Effects of tunicamycin, mannosamine, and other inhibitors of glycoprotein processing on skeletal alkaline phosphatase in human osteoblast-like cells
Effects of tunicamycin, mannosamine, and other inhibitors of glycoprotein processing on skeletal alkaline phosphatase in human osteoblast-like cells
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DOI：
10.1007/s00223-004-0023-2
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发表时间：
2005-01-01
影响因子：
4.2
通讯作者：
Magnusson, P
中科院分区：
文献类型：
--
作者：
Farley, JR;Magnusson, P
骨碱性磷酸酶（sALP）是一种糖蛋白，其糖基化位点为5个，糖脂结构（糖基磷脂酰肌醇，GPI）的羧基末端位点将sALP锚定在成骨细胞的外表面。目前的研究旨在表征抑制糖基化和糖基化加工对人骨肉瘤（SaOS-2）细胞中sALP的合成、质膜附着、细胞外分布和反应动力学的影响。sALP合成、糖基化和GPI-锚附着被评估为总蛋白质合成/免疫特异性sALP合成，唾液酸含量（即，小麦胚芽凝集素沉淀），和不溶性（即，温度依赖性相分离）。sALP反应动力学通过分析磷酰基底物的剂量依赖性初始速度数据来表征。这些研究的结果表明，抑制N-连接的糖基化或寡糖合成的GPI锚添加可以影响合成和分布的sALP，但不影响磷酸酶反应的动力学。通过抑制核心寡糖合成来阻断N-连接糖基化的衣霉素降低了细胞中的细胞层蛋白和sALP的总量，同时增加了细胞条件培养基（CM，即，释放的sALP的量）。这些作用归因于sALP合成和N-连接糖基化的剂量和时间依赖性降低以及凋亡性细胞死亡的增加（各P < 0.001）。与衣霉素对N-连接糖基化的影响相反，甘露糖胺抑制GPI锚定糖基化/形成，其作用包括（1）细胞层蛋白的增加，（2）细胞和CM中sALP比活性的降低，（3）细胞内和CM中sALP比活性的降低。和（3）增加培养基中无锚定和麦胚凝集素（WGA）可溶性sALP的百分比，而在细胞内则无（P < 0.005）。据推测，甘露糖胺的这些作用是抑制sALP插入/附着到质膜表面外部的结果。甘露糖胺或衣霉素对sALP的反应动力学或对磷酰底物的表观亲和力（K-M值）均无任何影响。
Skeletal alkaline phosphatase (sALP) is a glycoprotein similar to20% 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. Tunica-mycin 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.