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 beta1,3-galactosyltransferase or a terminal sialic acid by a N-acetylgalactosamine-specific alpha2,6-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 beta1,3-galactosyltransferase activity and an increase in N-acetylgalactosamine-specific alpha2,6-sialyltransferase activity. Also, expression of beta1,3-galactosyltransferase was significantly lower, and that of N-acetylgalactosamine-specific alpha2,6-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.

Figure 1. IgA1 secreted by IgA1-producing cell lines from patients with IgAN is Gal deficient.

(A) Degree of Gal deficiency of O-glycans on IgA1 secreted by IgAN-IgA1S cell lines (n = 11; filled circles) and HC-IgA1S cell lines (n = 11; open circles) was measured by lectin ELISA. The results were calculated relative to HAA reactivity of the standard naturally Gal-deficient IgA1 (Mce) myeloma protein (its relative HAA reactivity is set to 100%). IgA1 secreted by the IgAN-IgA1S cell lines showed higher reactivity with HAA than that secreted by the HC-IgA1S cell lines (P = 0.0001). (B) Serum levels of Gal-deficient IgA1 correlate with Gal deficiency of IgA1 secreted by the corresponding cell lines (P < 0.001; R² = 0.883). The mean values were expressed relative to HAA reactivity of the standard Gal-deficient IgA1 (Mce) myeloma protein, as in A.

Figure 2. Analysis of molecular form of IgA1 secreted by IgA1-producing cell lines.

Supernatants from randomly selected cell lines from 3 IgAN patients and 3 healthy controls were separated by SDS-PAGE under nonreducing (A) and reducing (B) conditions. The loaded samples were normalized to total IgA content (10 ng/well). (C) Densities of the bands in A were quantitated by densitometry. Cell lines from IgAN patients secreted predominantly polymeric IgA1 (about 70% of total IgA1 was dimeric [d] and trimeric [t]) in contrast to only 20%–30% as polymeric forms for the IgA1 secreted by the cell lines from healthy controls. mIgA1, monomeric IgA1.

Figure 3. Analysis of IgA1 fractionated by size-exclusion chromatography.

Culture supernatants from the 3 randomly selected IgAN-IgA1S cell lines (filled circles) and 2 HC-IgA1S cell lines (open circles) were separated by size-exclusion chromatography. Concentration of IgA1 in the fractions was determined by ELISA (A). Western blotting analysis after SDS-PAGE separation under nonreducing conditions confirmed distinct molecular forms of IgA in various fractions (inset in A). (B) Gal deficiency of IgA1 was determined by HAA-ELISA. Trimeric and dimeric IgA1 secreted by IgAN-IgA1S cell lines had high reactivity with HAA, while monomeric IgA1 did not react. Furthermore, IgA1 secreted by HC-IgA1S cell lines was mostly monomeric and did not react with HAA. Data are expressed as mean ± SD.

Figure 4. IgA1 secreted by cell lines from IgAN patients has Gal-deficient O-linked glycans with terminal or sialylated GalNAc.

(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).

Figure 5. Immunofluorescence analysis of IgA1-producing cell lines.

(A) HAA lectin bound to neuraminidase-treated IgA1 in the cytoplasm of cells secreting Gal-deficient IgA1 (IgAN-IgA1S cell lines from 3 IgAN patients). (B) IgA1 in HC-IgA1S cell lines from 3 healthy controls did not react with HAA, even after treatment with neuraminidase to remove terminal sialic acid residues. Staining for nuclei is shown in blue; for IgA, in green; and for HAA, in red. N+, neuraminidase treated. Scale bars: 10 μm.

Figure 6. HAA bound to Gal-deficient IgA1 in the Golgi apparatus.

(A) Representative example of immunostaining with anti-human IgA antibody (violet), HAA lectin (after neuraminidase treatment) (red), and Golgi marker (Golgin 97) antibody (green) in cell from IgAN-IgA1S cell lines. IgA and HAA were colocalized in the Golgi apparatus. Fluorescence intensity profile from the confocal microscope is shown in the right-hand bottom panel (left bottom panel: line with arrow marks the cross section). (B) Same staining, but without neuraminidase treatment. Nuclear staining is shown in blue. N+, neuraminidase treated; N–, not treated with neuraminidase.

Figure 7. Gene transcriptional levels in IgA1-producing cell lines from IgAN patients and healthy controls.

(A) Gene transcriptional levels of specific glycosyltransferases were quantitated by real-time RT-PCR, normalized to β-actin, and compared between patients (n = 11; black bars) and controls (n = 11; white bars) using the E-method (55) as mean relative expression (values for controls were set to 1.0). Transcription levels of C1GalT1 and its molecular chaperone Cosmc were lower in IgAN-IgA1S cell lines, while that of ST6GalNAcII was higher. (B) Gene expression of J chain and α chain was similar in the IgAN-IgA1S and HC-IgA1S cell lines. Data are presented as mean ± SD. *P < 0.01; **P < 0.001.

Figure 8. Enzyme activities of β1,3-galactosyltransferase and ST6GalNAc in IgA1-producing cell lines.

(A) β1,3-galactosyltransferase activity in Golgi-enriched fractions from IgAN-IgA1S cell lines was lower compared with that from HC-IgA1S cell lines (P = 0.006). (B) HAA Western blotting confirmed that HAA reactivity of the acceptor IgA1 decreased due to attachment of Gal to GalNAc, irrespective of neuraminidase treatment. (C) ST6GalNAc activity in Golgi-enriched fraction from IgAN-IgA1S cell lines was higher than that in the HC-IgA1S cell lines (P = 0.011). (D) HAA–Western blotting confirmed that HAA reactivity of the acceptor IgA1 decreased due to attachment of NeuAc to GalNAc (HAA reactivity increased after treatment with neuraminidase). N+, neuraminidase treated; N–, not treated with neuraminidase; IgA, load control developed with anti-IgA α chain–specific antibody.

Figure 9. Complex changes in biosynthetic pathways of O-linked glycans in IgA1-producing cell lines from IgAN patients.

Transcription of C1GalT1 and Cosmc and enzyme activity of C1GalT1 was lower in the IgAN-IgA1S cell lines than in the HC-IgA1S cell lines, whereas the transcription and enzymatic activity of ST6GalNAcII was higher. Red arrows in front of enzyme names denote increased or decreased enzymatic expression/activity in the cell lines from IgAN patients compared with that in cell lines from healthy controls. Gal-containing glycans are shown in blue, Gal-deficient glycans, in red.