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

Alkaline phosphatase (ALP) is a glycoenzyme that is highly expressed during carcinogenesis and is induced by retinoic acid (RA) in various cells. We i nvestigated the effects of RA on N-linked glycosylation of the tissue nonsp ecific liver/bone/kidney-type of ALP (L/B/K ALP), on ALP transcripts, and o n total protein glycosylation in two neuronal cell lines, P19 and NG108CC15 , and in primary cultures of neonatal rat brain. ALP activity was determine d in cell extracts and round to be induced by RA. Tunicamycin was used at v arious concentrations to inhibit protein N-glycosylation. After treatment o f cells with low concentrations (0.1 and 0.125 mu g/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 activit y. Interestingly, ALP activity in RA-treated cultures was less inhibited by tunicamycin compared to unheated controls suggesting that RA may have an i mpact on ALP N-glycosylation. To investigate effects of RA on ALP glycosyla tion further, incorporation of [C-14]-mannose and [S-35]methionine into ALP protein was determined in the presence or absence of RA. The ratio of mann osylation and biosynthesis demonstrate that incubation of cells with RA inc reased [C-14]mannose incorporation into ALP molecules. Also, the release of free [C-14]mannose from ALP molecules relative to the amount of protein by N-Glycanase was increased in RA-treated cultures. In addition, mannosylati on of total protein was round to be induced in cells after exposure to RA. Analysis of biosynthesized ALP monomers revealed that RA increased glycosyl ation 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 leve ls of this enzyme are affected by RA. The P19 cell line provides a useful m odel system to study the molecular mechanism(s) underlying the action of RA on glycosylation during neuronal differentiation further. J. Cell. Physiol . 182:50-61, 2000. (C) 2000 Wiley-Liss, Inc.