cefoxitin and Glomerulonephritis--IGA

Glycoproteins in animal cells contain a variety of glycan structures that are added co- and/or posttranslationally to proteins. Of over 20 different types of sugar-amino acid linkages known, the two major types are N-glycans (Asn-linked) and O-glycans (Ser/Thr-linked). An abnormal mucin-type O-glycan whose expression is associated with cancer and several human disorders is the Tn antigen. It has a relatively simple structure composed of N-acetyl-D-galactosamine with a glycosidic α linkage to serine/threonine residues in glycoproteins (GalNAcα1-O-Ser/Thr), and was one of the first glycoconjugates to be chemically synthesized. The Tn antigen is normally modified by a specific galactosyltransferase (T-synthase) in the Golgi apparatus of cells. Expression of active T-synthase is uniquely dependent on the molecular chaperone Cosmc, which is encoded by a gene on the X chromosome. Expression of the Tn antigen can arise as a consequence of mutations in the genes for T-synthase or Cosmc, or genes affecting other steps of O-glycosylation pathways. Because of the association of the Tn antigen with disease, there is much interest in the development of Tn-based vaccines and other therapeutic approaches based on Tn expression.

Topics: Animals; Antigens, Tumor-Associated, Carbohydrate; Galactosyltransferases; Glomerulonephritis, IGA; Humans; Mice; Molecular Chaperones; Neoplasms; Polysaccharides

Immunoglobulin A nephropathy (IgAN) is the most common form of glomerulonephritis worldwide and is histologically characterized by the deposition of IgA1 and consequent inflammation in the glomerular mesangium. Prior studies suggested that serum IgA1 from IgAN patients contains aberrant, undergalactosylated O-glycans, for example, Tn antigen and its sialylated version, SialylTn (STn), but the mechanisms underlying aberrant O-glycosylation are not well understood. Here we have used serial lectin separation technologies, Western blot, enzymatic modifications, and mass spectrometry to explore whether there are different glycoforms of IgA1 in plasma from patients with IgAN and healthy individuals. Although total plasma IgA in IgAN patients was elevated ∼ 1.6-fold compared with that in healthy donors, IgA1 in all samples was unexpectedly separable into two distinct glycoforms: one with core 1 based O-glycans, and the other exclusively containing Tn/STn structures. Importantly, Tn antigen present on IgA1 from IgAN patients and controls was convertible into the core 1 structure in vitro by recombinant T-synthase. Our results demonstrate that undergalactosylation of O-glycans in IgA1 is not restricted to IgAN and suggest that in vivo inefficiency of T-synthase toward IgA1 in a subpopulation of B or plasma cells, as well as overall elevation of IgA, may contribute to IgAN pathogenesis.

Topics: Adult; Antigens, Tumor-Associated, Carbohydrate; B-Lymphocytes; Female; Galactose; Galactosyltransferases; Glomerular Mesangium; Glomerulonephritis, IGA; Glycosylation; Humans; Immunoglobulin A; Inflammation; Lectins; Male; Peanut Agglutinin; Polysaccharides; Sialyltransferases; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

Incomplete or aberrant glycosylation leading to Tn antigen (GalNAcalpha1-Ser/Thr) expression on human glycoproteins is strongly associated with human pathological conditions, including tumors, certain autoimmune diseases, such as the idiopathic IgA nephropathy, and may modulate immune homeostasis. In addition, the Tn antigen is highly expressed by certain pathogens and plays a role in host-pathogen interactions. To enable experimental approaches to study interactions of the Tn antigen with the immune system and analyze anti-Tn antibody responses in infection or disorders, we generated a Tn-expressing resource that can be used for high-throughput screening. In consideration of IgA nephropathy in which the hinge region is incompletely glycosylated, we used this hinge sequence that encodes five potential glycosylation sites as the ideal template for the synthesis of a Tn antigen-expressing glycopeptide. Inclusion of an N-terminal biotin in the peptide enabled binding to streptavidin-coated ELISA plates as monitored using Helix pomatia agglutinin or anti-Tn monoclonal antibody. We also found that the biotinylated IgA-Tn peptide is a functional acceptor for beta1-3-galactosylation using recombinant T-synthase (beta1-3-galactosyltransferase). Besides its immunochemical functionality as a possible diagnostic tool for IgA nephropathy, the peptide is an excellent substrate for glycan elongation and represents a novel template applicable for glycan-antigen-associated diseases.

Topics: Antigens, Tumor-Associated, Carbohydrate; Biotinylation; Glomerulonephritis, IGA; Glycopeptides; Humans; Immunoglobulin A; Lectins

Abnormal O-glycosylation of IgA1 may contribute to pathogenic mechanisms in IgA nephropathy (IgAN). Observations of altered lectin binding to IgA1 in IgAN suggest that the O-glycan chains may be undergalactosylated, but precise structural definition of the defect has proved technically difficult, and it remains unconfirmed. This is the first study using fluorophore-assisted carbohydrate electrophoresis (FACE) to analyze IgA1 O-glycans in IgAN and controls. IgA1 was purified from serum, and the intact O-glycans were released by hydrazinolysis at 60 degrees C. After re-N-acetylation, the glycans were fluorophore-labeled and separated by polyacrylamide gel electrophoresis. Sequential exoglycosidase digestions of IgA1 allowed identification of the different O-glycan bands on FACE gels, and their relative frequencies in IgA1 samples were measured by ultraviolet densitometry. Lectin binding of the IgA1 samples was also measured. In some patients with IgAN, FACE analysis demonstrated a significant increase in the percentage of IgA1 O-glycan chains consisting of single N-acetyl galactosamine (GalNAc) units rather than the more usual galactosylated and sialylated forms. This finding was confirmed using both desialylated IgA1 and enzymatically released O-glycans. Good correlation was also found between O-glycan agalactosylation on FACE analysis and IgA1 lectin binding in IgAN, supporting the value of lectins as tools for detection of this abnormality. This is the first study in which all of the predicted O-glycan forms of IgA1 have been analyzed simultaneously, and demonstrates that in IgAN, the IgA1 Oglycan chains are truncated, with increased terminal GalNAc. This abnormality has the potential to significantly affect IgA1 behavior and handling with pathogenic consequences in IgAN.

Topics: Adult; Aged; Amino Acid Sequence; Antibodies; Antigens, Tumor-Associated, Carbohydrate; Electrophoresis; Female; Glomerulonephritis, IGA; Glycosylation; Hot Temperature; Humans; Hydrazines; Immunoglobulin A; Lectins; Male; Middle Aged; Molecular Sequence Data; Neuraminidase; Plant Lectins; Polysaccharides; Reference Values