IgA1-secreting cell lines from patients with IgA nephropathy produce aberrantly glycosylated IgA1
Hitoshi Suzuki, Zina Moldoveanu, Stacy Hall, Rhubell Brown, Huong L. Vu, Lea Novak, Bruce A. Julian, Milan Tomana, Robert J. Wyatt, Jeffrey C. Edberg, Graciela S. Alarcón, Robert P. Kimberly, Yasuhiko Tomino, Jiri Mestecky, Jan Novak
Hitoshi Suzuki, Zina Moldoveanu, Stacy Hall, Rhubell Brown, Huong L. Vu, Lea Novak, Bruce A. Julian, Milan Tomana, Robert J. Wyatt, Jeffrey C. Edberg, Graciela S. Alarcón, Robert P. Kimberly, Yasuhiko Tomino, Jiri Mestecky, Jan Novak
View: Text | PDF
Research Article Nephrology

IgA1-secreting cell lines from patients with IgA nephropathy produce aberrantly glycosylated IgA1

Abstract

Aberrant glycosylation of IgA1 plays an essential role in the pathogenesis of IgA nephropathy. This abnormality is manifested by a deficiency of galactose in the hinge-region O-linked glycans of IgA1. Biosynthesis of these glycans occurs in a stepwise fashion beginning with the addition of N-acetylgalactosamine by the enzyme N-acetylgalactosaminyltransferase 2 and continuing with the addition of either galactose by β1,3-galactosyltransferase or a terminal sialic acid by a N-acetylgalactosamine–specific α2,6-sialyltransferase. To identify the molecular basis for the aberrant IgA glycosylation, we established EBV-immortalized IgA1-producing cells from peripheral blood cells of patients with IgA nephropathy. The secreted IgA1 was mostly polymeric and had galactose-deficient O-linked glycans, characterized by a terminal or sialylated N-acetylgalactosamine. As controls, we showed that EBV-immortalized cells from patients with lupus nephritis and healthy individuals did not produce IgA with the defective galactosylation pattern. Analysis of the biosynthetic pathways in cloned EBV-immortalized cells from patients with IgA nephropathy indicated a decrease in β1,3-galactosyltransferase activity and an increase in N-acetylgalactosamine–specific α2,6-sialyltransferase activity. Also, expression of β1,3-galactosyltransferase was significantly lower, and that of N-acetylgalactosamine–specific α2,6-sialyltransferase was significantly higher than the expression of these genes in the control cells. Thus, our data suggest that premature sialylation likely contributes to the aberrant IgA1 glycosylation in IgA nephropathy and may represent a new therapeutic target.

Authors

Hitoshi Suzuki, Zina Moldoveanu, Stacy Hall, Rhubell Brown, Huong L. Vu, Lea Novak, Bruce A. Julian, Milan Tomana, Robert J. Wyatt, Jeffrey C. Edberg, Graciela S. Alarcón, Robert P. Kimberly, Yasuhiko Tomino, Jiri Mestecky, Jan Novak

×

Figure 4

IgA1 secreted by cell lines from IgAN patients has Gal-deficient O-linked glycans with terminal or sialylated GalNAc.

Options: View larger image (or click on image) Download as PowerPoint
IgA1 secreted by cell lines from IgAN patients has Gal-deficient O-linke...
(A) Gal-deficient IgA1 was measured by HAA-ELISA as the ratio of HAA binding IgA1 to total IgA1 with (N+) or without (N–) neuraminidase treatment. The values were expressed relative to HAA reactivity of the standard Gal-deficient IgA1 (Mce) myeloma protein, as described in Figure 1. Reactivity of IgA1 with HAA increased after neuraminidase treatment of IgA1 from cell lines from 5 IgAN patients but not from 5 healthy controls. Western blots obtained after SDS-PAGE under reducing conditions were developed with HAA before (N–) or after (N+) neuraminidase treatment (B) or with SNA (α2,6-NeuAc-specific lectin) (C). The loaded samples were normalized to total IgA (10 ng/well) (load control; lower panels developed with IgA-specific antibody). IgA1 secreted by IgAN-IgA1S cell lines reacted with HAA, and this reactivity was enhanced by neuraminidase treatment; in contrast, the IgA1 secreted by HC-IgA1S cell lines only marginally reacted with HAA (B). (C) SNA Western blotting of IgA1 not treated with neuraminidase. The results confirmed that the IgA1 secreted by IgAN-IgA1S cell lines was highly sialylated, whereas the IgA1 secreted by HC-IgA1S cell lines was less sialylated. Bands were densitometrically quantified and expressed as ratio of SNA-binding IgA to total IgA (bar graph shows mean ± SD).