Quantitative analysis of IgA1 binding protein in human serum by hypoglycosylated IgA1/Sepharose chromatography

Abstract

IgA nephropathy (IgAN) is a common disease characterized by predominant IgA deposits in the renal mesangium. It is well‐known that IgA1 among two subclasses, IgA1 and IgA2, is the dominant deposit in the glomeruli during IgAN. The most prominent structural difference between the IgA1 and IgA2 subclasses was the duplicated proline‐rich hinge portion and the characteristic O‐linked oligosaccharide chains on the IgA1 hinge portion. There are many reports on the presence of an incompletely glycosylated O‐linked oligosaccharide (s) on the IgA1 hinge region in some of the IgAN patients. It was also reported that the artificially deglycosylated IgA1 acquired binding ability for fibronectin, type IV collagen and laminin.In this experiment, preparation and analysis of binding protein (IgA1‐BP) to the hypoglycosylated IgA1/Sepharose column were carried out. In order to examine the presence of the specific binding protein to the hypoglycosylated IgA1 in human serum, IgA1, asialo‐IgA1 and asialo‐agalacto‐IgA1/Sepharose column chromatography of normal human serum was carried out. A portion of the serum protein was bound to those columns and eluted with the buffer containing 1.0 M NaCl. About four times the amount of protein was eluted from the hypoglycosylated IgA1/Sepharose column than that from IgA1/Sepharose. Most of the eluted protein was IgG, and the IgG1 and IgG3 subclasses but neither IgG2 nor IgG4 was dominant (Fig. 1). The Protein A passed‐fraction (PAP fraction) of the IgA1‐BP seemed to contain proteins other than IgG, a detailed analysis of the PAP fraction was carried out. The PAP fraction was composed of a relatively large amount of IgA, IgM and complement C3 besides IgG. The relative content of IgG : IgA : IgM : C3 : C4 was 25 : 10 : 41 : 22 : 2 in the PAP fraction. Meanwhile, the Protein A bound‐fraction of IgA1‐BP was essentially composed of IgG (78%) and IgM (19%) (Fig. 2). Since the deposited IgA1 in IgA nephropathy patient was reported as hypoglycosylated IgA1, the obtained results might explain well the high frequency of copresence of IgM and C3 with the deposited IgA1 in IgAN patients. With respect to the IgA content in the IgA1‐BP from IgAN patients, it was significantly higher than that from other nephropathy patients (Fig. 3). Both of the samples contained a few microgram of abberant IgA per millilitre of serum.IgG subclass ratio in normal serum and IgA‐BP.imageRelative content of IgG, IgA, IgM, C3 and C4 in the PAP and PAB fractions of IgA1‐BP.imageTotal amount of IgA1‐BP and content of IgA in IgA1‐BP prepared from nephropathy patients.imageThus, the obtained results indicated hypoglycosylated IgA‐BP were IgG1, IgG3, IgA, IgM, C3.