Age-dependent glycosylation of the sodium taurocholate co-transporte polypeptide: from neonates to adult human primary hepatocytes

Abstract

Background and Aims: The sodium taurocholate cotransporter polypeptide (NTCP) is the major hepatobiliary transporter responsible for bile acids portal vein re-uptake in hepatocytes. In humans, NTCP ontogeny has not been sufficiently investigated because of limited access to human tissue. Based on rodent models, NTCP glycosylation requires four weeks to be completed (Hardikar et al., 1995; Gao et al., 2004). Therefore, the aim of this study was to examine in humans the maturational profile of NTCP glycosylation levels from birth up to adulthood. Methods: Adult and neonatal primary human hepatocytes (PHH) were obtained from UCL-St Luc biobank with ethical consent for research purposes. PHH were isolated by a one stage collagenase perfusion and cryopreserved in liquid nitrogen (Ref). Donors were selected and approved by the medical ethical committee of our institution. Two groups of PHH were established: PHH donors younger than 1 year old were considered as early infants, whereas, PHH donors older than 1 years were considered as mature hepatocytes. The two groups were tested for NTCP transcriptional, translational and glycosylation age-dependent modulation levels by RT-qPCR and SDS-PAGE analysis. Characterization of NTCP glycosylation bands was conducted with PNGase F de-glycosylation treatment. Results: As a result, we show that NTCP mature complex-glycosylated form (55 kDa) was significantly increased in an age-dependent manners as compared to non-glycosylated NTCP (38 kDa) approximately at one year of age, whereas, NTCP mRNA and native protein levels were not significantly regulated, but had a slight tendency to increase with age. The complete deglycosylation of NTCP allowed us to then quantify the amount of de-glycosylated NTCP that shift at 38 kDa band. Here we show that NTCP 38 kDa band in adults was significantly increased by PNGase F treatment as compared to non treated, whereas in infants, no significant difference was shown. Conclusions: In conclusion, our data suggests that in human neonates, NTCP complex-glycosylation maturation process requires several months to up to one year to be completed,whereas, trascriptional and translational profiles were less influenced by age