Human α-galactosidase A:: glycosylation site 3 is essential for enzyme solubility
Human α-galactosidase A:: glycosylation site 3 is essential for enzyme solubility
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DOI：
10.1042/bj3320789
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发表时间：
1998-06-15
影响因子：
4.1
通讯作者：
Desnick, RJ
中科院分区：
文献类型：
--
作者：
Ioannou, YA;Zeidner, KM;Desnick, RJ
人α-半乳糖苷酶A（EC 3.2.1.22; alpha-Gal A）是从糖脂和糖蛋白水解末端α-半乳糖基部分的同型二聚体糖蛋白。确定了这种溶酶体水解酶上N-连接寡糖链的类型、位点占有率和功能。纯化的重组酶的内切糖苷酶处理和诱变研究表明，三个（Asn-139，Asn-192和Asn-215）的四个潜在的N-糖基化的共识序列分别被复杂，高甘露糖和混合型寡糖。当在COS-1细胞中表达时，糖基化位点1或2被消除的糖型具有野生型活性的70%以上，并且两种糖型都被分泌。相比之下，仅位点3被消除的糖型具有降低的活性（小于40%）;几乎没有（如果有的话）分泌。表达的突变体糖型，其中网站3和网站1或2被抹杀有很少的，如果有的话，细胞内或分泌的酶活性，和免疫荧光显微镜显示，表达的突变体糖型保留在内质网，大概在那里他们被降解。因此，位点3的糖基化对于可溶性活性酶的形成以及向溶酶体的转运至关重要。缺乏网站3杂交型寡糖暴露相邻的，通常受保护的，疏水区域，导致聚集的酶多肽在内质网。为了支持这一概念，内切糖苷酶H处理的酶或在苜蓿银纹草细胞中表达的甘露糖终止的酶在浓缩时也聚集，强调位点3被混合型寡糖占据是酶溶解度所需的。
Human alpha-galactosidase A (EC 3.2.1.22; alpha-Gal A) is the homodimeric glycoprotein that hydrolyses the terminal a-galactosyl moieties from glycolipids and glycoproteins. The type, site occupancy and function of the N-linked oligosaccharide chains on this lysosomal hydrolase were determined. Endoglycosidase treatment of the purified recombinant enzyme and mutagenesis studies indicated that three (Asn-139, Asn-192 and Asn-215) of the four potential N-glycosylation consensus sequences were occupied by complex, high-mannose and hybrid-type oligosaccharides respectively. When expressed in COS-1 cells, glycoforms with glycosylation site 1 or 2 obliterated had more than 70 % of wild-type activity, and both glycoforms were secreted. In contrast, the glycoform with only site 3 eliminated had decreased activity (less than 40 %); little, if any, was secreted. Expressed mutant glycoforms in which site 3 and site 1 or 2 were obliterated had little, if any, intracellular or secreted enzymic activity, and immunofluorescence microscopy revealed that the expressed mutant glycoforms were retained in the endoplasmic reticulum, presumably where they were degraded. Thus glycosylation at site 3 was crucial to the formation of soluble, active enzyme, as well as transport to the lysosome. Absence of the site 3 hybrid-type oligosaccharide exposed an adjacent, normally protected, hydrophobic region, resulting in aggregation of the enzyme polypeptide in the endoplasmic reticulum. In support of this concept, endoglycosidase H-treated enzyme or mannose-terminated enzyme expressed in Autographa californica cells also aggregated when concentrated, emphasizing that site 3 occupancy by a hybrid-type oligosaccharide was required for enzyme solubility.