Ameen M, Lazzarino D A, Kelly B M, Gabel C A, Chang P L
Department of Pediatrics, McMaster University, Ontario, Canada.
Mol Cell Biochem. 1990 Feb 9;92(2):117-27. doi: 10.1007/BF00218129.
Deficient arylsulfatase-A activity is diagnostic of a neurodegenerative human lysosomal storage disease, metachromatic leukodystrophy. Paradoxically, similar enzyme deficiency also occurs in normal individuals, who are known as being pseudo arylsulfatase-A deficient. We showed previously that this phenotype is associated with a structural gene mutation that produces an exceptionally labile enzyme. We now report on the nature and consequence of this mutation. When the mutant arylsulfatase-A is deglycosylated by endoglycosidase H, only one smaller molecular species was generated, instead of the two from the normal enzyme. This is consistent with the loss of one of the two N-linked oligosaccharide side chains known to be present on the wild-type enzyme. Quantitative analysis of mannose and leucine incorporation showed that the mutant enzyme incorporated two- to tenfold less mannose than the normal enzyme on a molar basis. This deficient glycosylation was specific to arylsulfatase-A. Another lysosomal enzyme not affected in this mutation, beta-hexosaminidase, was glycosylated normally in the mutant cells. The remaining single oligosaccharide side chain released from the mutant arylsulfatase-A by pronase digestion was normally processed to complex and high-mannose forms. However, the high-mannose side chains contained 30% fewer phosphorylated residues than those of the normal enzyme. Nevertheless, this reduced level of phosphorylation did not prevent targeting of the mutant enzyme to the lysosomes, a process normally mediated through phosphorylated mannose residues. In conclusion, pseudo arylsulfatase-A deficiency is a unique human mutation associated with reduced glycosylation and phosphorylation of a lysosomal enzyme with the loss of one of the two carbohydrate side chains. The mutation results in greatly reduced enzyme stability, thus indicating a role for oligosaccharides in maintaining enzyme stability within the degradative environment of the lysosomes. However, the residual catalytic activity or subcellular targeting of the mutant enzyme was not affected. These properties probably account for the benign clinical presentation of pseudo arylsulfatase-A deficiency.
芳基硫酸酯酶A活性缺乏是人类神经退行性溶酶体贮积病——异染性脑脑脑营养不良症的诊断依据。矛盾的是，正常个体中也会出现类似的酶缺乏情况，这些个体被称为假性芳基硫酸酯酶A缺乏者。我们之前表明，这种表型与一种导致酶异常不稳定的结构基因突变有关。我们现在报告这种突变的性质和后果。当突变型芳基硫酸酯酶A被内切糖苷酶H去糖基化时，只产生了一种较小的分子形式，而正常酶会产生两种。这与野生型酶已知存在的两个N - 连接寡糖侧链之一的缺失一致。对甘露糖和亮氨酸掺入的定量分析表明，突变型酶在摩尔基础上掺入的甘露糖比正常酶少两到十倍。这种糖基化缺陷是芳基硫酸酯酶A特有的。另一种在该突变中不受影响的溶酶体酶——β - 己糖胺酶，在突变细胞中糖基化正常。经链霉蛋白酶消化从突变型芳基硫酸酯酶A释放的剩余单个寡糖侧链通常被加工成复合和高甘露糖形式。然而，高甘露糖侧链中的磷酸化残基比正常酶少30%。尽管如此，这种磷酸化水平的降低并没有阻止突变型酶靶向溶酶体，这一过程通常是通过磷酸化的甘露糖残基介导的。总之，假性芳基硫酸酯酶A缺乏是一种独特的人类突变，与溶酶体酶的糖基化和磷酸化减少以及两个碳水化合物侧链之一的缺失有关。该突变导致酶稳定性大大降低，从而表明寡糖在维持溶酶体降解环境中酶的稳定性方面发挥作用。然而，突变型酶的残余催化活性或亚细胞靶向不受影响。这些特性可能解释了假性芳基硫酸酯酶A缺乏的良性临床表现。
