Transport and metabolism of 5′‐nucleotidase in a rat hepatoma cell line

Abstract

The biosynthesis of the ectoenzyme 5'-nucleotidase in the rat hepatoma cell line H4S has been studied by pulse-labeling with [35S]methionine and subsequent immunoprecipitation of the cell lysate. 5'-Nucleotidase is a membrane glycoprotein with an apparent molecular mass on SDS-gels of 72 kDa. The enzyme is initially synthesized as a 68-kDa precursor which is converted to the mature 72-kDa form in 15 -60 min (t1,2 = 25 min). The molecular mass of the unglycosylated enzyme is approximately 58 kDa. Culturing the cells in the presence of varying concentrations of tunicamycin, an inhibitor of N-glycosylation, revealed six species of 5'-nucleotidase after sodium dodecyl sulfate/polyacrylamide electrophoresis. This indicates the presence of five N-linked oligosaccharide chains accounting for the difference between the 58-kDa polypeptide backbone and the 68-kDa species. The 68-kDa precursor is susceptible to cleavage by endo-b-N-acetylglycosaminidase H ; the 72-kDa mature protein is converted to several bands upon this treatment. This result indicates that part of 5'-nucleotidase keeps one or two high-mannose or hybrid chains in the mature form, even after prolonged pulse-chase labeling. The newly synthesized mature enzyme reaches the cell surface after 20 -30 min. The half-life of 5'-nucleotidase is about 30 h in H4S cells. No immunoprecipitable 5'-nucleosidase is released into the culture medium.

Unveiling the Role of Ecto-5′-Nucleotidase/CD73 in Astrocyte Migration by Using Pharmacological Tools

“…Since CD73 has no structural isoforms ( Zimmermann, 1992 ), these functional variations are exhibited by CD73 molecules which are identical at protein, mRNA and cDNA level ( Airas et al, 1995 ) but differ in their carbohydrate content. Based on the carbohydrate content, CD73 may be classified as a high-mannose type, complex carbohydrates type and hybrid type, which contains both complex carbohydrates and sialic acid residues ( Meflah et al, 1984 ; van den Bosch et al, 1986 , 1988 ; Wada et al, 1986 ; Baron and Luzio, 1987 ). As it is quite clear that the differences in glycosylation may be responsible for the variations found in apparent molecular weight of CD73 isolated from different sources ( Turnay et al, 1989 ; Olmo et al, 1992 ; Zimmermann, 1992 ; Navarro et al, 1998 ; Grkovic et al, 2014 ; Lavrnja et al, 2015 ), it has been increasingly evident that the variations in glycan content may be responsible for the functional differences of CD73 found in different cell types and tissues.…”