Effects of Tunicamycin, Mannosamine, and Other Inhibitors of Glycoprotein Processing on Skeletal Alkaline Phosphatase in Human Osteoblast-Like Cells

Skeletal alkaline phosphatase (sALP) is a glycoprotein—~20% carbohydrate by weight, with five presumptive sites for N-linked glycosylation, as well as a carboxy-terminal site for attachment of the glycolipid structure (glycosylphosphatidylinositol, GPI), which anchors sALP to the outer surface of osteoblasts. The current studies were intended to characterize the effects of inhibiting glycosylation and glycosyl-processing on the synthesis, plasma membrane attachment, cellular–extracellular distribution, and reaction kinetics of sALP in human osteosarcoma (SaOS-2) cells. sALP synthesis, glycosylation, and GPI-anchor attachment were assessed as total protein synthesis/immunospecific sALP synthesis, sialic acid content (i.e., wheat germ agglutinin precipitation), and insolubility (i.e., temperature-dependent phase-separation), respectively. sALP reaction kinetics were characterized by analysis of dose-dependent initial velocity data, with a phosphoryl substrate. The results of these studies revealed that the inhibition of either N-linked glycosylation or oligosaccharide synthesis for GPI-anchor addition could affect the synthesis and the distribution of sALP, but not the kinetics of the phosphatase reaction. Tunicamycin—which blocks N-linked glycosylation by inhibiting core oligosaccharide synthesis—decreased cell layer protein and the total amount of sALP in the cells, while increasing the relative level of sALP in the cell-conditioned culture medium (CM, i.e., the amount of sALP released). These effects were attributed to dose- and time-dependent decreases in sALP synthesis and N-linked glycosylation, and an increase in apoptotic cell death (P < 0.001 for each). In contrast to the effects of tunicamycin on N-linked glycosylation, the effects of mannosamine, which inhibits GPI-anchor glycosylation/formation, included (1) an increase in cell layer protein; (2) decreases in sALP specific activity, in the cells and in the CM; and (3) increases in the percentages of both anchorless and wheat germ agglutinin (WGA)-soluble sALP in the medium, but not in the cells (P < 0.005 for each). These effects of mannosamine were, presumably, a consequence of inhibiting the insertion/attachment of sALP to the outside of the plasma membrane surface. Neither mannosammine nor tunicamycin had any effect on the reaction kinetics of sALP or on the apparent affinity (the value of K_M) for the phosphoryl substrate.