Galectin-8 in IgA nephritis: decreased binding of IgA by galectin-8 affinity chromatography and associated increased binding in non-IgA serum glycoproteins

Abstract

Background: Immunoglobulin A nephritis (IgAN) is the most common primary glomerulonephritis worldwide. It is caused by accumulation of IgA1-containing immune complexes in the kidney resulting in renal failure, which is thought to be due to altered glycosylation of IgA with a decrease of 2-3-sialylated galactosides (NeuAcα2-3Gal).

Purpose: The purpose of this study was to analyze whether altered glycosylation of IgA would lead to an altered binding to galectin-8, an endogenous lectin with strong affinity for 2-3-sialylated galactosides. Galectins are a family of β-galactoside-binding proteins; by binding various glycoproteins, they play important roles in the regulation of cellular functions in inflammation and immunity. Hence, an altered binding of IgA to galectin-8 could lead to pathologic immune functions, such as glomerulonephritis.

Methods: Affinity chromatography of serum glycoproteins on the human sialogalactoside-binding lectin galectin-8N permitted quantitation of bound and unbound fractions, including IgA.

Results: Analysis of ~100 IgA nephritis sera showed that the galectin-8N unbound fraction of IgA increased compared to ~100 controls, consistent with the known loss of galactosylation. A subgroup of ~15% of the IgAN patients had a ratio of galectin-8 bound/unbound IgA <0.09, not found for any of the controls. Unexpectedly, the galectin-8N-binding fraction of serum glycoproteins other than IgA increased in the sera of IgAN patients but not in controls, suggesting a previously unrecognized change in this disease.

Conclusion: This is the first study that relates a galectin, an endogenous lectin family, to IgA nephritis and thus should stimulate new avenues of research into the pathophysiology of the disease.

Fig. 1

Simplified schematic of pathological O-glycosylation changes of IgA1 in IgAN and predicted binding of galectin-8. The two top O-glycans NeuAcα2,3Galβ1,3GalNAcα and Galβ1,3GalNAc are predominant (30–40% each) in IgA1 of normal sera [35], whereas the bottom two, GalNAcα and Neuα2,6GalNAcα, increase in IgA1 from IgAN patients, due to decreased activity of the galactosyltransferase adding the Galβ1,3 (black circle in top two glycans) and increased activity of the sialyltransferase adding the NeuAcα2,6 (vertical diamond in bottom glycan) to GalNAc (open square). Galectin-8N binds the top O-glycan with high affinity, but not the others [24]. The NeuAcα2,6 sialyltransferase may also act on the top structure to make NeuAcα2,3Galβ1,3(NeuAcα2,6)GalNAc, but also this structure has strongly reduced affinity for galectin-8N [24]. Thus, galectin-8N binding of IgA1 is expected to decrease in IgAN

Fig. 2

Affinity chromatography of untreated or 2,3-neuraminidase-treated serum on immobilized human galectin-8N. Chromatograms of serum from a healthy individual treated with specific neuraminidase (NA) or untreated (UT) and subjected to affinity chromatography with immobilized galectin-8N or the galectin-8 Q47A mutant, deficient in binding to sialylated galactosides. The protein concentration of each fraction (0.2 or 1 ml) is given on the Y-axis. Elution with lactose (150 mM) started after washing with 32-ml PBS (arrow heads). Each chromatogram has been moved for clarity by +0.1 on the Y-axis and by +5 on the X-axis

Fig. 3

Identification of major galectin-8N binding serum glycoproteins. a SDS-PAGE (4–20% stained with Coomassie) of galectin-8N bound glycoproteins with major proteins indicated as previously identified by Western blot and MALDI-TOF-MS [25]. b Estimated relative amounts of galectin-8N bound serum proteins in four samples by LC–MS/MS analysis of pooled tryptic peptides. The serum concentrations of galectin-8N bound total protein and IgA were determined by protein assay and nephelometry as given in Table SI. The relative amounts of the remaining galectin-8N bound proteins were determined by LC–MS/MS assuming a linear relationship between sample protein concentration and the summed abundances for the peptides uniquely mapping to each protein (Tables SII and SIII). c SDS-PAGE of galectin-8N unbound (UB) and bound (B) fractions with the highest protein concentrations from four healthy individuals, four IgAN patients, and three controls with other histological patterns of glomerulonephritis. Indicated to the left are the mobilities of known size markers. The major visible bands of the unbound fractions correspond to albumin (at 67 kDa), transferrin (above the albumin), and IgG heavy chain (below the albumin)

Fig. 4

Significant increase of serum proteins binding to galectin-8N in IgAN patients. Yields of galectin-8N binding serum glycoproteins (sum of amount in bound fractions multiplied by 10 to give milligram per milliliter of original serum) for sera from 100 IgAN patients, 20 healthy subjects, and 92 controls with IgAN symptoms. Horizontal lines mark the mean for each group (mean/median for each group (mg/ml sera): 3.3/3.1, 2.2/2.1, 2.1/2.1); the difference between IgAN patients and the two other groups was statistically significant (p < 0.0001) as calculated by one-way ANOVA. Yields from all sera are found in supplementary Table I

Fig. 5

Significant increase of galectin-8N non-binding IgA in sera from IgAN patients compared to healthy controls. a Total IgA (mean/median for each group (mg/ml sera): 3.4/3.3, 1,7/2.1, 2.3/2.4), b galectin-8N unbound (total bound) IgA (mean/median for each group (mg/ml sera): 2.6/2.4, 1.5/1.0, 1.6/1.7), and c ratio of galectin-8N bound/unbound serum IgA (logarithmic scale on the Y-axis) in sera from 98 IgAN patients, 17 healthy, and 76 kidney disease controls. d Scatter plot of ratio from c (Y-axis) vs. total IgA (X-axis). IgA in unfractionated serum (total) or galectin-8N bound fractions was quantitated using nephelometry. Horizontal lines mark the average for each group in a–c. The difference between groups was statistically significant (p < 0.0001) as calculated by one-way ANOVA

Fig. 6

Indication of increased rate of disease progress to end stage renal disease for IgAN patients with low galectin-8 bound/unbound IgA ratio. Kaplan–Meier cumulative analysis of end stage renal disease for 87 IgAN patients, divided according to the ratio of galectin-8N bound/unbound IgA with a cutoff at a ratio 0.50 based on the mean in the control group. Patients with a ratio >0.50 may have better early (the first 9 years) renal survival than patients with a binding ratio <0.50, although statistical significance is slightly weaker than 0.05 level (p = 0.059, log rank test). Forty-one patients were lost from the follow-up group and are censored as indicated by vertical lines