Immunoglobulin A Glycosylation Differs between Crohn's Disease and Ulcerative Colitis

Abstract

Inflammatory bowel diseases (IBD), such as Crohn's disease (CD) and ulcerative colitis (UC), are chronic and relapsing inflammations of the digestive tract with increasing prevalence, yet they have unknown origins or cure. CD and UC have similar symptoms but respond differently to surgery and medication. Current diagnostic tools often involve invasive procedures, while laboratory markers for patient stratification are lacking. Large glycomic studies of immunoglobulin G and total plasma glycosylation have shown biomarker potential in IBD and could help determine disease mechanisms and therapeutic treatment choice. Hitherto, the glycosylation signatures of plasma immunoglobulin A, an important immunoglobulin secreted into the intestinal mucin, have remained undetermined in the context of IBD. Our study investigated the associations of immunoglobulin A1 and A2 glycosylation with IBD in 442 IBD cases (188 CD and 254 UC) and 120 healthy controls by reversed-phase liquid chromatography electrospray-ionization mass spectrometry of tryptic glycopeptides. Differences of IgA O- and N-glycosylation (including galactosylation, bisection, sialylation, and antennarity) between patient groups were associated with the diseases, and these findings led to the construction of a statistical model to predict the disease group of the patients without the need of invasive procedures. This study expands the current knowledge about CD and UC and could help in the development of noninvasive biomarkers and better patient care.

Keywords: Crohn’s disease; IgA1; biomarker; glycosylation; inflammatory bowel disease; ulcerative colitis.

Figure 1

Symbolic representation of IgA1 and IgA2 glycosylation. The acronyms HYT, LSL, TPL, LAG(C/Y), and MAG(C/Y), which refer to the different glycopeptides, come from the first three amino acids of the corresponding tryptic digest sequence of IgA1 and IgA2. (A) IgA1 has two N-glycosylation sites at Asn₁₄₄ and Asn₃₄₀ that are occupied by complex-type N-glycans. IgA1 also contains six O-glycosylation sites in the hinge region at Thr₁₀₆, Thr₁₀₉, Ser₁₁₁, Ser₁₁₃, Thr₁₁₄, and Thr₁₁₇. The O-glycosylation of IgA1 is represented as one combined monosaccharide composition, as no information about specific glycan structures and attachment sites was obtained. (B) The observed glycosylation sites of IgA2 at Asn₁₃₁, Asn₂₀₅, and Asn₃₂₇ are occupied with complex-type N-glycans. * The IgA2 glycosylation sites Asn₄₇ and Asn₉₂ and Asn₃₂₇ did not pass quality curation criteria due the low intensity of analytes. (C) Symbols used for representing the glycans and their general antennary structure (N-linked glycans) and example structures for the O-linked glycans.

Figure 2

Description of the glycopeptide clusters. (A) IgA1 and IgA2 glycopeptides overview. Glycosylation sites, short peptide names, number of extracted glycoforms, and peptide sequences are given for each glycopeptide cluster. (B) Extracted Ion Chromatograms (EIC) of one selected glycopeptide per cluster with representative retention time of the whole glycopeptide cluster.

Figure 3

Assignment of the major N-glycoforms of IgA1 and IgA2. (A) Peptide cluster TPL at Asn₂₀₅ of IgA2, zoomed-in view of the 3⁺ charged glycopeptides. (B) Truncated peptide cluster LAGC at Asn₃₄₀ of IgA1, zoom-in of the 4⁺ charged glycopeptides. The two peaks marked with an asterisk are residues from another peptide without glycan eluting at the same time and in the charge state 2⁺. (C) Peptide cluster LAGY at Asn₃₄₀ of IgA1, zoom-in of the 4⁺ charged glycopeptides. (D) Peptide cluster LSL at Asn₁₄₄/Asn₂₀₅ of IgA1 and IgA2 and zoom-in of the 4⁺ charged glycopeptides. All of the glycoforms are described in Table S1.

Figure 4

Assignment of O-glycoforms of peptide cluster HYT. Assignment of O-glycoforms of peptide cluster HYT at Ser_89–126 of IgA1 and zoom-in of the 5⁺ charged glycopeptides. The pictograms represent the combined O-glycopeptide monosaccharide composition from all sites and do not convey information on specific glycan structures of attachment sites. Overall, the glycopeptides covered a wide range of compositions (H₃–₅N₃–₆S_1–5) containing up to 6 N. Not all glycoforms are labeled on the figure, as the complete list of the 25 IgA1 hinge region O-glycopeptide species identified and quantified is reported in Table S1.

Figure 5

Main associations between IgA glycosylation-derived traits and IBD per patient groups (HC = healthy controls, UC = ulcerative colitis, and CD = Crohn’s disease). The derived traits were corrected age, sex, and age*sex and are shown in boxplots. The 25th, 50th, and 75th percentiles are represented in the boxes, and the whiskers are at the 1st quartile −1.5 × IQR (interquartile range) and at the 3rd quartile +1.5 * IQR. P-values, ORs, and confidence intervals are reported in Table S4. Statistically significant associations (multiple testing corrected threshold 3.70 × 10^–4 as described in the supplementary tables) are marked with an asterisk.

Figure 6

Receiver operating characteristic curves showing the power of selected glycan traits to predict CD and UC. The mean and SE of 20 predictions are reported for the area under the curve. The prediction model included the interactions of age*sex *(LAGCb_CB + LAGCb_CA3 + LAGY_A2SB + HYT_S > G).

Figure 7

Main associations between IgA glycosylation derived traits and medication in CD and UC. The derived traits were corrected age, sex, and age*sex and are shown in boxplots. The 25th, 50th, and 75th percentiles are represented in the boxes and the whiskers are at the 1st quartile −1.5 * IQR (interquartile range) and at the 3rd quartile +1.5 * IQR. P-values, ORs, and confidence intervals are reported in Table S9. Statistically significant associations (multiple testing corrected threshold 1.85 × 10^–4 as described in the Supplementary Tables) are marked with an asterisk.

Figure 8

Comparison of associations found between IgA glycosylation and IBD in this study with IgA and IgG glycosylation associated with IBD, IgA Nephropathy, RA and pregnancy associated changes in the recent literature. A = Antennarity, B = Bisection, G = galactosylation, GN = GalNAc, S= Sialylation. ↑ = Trait elevated in cases vs HC, ↓ = Trait lower in cases vs HC. In this study, the associations found between IgA glycosylation and IBD are marked in black. In the other studies, the color green indicates a significant association in the same direction (cases vs HC) as reported here, red indicates association in the opposite direction, and blue indicates reported associations that were not found in our study. The studies used for comparison are part of the bibliography cited in this article.