Alcohol induces α2-6sialo mucin O-glycans that kill U937 macrophages mediated by sialic acid-binding immunoglobulin-like lectin 7 (Siglec 7)

Abstract

Alcohol misuse increases infections and cancer fatalities, but mechanisms underlying its toxicity are ill-defined. We show that alcohol treatment of human tracheobronchial epithelial cells leads to inactivation of giantin-mediated Golgi targeting of glycosylation enzymes. Loss of core 2 N-acetylglucosaminyltransferase 1, which uses only giantin for Golgi targeting, coupled with shifted targeting of other glycosylation enzymes to Golgi matrix protein 130-Golgi reassembly stacking protein 65, the site normally used by core 1 enzyme, results in loss of sialyl Lewis x and increase of sialyl Lewis a and α2-6sialo mucin O-glycans. The α2-6sialo mucin O-glycans induced by alcohol cause death of U937 macrophages mediated by sialic acid-binding immunoglobulin-like lectin 7. These results provide a mechanistic insight into the cause of the toxic effects of alcohol and might contribute to the development of therapies to alleviate its toxicity.

Keywords: Siglec 7; U937 macrophages; ethanol; giantin; α2‐6sialo mucin O‐glycans.

Fig. 1

Ethanol concentration effects on Golgi morphology, and intracellular distribution and levels of C2GnT1 in HTBE cells. (A) Confocal immunofluorescence images of Golgi (giantin‐red) and C2GnT1 (green) in the paraffin sections of ALI cultures of HTBE cells exposed to 30–100 mm ethanol for 72 h, (B) % cells with fragmented Golgi, and (C) intracellular levels of C2GnT1 measured by imagej program at five randomly chosen regions and expressed as mean ± SD. Statistical analysis was performed by ANOVA.

Fig. 2

Confocal immunofluorescence images of ALI cultures of HTBE cells treated with alcohol or ACH with or without inhibitors or activator. Paraffin sections of HTBE cells were treated with anti‐giantin Ab (red), anti‐C2GnT1 Ab (green), and DAPI after the cultured cells had been treated with (A) 50 mm ethanol in the absence or presence of 5 mm 4‐methylpyrazole or 50 μm Alda‐1, or with 50 μm ACH, and (B) 100 mm ethanol ± 5 mm 4‐methylpyrazole or 35 μm blebbistatin, or 100 μm ACH ± 35 μm blebbistatin.

Fig. 3

Confocal immunofluorescence images of in situ Proximity Ligation Assay of Man IA colocalization with giantin vs. GM130 and GRASP65 in HTBE cells with or without ethanol treatment. The analysis was performed on paraffin sections of ALI cultures of HTBE cells exposed to 0 or 50 mm ethanol.

Fig. 4

VVA and PNA lectin stain of HTBE cells treated with EtOH. Confocal lectin fluorescence images of (A) FITC‐PNA and (B) FITC‐PNA ± neuraminidase‐treated paraffin sections of HTBE cells that had been exposed to 0, 30, or 80 mm EtOH. The fluorescence intensity in (A) and (B) was measured at five randomly chosen areas and expressed as mean ± SD shown in (C) and (D), respectively. The statistical analysis was performed by ANOVA.

Fig. 5

Immunostaining of sLe^x and sLe^a in HTBE cells treated with EtOH. (A) Confocal immunofluorescence images of sLe^x and sLe^a in paraffin sections of ALI cultures HTBE cells treated with anti‐sLe^x and sLe^a antibodies and DAPI after the cultured cells had been exposed to 0, 30, or 80 mm EtOH for 72 h. The immunofluorescence intensities of (B) sLe^x and (C) sLe^a were measured at five randomly chosen areas by imagej and expressed as mean ± SD. The statistical analysis was performed by ANOVA.

Fig. 6

Confocal SNA‐I lectin fluorescence images of 50 mm alcohol‐treated HTE cells stained with (A) FITC‐SNA‐I and measured for fluorescence intensity at five random areas by imagej and expressed as (B) mean ± SD. The statistical analysis was performed by ANOVA.

Fig. 7

(A) SNA‐I inhibition of the killing of U937 macrophages by OSM and (B) Siglec 7‐Fc inhibition of the killing of U937 macrophages by OSM, sTn‐ser, or DisT‐ser. The viability of U937 cells was measured by trypan blue exclusion after exposure for 3–4 h to (A) culture medium or OSM with or without SNA‐I or (B) culture medium, OSM, sTn‐ser, or DisT‐ser with or without Siglec 7‐Fc. Statistical analysis (n = 3) was performed by ANOVA.

Fig. 8

Altered biosynthesis of mucin O‐glycans in alcohol‐treated HTBE cells. (A) HTBE cells synthesize sLe^x extended from core 2 branch produced by C2GnT1. (B) In EtOH‐treated HTBE cells, loss of C2GnT1 coupled with shifted Golgi targeting of other glycosylation enzymes as the result of loss of giantin functions leads to loss of sLe^x but increase of Tn, sTn, T antigen, 6sT, DisT, and sLe^a.