Effects of retinoic acid on N‐glycosylation and mRNA stability of the liver/bone/kidney alkaline phosphatase in neuronal cells

Abstract

Alkaline phosphatase (ALP) is a glycoenzyme that is highly expressed during carcinogenesis and is induced by retinoic acid (RA) in various cells. We investigated the effects of RA on N-linked glycosylation of the tissue nonspecific liver/bone/kidney- type of ALP (L/B/K ALP), on ALP transcripts, and on total protein glycosylation in two neuronal cell lines, P19 and NG108CC15, and in primary cultures of neonatal rat brain. ALP activity was determined in cell extracts and found to be induced by RA. Tunicamycin was used at various concentrations to inhibit protein N-glycosylation. After treatment of cells with low concentrations (0.1 and 0.125 microgram/ml) of tunicamycin for 48 h, uninduced and RA-induced ALP activity declined while incubation with a protease inhibitor restored activity, indicating that the L/B/K ALP bear N-linked oligosaccharide chains important for maintaining enzymatic activity. Interestingly, ALP activity in RA-treated cultures was less inhibited by tunicamycin compared to untreated controls suggesting that RA may have an impact on ALP N-glycosylation. To investigate effects of RA on ALP glycosylation further, incorporation of [(14)C]mannose and [(35)S]methionine into ALP protein was determined in the presence or absence of RA. The ratio of mannosylation and biosynthesis demonstrate that incubation of cells with RA increased [(14)C]mannose incorporation into ALP molecules. Also, the release of free [(14)C]mannose from ALP molecules relative to the amount of protein by N-Glycanase was increased in RA-treated cultures. In addition, mannosylation of total protein was found to be induced in cells after exposure to RA. Analysis of biosynthesized ALP monomers revealed that RA increased glycosylation of the polypeptides. Furthermore, tunicamycin decreased the stability of ALP mRNA, an effect that was reduced by cotreatment with RA. Thus, the degree of N-glycosylation of the L/B/K ALP as well as mRNA and protein levels of this enzyme are affected by RA. The P19 cell line provides a useful model system to study the molecular mechanism(s) underlying the action of RA on glycosylation during neuronal differentiation further.

Regulation of ornithine aminotransferase gene expression and activity by all-transretinoic acid in Caco-2 intestinal epithelial cells

“…Although the mechanism for the RA-mediated increase in OAT expression is not understood, one potential mechanism may be a RAinduced increase in OAT mRNA stability. This idea is further supported by previous reports of RA-mediated increases in mRNA stability of bone-type alkaline phosphatase [45,46], surfactant proteins [47] and IGF-binding proteins [48]. Additional studies are necessary to fully elucidate the mechanism by which RA increases OAT expression.…”

Differential expression of the bone and the liver tissue non-specific alkaline phosphatase isoforms in brain tissues

“…Second, TNAP activity has also been shown to be modulated by post-translational modification, such as glycosylation (Halling Linder et al 2009;Kiledjian and Kadesch 1991;Mueller et al 2000). The impact of glycosylations on protein biosynthesis makes it unclear whether their effects on TNAP activity are only direct.…”

Murine synaptosomal lipid raft protein and lipid composition are altered by expression of human apoE 3 and 4 and by increasing age

“…Furthermore, activity of ALP was significantly lower in the three age groups of apoE4 mice than activity of age-matched apoE3 mice. ALPs are found in numerous tissues but their functions are not well-understood [35]. There is evidence that ALPs may play a role in mineralization, act as a tyrosine-specific phosphoprotein phosphatase, involvement in transport of inorganic orthophosphate and maintenance of cellular permeability [34,35].…”