Immunoglobulin A (IgA) nephropathy (IgAN) or Berger's disease is an autoimmune disease resulting from the abnormal deposit of IgA in the glomerulus. Primary IgAN is the most common form of primary glomerulonephritis, leading to progressive renal failure in almost one third of the patients. Numerous studies have suggested that IgAN results from abnormal O-glycosylation of IgA1. Normal IgA1 is unique among immunoglobulins in that it contains ~5 Ser/Thr-linked O-glycans with mono-sialylated and di-sialylated core 1 structures. The pathogenesis of the IgAN is associated with a deficiency of galactose in O-glycans and concomitant expression of the Tn antigen (GalNAc?-Ser/Thr) in its hinge region of IgA1. Tn antigen is normally masked through addition of galactose by the T-synthase to generate the core 1 structure (Gal(3GalNAc-R). We discovered that T-synthase requires a unique molecular chaperone we termed Cosmc that helps to fold the T-synthase during its biosynthesis in the ER. Cosmc is encoded on Xq24 and is an ER chaperone that prevents the aggregation/proteasomal degradation of the T-synthase. Preliminary studies show that somatic mutations in Cosmc cause expression of the Tn antigen. We now hypothesize that somatic mutations of Cosmc in IgA-secreting B cells cause undergalactosylation of IgA1 and result in non-familial or sporadic IgAN. In this proposal, we will focus on three Specific Aims.
Aim 1 : We will analyze Cosmc in Tn/CD19(+) B lymphocytes from peripheral blood of patient with IgAN. For all mutations found, the effects of mutation on Cosmc chaperone toward the T-synthase will be examined by in vitro complementation assays. We will isolate, immortalize and establish the Tn/CD19(+) or Tn/IgA(+) Blymphocytes from patients with IgAN, examine the cDNA and gene for Cosmc;introduce wild type Cosmc into the B cells to restore T-synthase activity;and test whether Cosmc expression corrects O-glycosylation of cell surface glycoproteins and IgA1.
Aim 2 : We will define the structures of O-glycans on each site of the hinge region in IgA from patients with IgAN.
Aim 3 : We will characterize the autoantibodies in sera of patients with IgAN using a glycan microarray including various glycans and synthetic glycopeptides representing the IgA1 hinge region, and establish an ELISA method for potential diagnosis of IgAN from serum samples. Understanding the genetic basis for IgAN will shed light on developing new diagnostics and therapeutic treatments for IgAN.
Our goal of this project is to investigate the genetic basis or molecular mechanism of non-familial IgA nephropathy (IgAN), the most common form of primary glomerulonephritis leading to progressive renal failure in almost one third of the patients. IgAN is caused by the deposit of IgA1 in glomerulus because the IgA1 carries aberrant O-glycans such as Tn antigen. O-glycan biosynthesis is regulated by Cosmc, core 1 (3-galactosyltransferase specific molecular chaperone, through assisting the folding of core 1 (3- galactosyltrasferase (T-synthase) which is a key enzyme guarding the pathway. Human Cosmc is encoded by a single-Exon gene Cosmc on Xq24. Somatic mutation in Cosmc is responsible for the abnormal expression of Tn and sialylTn antigens in human tumor cell lines and blood cells of patients with Tn syndrome. We hypothesize that the somatic mutation of X-linked Cosmc in IgA1 secreting B cell is associated with IgAN. To test our hypothesis, we will focus on three Aims for this proposal: 1) Examine Cosmc gene in Tn(+), IgA1 secreting B cells or plasma cells isolated from patients with IgAN and matched healthy controls;examine the Cosmc gene from immortalized Tn(+), IgA1 secreting B cells or plasma cells;correct the O-glycan structure on IgA1 by introducing the wild type Cosmc into these cells;2) Define the O-glycan structure on each site of hinge region in IgA1;3) Characterize the autoimmune antibody in sera of patients with IgAN. These studies will directly address the molecular mechanism of IgAN, which will have an important impact on our understanding of the pathogenesis of IgA1, and provide new diagnostics and avenues of treatment or prevention for sporadic IgAN.
|Lehoux, Sylvain; Mi, Rongjuan; Aryal, Rajindra P et al. (2014) Identification of distinct glycoforms of IgA1 in plasma from patients with immunoglobulin A (IgA) nephropathy and healthy individuals. Mol Cell Proteomics 13:3097-113|
|Ju, Tongzhong; Cummings, Richard D (2013) A fluorescence-based assay for Core 1 *3galactosyltransferase (T-synthase) activity. Methods Mol Biol 1022:15-28|
|Ju, Tongzhong; Xia, Baoyun; Aryal, Rajindra P et al. (2011) A novel fluorescent assay for T-synthase activity. Glycobiology 21:352-62|
|Wang, Yingchun; Ju, Tongzhong; Ding, Xiaokun et al. (2010) Cosmc is an essential chaperone for correct protein O-glycosylation. Proc Natl Acad Sci U S A 107:9228-33|
|Ju, Tongzhong; Cummings, Richard D (2010) Functional assays for the molecular chaperone cosmc. Methods Enzymol 479:107-22|
|Bolscher, Jan G M; Brevoord, Judith; Nazmi, Kamran et al. (2010) Solid-phase synthesis of a pentavalent GalNAc-containing glycopeptide (Tn antigen) representing the nephropathy-associated IgA hinge region. Carbohydr Res 345:1998-2003|