Lazzarino D A, Gabel C A
Department of Anatomy and Cell Biology, Columbia University, College of Physicians and Surgeons, New York, New York 10032.
J Biol Chem. 1988 Jul 25;263(21):10118-26.
The biosynthesis of the mannose 6-phosphate recognition marker has been studied in transport-impaired mouse lymphoma cells to determine the subcellular location of the processing enzymes and to characterize the biosynthetic intermediates. Cells were labeled with [2-3H]mannose and chased at a low temperature (15 or 20 degrees C) or at 37 degrees C in the presence of m-chlorocarbonylcyanide phenylhydrazone to disrupt transport of the pulse-labeled molecules within the secretory apparatus. Both treatments inhibited the migration of the pulse-labeled glycoproteins to the Golgi apparatus as measured by the production of complex-type asparagine-linked oligosaccharides. Despite this inhibition in protein transport, acid hydrolases were phosphorylated. Structural analysis of the phosphorylated oligosaccharides indicated that the transport-impaired cells produced a single species of phosphorylated high mannose oligosaccharide; essentially all of the molecules contain a single phosphodiester group that is restricted to the alpha 1,6 branch of the oligosaccharide. The results suggest that synthesis of mannose 6-phosphate-bearing high mannose oligosaccharides occurs in an ordered, compartmentalized posttranslational process. The initial phosphorylation of newly synthesized acid hydrolases occurs at a pre-Golgi site and results in the production of high mannose-type units that contain a single phosphodiester group. In a subsequent compartment, probably within the Golgi apparatus, the monophosphorylated units may be converted to diphosphorylated forms. Finally, at a site distal to the phosphorylation reactions the diesters are hydrolyzed to reveal the mannose 6-phosphate recognition marker.
为了确定加工酶的亚细胞定位并对生物合成中间体进行表征，研究人员在运输功能受损的小鼠淋巴瘤细胞中对甘露糖6 - 磷酸识别标记的生物合成进行了研究。用[2 - 3H]甘露糖标记细胞，并在低温（15或20摄氏度）下或在间氯羰基氰化物苯腙存在的37摄氏度下进行追踪，以破坏脉冲标记分子在分泌装置内的运输。通过产生复合型天冬酰胺连接寡糖来测量，这两种处理均抑制了脉冲标记糖蛋白向高尔基体的迁移。尽管蛋白质运输受到这种抑制，但酸性水解酶仍被磷酸化。对磷酸化寡糖的结构分析表明，运输功能受损的细胞产生了单一类型的磷酸化高甘露糖寡糖；基本上所有分子都含有一个单一的磷酸二酯基团，该基团仅限于寡糖的α1,6分支。结果表明，含甘露糖6 - 磷酸的高甘露糖寡糖的合成发生在一个有序的、分隔的翻译后过程中。新合成的酸性水解酶的初始磷酸化发生在高尔基体前位点，并导致产生含有单一磷酸二酯基团的高甘露糖型单元。在随后的区室中，可能是在高尔基体内部，单磷酸化单元可能会转化为双磷酸化形式。最后，在磷酸化反应远端的一个位点，二酯被水解以暴露出甘露糖6 - 磷酸识别标记。
