ACRT Journal

Introduction

Glycosylation is one of the most important post-translational modifications of proteins. Among the different types of glycosylation, N-glycosylation and O-glycosylation are essential for the regulation of glycoproteins that promote optimal functioning [1]. The glycan structure is well known as remarkably diverse and is often modified in pre-disease and disease states. For example, the glycan structures known as AFP-L3 [2] in primary hepatoma and CA19-9 (sialyl-Lewis A) [3] in gastrointestinal cancers are often used for the diagnosis and monitoring of these diseases.

The N-glycans of IgG are localized at the Asn297 amino acid position [4]. Agalactosylated IgG was found in early studies on rheumatoid arthritis, and reduced galactosylation of IgG glycans is now regarded as a sign of inflammation [5]. Moreover, recent studies have shown that the glycan structure of N-glycan in IgG is altered also via sialylation and fucosylation in several diseases. For example, patients with ulcerative colitis show a reduction in the level of fucose structures in IgG [6]. On the other hand, patients with pancreatic cancer or renal cancer show an increased level of sialic acid structures, bisecting GlcNAc structures, and agalactose (non-galactosylation) structures in IgG [7,8]. A decrease in fucose structures in IgG in the early stages following infection with influenza virus has also been reported [9]. These reports strongly suggest that modification of the glycan structure in IgG would be a useful biomarker for diagnosing and monitoring some diseases. A lack of core fucose in IgG is well known to enhance antibodydependent cellular cytotoxicity (ADCC) via increased FcgRIIIA binding and signalling [10,11].

In this study, we attempted to analyse the glycan structure of IgG, in particular the core fucose structure in N-glycans (Supplementary Figure S1), in a patient before and after the onset of interstitial pneumonia. For detection via ELISA, we used a monoclonal antibody against core fucosylated IgG that was recently developed in our laboratory [12].

Materials and Methods

Preparation of plasma samples

Surplus blood samples were collected into tubes that included an anti-coagulant. After gentle mixing by inversion, the tubes were centrifuged at 1,200 g for 10 min at room temperature and the supernatants served as plasma samples. The resultant samples were stored at -80°C.

Enzyme-linked immunosorbent assay (ELISA)

The patient’s plasma was diluted in PBS (1:20,000 or 1:40,000), and 100 µl of the diluted plasma was added to the wells of a 96well plate (Sumitomo Bakelite Co., Ltd., Tokyo, Japan). After incubation overnight at 4 ℃, the wells were washed three times with 0.05% Tween20 in PBS. Then 100 µl of 5% (w/v) bovine serum albumin (BSA) (Sigma-Aldrich, Saint Louis, MO) in PBS was added and the mixture was incubated for 1 hr at RT for blocking. After washing the wells again three times with 0.05% Tween20 in PBS, 100 µl of an anti-core fucosylated human IgG antibody [1.25 µg/ml in 1% (w/v) of BSA in PBS] was added and the mixture was incubated for 2 hr at RT. The wells were then washed five times and incubated with an anti-mouse IgG antibody labelled with HRP (GE Healthcare, Chicago, IL) (1:2,000). After incubation for 1 hr at RT, the wells were washed again five times and the substrate solution [0.26 mg/ml of o-Phenylenediamine in 0.1 M citrate buffer (pH 5.0) including 0.009% H2O2] was added.

After incubation for 30 min in the dark, the absorbance values at 492 nm were measured using an Infinite M Plex plate reader (TECAN, Männedorf, Switzerland). For detection of total IgG, an anti-human IgG antibody labelled with HRP (GE Healthcare) was used. For detection of SP-D, Human SP-D Quantikine ELISA Kit (R&D Systems, Minneapolis, MN) was used following the manufacturer’s protocol.

Case Presentation

The patient was in his 70s and had undergone surgery for esophageal cancer in our hospital and was then transferred to the intensive care unit (ICU) due to the occurrence of respiratory insufficiency (day 1). The patient’s blood was collected in the ICU early each morning (usually around 5 – 6 am) and immediately prepared as plasma samples. Tracheal intubation was performed on day 5 and respiratory care was started using an artificial ventilator. On day 8, the patient was suspected of having interstitial pneumonia, and by day 9, CT analysis revealed ground-glass opacity in his right lung. Therefore, pulse therapy with a steroid (methylprednisolone sodium succinate) was performed on days 9 – 11. Following the pulse therapy, administration of another steroid (prednisolone) was started, and the patient’s respiratory function was stabilized at a certain level (Supplementary Table S1). By day 18, CT analysis showed that the disease state of the patient’s lung had worsened. During his stay in the ICU, the patient was administered other drugs including diuretics, psychotropics, and antibiotics. Infection with Pseudomonas aeruginosa was determined at certain time points via testing of the bacterial culture of the patient’s sputum.

Results

Core fucosylated IgG and total IgG in plasma samples were analysed by an enzyme-linked immunosorbent assay (ELISA). Both the amount of core fucosylated IgG and total IgG were decreased following diagnosis (days 11 – 14 and 17 – 24) (Figure 1A and 1B). The levels of core fucosylated IgG, which is the ratio of core fucosylated IgG relative to total IgG, were approximately 80% – 100% during days 1 – 10 but had decreased dramatically to approximately 40% by days 17 – 24 (Figure 2A). The steroid pulse treatment on days 9 – 11 resulted in temporary recovery of the level of core fucosylated IgG (days 15 and 16). The high level of acore fucosylated IgG (non-core fucosylated IgG) following the onset of interstitial pneumonia is shown in Figure 2B. The levels of C-reactive protein (CRP) and surfactant protein-D (SP-D) were also monitored during the patient’s stay in the ICU and they are shown in Supplementary Figure S2.

 

Figure 1: Levels of core fucosylated IgG and total IgG in the patient before and after the onset of interstitial pneumonia. The amounts of core fucosylated IgG (A) and total IgG (B) in plasma were measured by ELISA. Plasma samples were prepared and analyzed every other day. ELISA was performed in triplicate with values as means ± the standard deviation. The arrows indicate the day of the onset of interstitial pneumonia. Plasma samples were diluted in PBS at a ratio of 1:20,000 or 1:40,000 and analyzed in triplicate. The absorbance values were very low in the final detection step.

Figure 2: Levels of core fucosylated IgG and non-core fucosylated IgG relative to total IgG in the patient before and after the onset of interstitial pneumonia. The levels of core fucosylated IgG (A) and non-core fucosylated IgG (B) relative to total IgG in the plasma were calculated using the results shown in Figure 1. Values are reported as means ± the standard deviation. The arrows indicate the day of the onset of interstitial pneumonia.

 

Supplementary Figure S1: Core fucose structure in human immunoglobulin G (IgG). This figure is a modification of Figure 1A in our previously published paper [12].

 

Supplementary Figure S2: C-reactive protein (CRP) and surfactant protein-D (SP-D) in the patient’s plasma. (A) The amount of CRP in the plasma was measured as a daily bedside routine. (B) The amount of SP-D in the plasma was measured via ELISA, which was performed in triplicate with values reported as the mean ± the standard deviation. The allows indicates the day of the onset of interstitial pneumonia.