Biosynthesis, processing, and secretion of alpha-L-fucosidase in lymphoid cells from patients with I-cell disease and pseudo-Hurler polydystrophy.
Biosynthesis, processing, and secretion of alpha-L-fucosidase in lymphoid cells from patients with I-cell disease and pseudo-Hurler polydystrophy.
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I 细胞病和假性 Hurler 多发性营养不良患者淋巴细胞中 α-L-岩藻糖苷酶的生物合成、加工和分泌。
DOI：
10.1093/glycob/1.6.595
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发表时间：
1991
期刊：
影响因子：
4.3
通讯作者：
Miller,AL
中科院分区：
文献类型：
--
作者：
DiCioccio,RA;Miller,AL
N-乙酰葡糖胺1-磷酸转移酶是合成甘露糖6-磷酸识别标记物所需的关键酶，该标记物被许多新制备的酸性水解酶用于将其转运至溶酶体。以前已经发现，尽管缺乏N-乙酰葡糖胺1-磷酸转移酶，但I细胞病和假性Hurler多营养不良患者的淋巴细胞具有几乎正常的几种溶酶体酸水解酶的细胞内和溶酶体内活性。这些结果表明，淋巴样细胞可能提供了一个重要的系统，以调查替代机制，针对新的酸性水解酶的溶酶体。本研究以I细胞和假Hurler淋巴细胞中α-L-岩藻糖苷酶的生物合成、加工和分泌为模型系统来研究此类机制的存在。在指数生长的I细胞或假Hurler淋巴培养物中，细胞内α-L-岩藻糖苷酶蛋白水平与19种对照培养物的平均值在统计学上无法区分。1.5 h [35 S]蛋氨酸脉冲实验表明，α-L-岩藻糖苷酶最初由I-细胞、假Hurler和对照培养物以胞内形式合成（Mr= 58000）。伴随培养物追逐蛋氨酸从2至21小时处理的酶的细胞内形式（先生= 60 000）和细胞外形式（先生= 62 000）。所有酶的形式与多肽链的Mr 52 000的糖蛋白。在与放射性无机磷酸盐（32 Pi）孵育的对照细胞中，掺入α-L-岩藻糖苷酶的32 Pi中，<1%与碳水化合物链结合，>99%与多肽链结合。在I-细胞病淋巴细胞中，掺入α-L-岩藻糖苷酶的32 Pi仅与多肽链结合。磷酸化残基的定性分析鉴定了对照和I细胞淋巴样细胞中α-L-岩藻糖苷酶中的磷酸丝氨酸。仅来自对照细胞的α-L-岩藻糖苷酶含有甘露糖6-磷酸。这些结果与I细胞淋巴样细胞可能使用甘露糖6-磷酸非依赖性机制来路由α-L-岩藻糖苷酶的提议一致。另外的代谢标记实验表明，在对照淋巴培养的细胞和培养基中均存在32 P标记的α-L-岩藻糖苷酶，但仅在I细胞淋巴培养的细胞中存在。相反，在对照和I细胞淋巴培养物的细胞和培养基中发现了[35 S]蛋氨酸标记的α-L-岩藻糖苷酶。由于磷酸丝氨酸仅存在于I细胞培养物的细胞内，而非细胞外α-L-岩藻糖苷酶中，我们推测磷酸丝氨酸可能参与了I细胞淋巴样细胞中α-L-岩藻糖苷酶的细胞内滞留。
N-Acetylglucosamine 1-phosphotransferase is a key enzyme required for synthesis of the mannose 6-phosphate recognition marker that is used by many newly made acid hydrolases for their transport to lysosomes. It has previously been found that lymphoid cells from patients with I-cell disease and pseudo-Hurler polydystrophy have nearly normal intracellular and intralysosomal activities of several lysosomal acid hydrolases, despite a deficiency ofN-acetylglucosamine 1-phosphotransferase. These results suggest that lymphoid cells may provide an important system to investigate alternate mechanisms for targeting newly made acid hydrolases to lysosomes. In the present study, the biosynthesis, processing and secretion of α-L-fucosidase in I-cell and pseudoHurler lymphoid cells was used as a model system to study the existence of such mechanisms. The level of intracellular α-L-fucosidase protein in exponentially growing I-cell or pseudo-Hurler lymphoid cultures was statistically indistinguishable from the mean of 19 control cultures. A 1.5 h [35S]methionine pulse experiment showed that α-L-fucosidase is initially sythesized by I-cell, pseudo-Hurler and control cultures as an intracellular form (Mr= 58 000). Companion cultures chased with methionine from 2 to 21 h processed the enzyme to an intracellular form (Mr= 60 000) and an extracellular form (Mr= 62 000). All enzyme forms were glycoproteins with polypeptide chains of Mr 52 000. In control cells incubated with radioactive inorganic phosphate (32Pi), <1% of the32Pi incorporated into α-L-fucosidase was associated with carbohydrate chains and >99% with polypeptide chains. In I-cell disease lymphoid cells, the32Pi incorporated into α-L-fucosidase was associated solely with polypeptide chains. A qualitative analysis of phosphorylated residues identified phosphoserine in α-L-fucosidase from control and I-cell lymphoid cells. Only α-L-fucosidase from control cells contained mannose 6-phosphate. These results are consistent with the proposal that I-cell lymphoid cells may use a mannose 6-phosphate-independent mechanism for routing α-L-fucosidase. Additional metabolic labelling experiments demonstrated the presence of32P-labelled α-L-fucosidase in both cells and medium of a control lymphoid culture, but only in cells of an I-cell lymphoid culture. In contrast, α-L-fucosidase labelled with [35S]methionine was found in cells and medium of control and I-cell lymphoid cultures. Since phosphoserine was only found to occur in intracellular, but not in extracellular α-L-fucosidase of the I-cell culture, we speculate that phosphoserine may be involved in intracellular retention of α-L-fucosidase in I-cell lymphoid cells.