Liver Galbeta1,4GlcNAc alpha2,6-sialyltransferase is down-regulated in human alcoholics: possible cause for the appearance of asialoconjugates

Abstract

Galbetal,4GlcNAc alpha2,6-sialyltransferase (ST6GalI) mediates the glycosylation of proteins and lipids to form functionally important glycoproteins and glycolipids in the Golgi compartment. Our previous work demonstrated that long-term ethanol feeding in rats caused a marked 59% decrease in ST6GalI activity as well as ST6GalI messenger RNA (mRNA) level in the liver that was due to decreased stability of the mRNA. Clinical observations show that down-regulation of ST6GalI gene and consequent impaired activity of ST6GalI seems to be the major cause for the appearance of asialoconjugates in the blood of long-term alcoholics. The plasma carbohydrate-deficient transferrin (CDT) and sialic acid index of plasma apolipoprotein J were also altered in the alcoholic group compared with the nondrinkers. We have now investigated how alcohol affects the gene regulation of ST6GalI and the possible mechanism in postmortem human liver specimens taken from nondrinkers, moderate alcohol drinkers, and heavy alcohol drinkers. Real-time polymerase chain reaction analyses of the liver RNA extract showed that ST6GalI mRNA level was progressively decreased by 49% in moderate drinkers (P < .01) and by 69% in heavy drinkers (P < .01) compared with nondrinkers. Western blot analysis showed that liver ST6GalI protein level was negligibly decreased in moderate drinkers but decreased by 30% (P < .05) in heavy drinkers compared with nondrinkers. We further demonstrated a single ST6GalI mRNA-binding protein complex in the normal human liver extract, which progressively decreased in the liver extracts of moderate and heavy alcohol drinkers. Thus, it is concluded that the appearance of asialoconjugates in alcoholics is possibly due to the down-regulation of ST6GalI gene expression.

Figure 1. Plasma CDT and SIJ in alcoholics and non drinkers

A suitable aliquot of each plasma sample was analyzed for CDT and SIJ as described by us previously (14). Plasma CDT is expressed as units/liter based on the Kabi Pharmacia Diagnostics, Uppsala, Sweden. Each value is the Mean ± SEM of 12 specimens from each group.

Figure 2. Real time RT- PCR analyses of ST6GalI in human liver specimens

Total RNA from the non drinkers group (ND), the moderate drinkers group (MD) and the heavy drinkers group (HD) was reverse transcribed and used in the real time PCR. The RNA levels were normalized to the level of β-actin on experiment to experiment basis. Each sample analysis was performed in triplicate independently and each bar graph represents the Mean ± SEM of 12 specimens in each group with the ND group set at 1 for convenience.

Figure 3. Western Blot analysis of ST6GalI in human liver specimens

50 μg of liver Golgi fraction extracts from the non drinkers group (ND), the moderate drinkers group (MD) and the heavy drinkers group (HD) was subjected to Western Blot analyses using the polyclonal anti ST6GalI and anti β-actin antibodies. Densitometry analysis of the intensity of each band was then performed. Western blot of two representative specimens from each group are shown in panel A. Bands on the top represent ST6GalI; bands on the bottom represent β-actin. In panel B, the relative intensity of ST6 Gal1 protein level in each specimen was normalized to its β-actin level. The bar graphs represent the relative level of ST6GalI in each group with the ND group set at 1 for convenience. The value in each group is the Mean ± SEM of 12 specimens.

Figure 4. RNA-protein Electrophoretic Gel Mobility Shift Assays (EMSA)

³²P-labeled RNA at a concentration of 2 nM was incubated with 20 μg of cytosolic proteins from each liver sample and the RNA-protein complexes were resolved on an 8% native polyacrylamide gel in 0.5X TBE buffer. The gel was then dried and exposed to XAR film with an intensifying screen at −80°C overnight. Two representative specimens from each group were shown in 4A. Arrow indicates the RNA-protein complex; Lane P represents RNA probe without the cytosolic protein. 4B: shows the average density of 12 specimens in each group. The value in each group is the Mean ± SEM of 12 specimens. The RNA-protein complex was progressively decreased both in MD and HD groups compared to the ND group.