Figure 1.
Structure of N-glycans in human IgG.
(A) N-glycan with attached monosaccharaides on IgG Fc region. The N-glycan that is attached to the conserved asparagine (Asn) residue at position 297 in the Fc comprises a core structure of N-acetylglucosamine and mannose, plus additional carbohydrate residues, which can vary, including fucose, galactose, sialic acid and bisecting N-acetylglucosamine. (B) Scheme showing the described N-glycan in human IgG and activities of the enzymes studied in this work. The arrows indicate the carbohydrate units added or hydrolyzed by the corresponding enzymes. The human glycans are mainly classified as 'biantennary complex' structure with a core fucose and are often terminated with sialic acid. The third N-acetylglucosamine (GlcNAc) bisecting arm represents around 10% of human IgGs glycoforms. Only the α2-6-linked sialic acid is associated with the anti-inflammatory effects of IVIG. Black square, GlcNAc; white triangle, fucose; white circle, mannose; gray circle, galactose; black diamond, α2-6 linkage sialic acid. ST6Gal-I: β-galactoside α2-6 sialyltransferase-1, Neu1: neuramidase 1, β4GALT1: β1,4-galactosyltransferase 1, GLB1: β-galactosidase 1, FUT8: α1-6 fucosyltransferase , FUCA1:α-fucosidase.
Figure 2.
Overview of different glycosylation assays.
PNGase F: peptide -N-Glycosidase F, DMB assay: 1,2-diamino-4,5-methylenoxybenzene (DMB)-labeled sialic acid by HPLC, 2AB: 2-aminobenzamide, PM: per-O-methylation, MALDI-TOF: matrix-assisted laser desorption-ionization time-of-flight. PMAA: partially methylated alditol acetate, GC-MS: gas chromatography–mass spectrometry, HPAEC-PAD: high performance anion exchange chromatography with pulsed amperometric detection.
Figure 3.
Levels of sialic acid, fucose, and galactose in infused IVIG and endogenous IgG from IVIG-responsive and -resistant KD subjects at acute and 1 year time points using four different assays.
(A) Levels of total sialic acid in IVIG and endogenous IgG, measured by DMB assay, (B) Levels of sialylated N-glycans in IVIG and endogenous IgG, measured by 2AB-labeling assay, (C) Levels of α2-6 linked sialic acid relative to 5 μg inositol in IVIG and endogenous IgG measured by GC-MS assay. (D) Galactose levels on IVIG and endogenous IgG measured by HPAEC-PAD assay. (E) Fucose levels on IVIG and endogenous IgG measured by HPAEC-PAD assay. The same subjects were used for all assays. Open symbols: Subjects with coronary aneurysms. Results shown as medians and IQR. p-values by Mann-Whitney U test. *p<0.05, **p<0.01, ***p<0.001. IVIG: intravenous immunoglobulin, KD: Kawasaki disease, DMB assay: 1,2-diamino-4,5-methylenoxybenzene (DMB)-labeled sialic acid by HPLC, 2AB: 2-aminobenzamide, GC-MS: gas chromatography–mass spectrometry. HPAEC-PAD: high performance anion exchange chromatography with pulsed amperometric detection.
Figure 4.
Microarray analysis of ST6GAL1 transcript.
(A) Illumina array (probe ID: 1260601). On this microarray, whole blood transcript levels from 110 IVIG-responsive and 30 IVIG-resistant subjects at the acute (pre-treatment) and convalescent (at least 3 weeks after treatment) time points were analyzed. (B) Affymetrix Human Genome U133 Plus 2.0 Array (probe ID: 201998_at), whole blood transcript levels from six IVIG-responsive and six -resistant subjects (all Japanese) at the acute and convalescent (36 hours after end of treatment) time points were analyzed. Microarray clones were from exon VI (conserved region) of ST6GAL1 mRNA. Open symbols: Subjects with coronary aneurysms. Results shown as medians and IQR. p-values by Mann-Whitney U test. *p<0.05, **p<0.01, ***p<0.001. KD: Kawasaki disease.
Figure 5.
Whole blood transcript levels of ST6GAL1 in KD patients using qRT-PCR.
Transcript levels of ST6GAL1 were analyzed with 4 different primer pairs. (A) Total ST6GAL1 transcript levels amplified with primers for conserved region spanning exons V and VI, (B) ST6GAL1 Transcript 1 levels encoded by the P1 promoter (hepatocytes). (C) ST6GAL1 Transcript 2 levels amplified with primer encoding exons X and I (B cells) (D) ST6GAL1 Transcript 3 levels encoding exons Y and Z (all cells). Results are relative units of ST6GAL1 transcripts normalized for expression of housekeeping gene, TAF1B. Open symbols: Subjects with coronary aneurysms. Bars are medians and IQR. p-values by Mann-Whitney U test. *p<0.05, **p<0.01, ***p<0.001. KD: Kawasaki disease.
Figure 6.
Expression transcript levels of ST6GAL1 in EBV-transformed B cell lines from IVIG-responsive and -resistant KD subjects at acute phase by qRT-PCR.
(A) Amplification with primers for the conserved region in exon VI in ST6GAL1 mRNA. (B) Amplification with primers for Transcript 1 initiated from the P1 promoter in ST6GAL1 (P1 promoter and exon I region). (C) Amplification with primers for Transcript 2 (exon X and I region) in ST6GAL1. qRT-PCR results are presented as relative units of ST6GAL1 amplicon normalized for the housekeeping gene TAF1B. Open symbols: Subjects with coronary aneurysms. Results are shown as medians and IQR. p-values by Mann-Whitney U test. *p<0.05. KD: Kawasaki disease.
Figure 7.
Soluble (s)ST6Gal-I in KD and febrile control subjects measured by ELISA assay.
(A) sST6Gal-I in serum from IVIG-responsive and IVIG-resistant KD subjects and febrile controls. (B) sST6Gal-I levels in plasma from independent cohort of IVIG-responsive and IVIG-resistant KD subjects and febrile controls. Open symbols: Subjects with coronary aneurysms. Results shown as medians and IQR. p-values by Mann-Whitney U test. *p<0.05, **p<0.01, ***p<0.001. KD: Kawasaki disease.