IgG Glycosylation Profiling of Peripheral Artery Diseases with Lectin Microarray

Abstract

Background: Inflammation plays a key role in the progression of atherosclerotic plaque for peripheral artery disease (PAD). Immunoglobulin G (IgG) glycosylation could modulate immunological effector functions and has been explored as biomarkers for various diseases. Methods: Lectin microarray was applied to analyze the expression profile of serum IgG glycosylation in patients with lower-extremity peripheral artery disease (LEPAD), carotid artery stenosis (CAS), abdominal aortic aneurysm (AAA), and healthy controls. Lectin blot was performed to validate the differences. Results: SNA (Sambucus nigra agglutinin) binding (preferred sialic acid) was significantly higher in the LEPAD (3.21 ± 2.06) and AAA (3.34 ± 2.42) groups compared to the CAS (2.47 ± 1.45) group. Significantly higher binding levels of ConA (Concanavalin A) (preferred mannose) and PSA (Pisum sativum agglutinin) (preferred fucose) were also observed in LEPAD compared to CAS patients. Among LEPAD patients, a significant lower binding level of Black bean crude (preferred GalNAc) was present for dyslipidemia patients. A higher binding level of MNA-M (Morniga M agglutinin) (preferred Mannose) and Jacalin-AIA (Artocarpus integrifolia agglutinin) (preferred Galβ3GalNAc) was observed for Fontaine severe patients. Higher binding levels of PHA-E (Phaseolus vulgaris Erythroagglutinin) and PHA-L (Phaseolus vulgaris Leucoagglutinin) (preferred Galβ4GlcNAc) were observed for diabetic patients, and higher binding of ASA (Allium sativum agglutinin) (preferred Mannose) was present in patients with hypertension. The level of high-sensitivity C-reactive protein (hsCRP) was positively associated with LTL (Lotus tetragonolobus lectin) (r = 0.44), PSA (r = 0.44), LCA (Lens Culinaris agglutinin) (r = 0.39), SNA (r = 0.57), and CSA (Cytisus sscoparius agglutinin) (r = 0.56). For CAS, symptomatic patients had lower binding levels of AAL (Aleuria aurantia lectin) (preferred fucose) and IAA (Iberis amara agglutinin) (preferred GalNAc). Blood total cholesterol level was positively associated with SNA-I (r = 0.36) and SBA (Soybean agglutinin) (r = 0.35). Creatinine levels were positively associated with lectins including, but not limited to, MNA-M (r = 0.42), CSA (r = 0.45), GHA (Glechoma hederacea agglutinin) (r = 0.42), and MNA-G (Morniga G agglutinin) (r = 0.45). Conclusion: LEPAD patients had increased IgG binding levels of SNA and ConA compared to CAS, which could provide potential diagnostic value. Fontaine severity was associated with Mannose-rich IgG N-glycan, while diabetic LEPAD correlated with bisecting GlcNAc. The levels of hsCRP and creatinine were positively associated with IgG fucosylation and galactosylation. Changes in IgG glycosylation may play important roles in PAD pathogenesis and progression.

Changes in serum immunoglobulin G glycosylation patterns for antiphospholipid syndrome patients with lectin microarray

“…To detect the glycan profile of serum IgG in 413 participants, a commercial lectin microarray (BCBIO Biotech, Guangzhou, China) with 56 lectins was used, the reliability of which had been proven in previous studies 12‐14 . First, lectin microarrays were taken from −80°C, placed at room temperature for 1.5 h and incubated with a blocking buffer composed of 3% bovine albumin (BSA) in phosphate‐buffered saline (PBS) for 2 h. Then, microarrays were washed with phosphate‐buffered saline Tween (PBST) three times and incubated with 200 μL of 1:1000 diluted serum samples at 4°C overnight.…”

“…To detect the glycan profile of serum IgG in 413 participants, a commercial lectin microarray (BCBIO Biotech, Guangzhou, China) with 56 lectins was used, the reliability of which had been proven in previous studies. [12][13][14] First, lectin microarrays were taken from −80°C, placed at room temperature for 1.5 h and incubated with a blocking buffer composed of 3% bovine albumin (BSA) in phosphate-buffered saline (PBS) for 2 h. Then, microarrays were washed with phosphate-buffered saline Tween (PBST) three times and incubated with 200 μL of 1:1000 diluted serum samples at 4°C overnight. After washing the microarrays three times with PBST, 5 mL of 1:1000 diluted Cy3-labelled goat anti-human IgG antibodies (Jackson Immuno Research Labs, Pennsylvania, PA, USA) were added to the microarrays, then incubated at room temperature away from direct sunlight for an hour.…”

Retrospective screening of serum IgG glycosylation biomarker for primary Sjögren’s syndrome using lectin microarray

“…The detailed glycan binding specificities and type of linkage for each lectin could be found in Supplemental File . Detailed procedure could be seen in our previous works ( Hu et al, 2020 ; Li et al, 2022a ; Li et al, 2022b ; Zeng et al, 2021 ). Briefly, lectin microarrays were taken out from −80 °C and warmed up at room temperature for half an hour, then they were incubated with a blocking buffer (3% BSA in PBS) at room temperature for 2 h. After washing three times with PBS, 200 μl of 1:1,000 diluted samples serum was added and incubated with the microarrays at 4 °C overnight.…”

“…For lectin array assays, the median foreground and background fluorescent intensity for each spot on the arrays were acquired using the GenePix Pro 6.0 software ( Li et al, 2022b ). We calculated the signal-to-noise ratio (S/N) (the medium intensity of the spot foreground relative to the background) of each lectin spot.…”

“…First, serum samples were diluted by 1 × PBS, mixed with gel electrophoresis loading buffer (CWbiotech, Beijing, China) to a final 1:100 ratio, and boiled for 10 min. Twenty microliters per sample were separated by 10% sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) and electrotransferred onto polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA) ( Li et al, 2022b ). After washing two times with PBS Tween, the membrane was incubated with 10× Carbo-Free Blocking Solution (1:10; Vector Laboratories Inc., Newark, CA, USA) at room temperature for 2 h. Then, the membranes were washed twice and incubated with 20 μg/mL of Cy3-labeled (1:1,000; GE Healthcare, Chicago, IL, USA) LCA and MNA-M lectins at 4 °C overnight in the dark.…”