangial cells with the antigen at 37℃ for 15 minutes resulted in higher fluorescence intensity in flow cytometry, when compared with incubation at 4℃. Treatment with a low dose of recombinant staphylococcal cell membrane antigen in the absence of anti-TLR antibodies did not accelerate incorporation of BrdU by the mesangial cells, whereas treatment with anti-TLR antibodies attenuated BrdU incorporation. A moderate to high dose of antigen without anti-TLR antibodies also attenuated BrdU incorporation in a dose-dependent manner. Genomic DNA from mesangial cells incubated with the antigen showed fragmentation.Conclusion : These results suggest that staphylococcal cell membrane antigen interacts with mesangial cells directly and transmits weak growth acceleration signals via TLRs and inhibitory signals via an apoptotic pathway.(2) A novel analysis of under-O-glycosylated IgA1 in IgA nephropathyAberrant glycosylation of IgA1 plays an essential role in the pathogenesis of IgA nephropathy(IgA-N). This abnormality is manifested by a deficiency of galactose in the hinge-region O-linked glycans of IgA1. When compared with the healthy control patients, a decrease in beta1,3-galactosyltransferase activity and an increase in N-acetylgalactosamine-specific alpha2,6-sialyltransferase activity were observed in IgA nephropathy patients. This abnormally O-glycosylated IgA1 is likely to be involved in the pathogenesis of IgA nephropathy. However, correlation between the clinilcal parameters, (proteinuria, hematuria, pathological findings and so on), clinical therapeutic efficacy (steroid therapy, tonsillectomy etc.) and this aberrant glycosylated IgA1 are still unclear.The purpose of this study is to determine how this aberrant glycosylation is effected by the treatment of IgA nephropathy.Fcα/μ receptor(Fcα/μR) is costitutively expressed on the majority of B lymphocytes and macrophages and its human homolog, which bind both IgA and IgM with high affinity. We established a method to capture serum IgA from whole serum by mouse Fcα/μR transfected cells. The immunoglobulin domain of Fcα/μR which can fix IgA and IgM has marked homology for the domain of polymeric immunoglobulin receptor (poly-IgR). We also hypothesized that this system can evaluate the underglycosylation of serum polymeric IgA mainly observed in the patients' glomeruli.BW5147 (parent) or moFcα/μR・BW5147 transfectant was treated with normal serum. Captured IgA was detected by FITC conjugated anti-human IgA in flow cytometry. We also measured lectin binding (Helix aspersa-phycoerythrin ; HA-PE) by this flow cytometry analysis. The transfectant did not bind to the monomeric IgA but bound to polymeric IgA. Observation by a confocal laser microscope revealed IgA binding of Fcα/μR grouped on the cell surface. In the HAA-PE binding assay between patients with IgA nephropathy (n=33) and healthy controls (n=22), the patients showed significantly higher HAA-binding, indicating underglycosylation of IgA, when compared with controls. Less