Brooks D A, Robertson D A, Bindloss C, Litjens T, Anson D S, Peters C, Morris C P, Hopwood J J
Department of Chemical Pathology, Women's and Children's Hospital, North Adelaide, Australia.
Biochem J. 1995 Apr 15;307 ( Pt 2)(Pt 2):457-63. doi: 10.1042/bj3070457.
The sulphatase family of enzymes have regions of sequence similarity, but relatively little is known about either the structure-function relationships of sulphatases, or the role of highly conserved amino acids. The sequence of amino acids CTPSR at position 91-95 of 4-sulphatase has been shown to be highly conserved in all of the sequenced sulphatase enzymes. The cysteine at amino acid 91 of 4-sulphatase was selected for mutation analysis due to its potential role in either the active site, substrate-binding site or part of a key structural domain of 4-sulphatase and due to the absence of naturally occurring mutations in this residue in mucopolysaccharidosis type VI (MPS VI) patients. Two mutations, C91S and C91T, altering amino acid 91 of 4-sulphatase were generated and expressed in Chinese hamster ovary cells. Biochemical analysis of protein from a C91S cell line demonstrated no detectable 4-sulphatase enzyme activity but a relatively normal level of 4-sulphatase polypeptide (180% of the wild-type control protein level). Epitope detection, using a panel of ten monoclonal antibodies, demonstrated that the C91S polypeptide had a similar immunoreactivity to wild-type 4-sulphatase, suggesting that the C91S substitution does not induce a major structural change in the protein. Reduced catalytic activity associated with normal levels of 4-sulphatase protein have not been observed in any of the MPS VI patients tested and all show evidence of structural modification of 4-sulphatase protein with the same panel of antibodies [Brooks, McCourt, Gibson, Ashton, Shutter and Hopwood (1991) Am. J. Hum. Genet. 48, 710-719]. The loss of enzyme activity without a detectable protein conformation change suggests that Cys-91 may be a critical residue in the catalytic process. In contrast, analysis of protein from a C91T cell line revealed low levels of catalytically inactive 4-sulphatase polypeptide (0.37% of the wild-type control protein level) which had missing or masked epitopes, suggesting an altered protein structure or conformation. Subcellular fractionation studies of the C91T cell line demonstrated a high proportion of 4-sulphatase polypeptide content in organelles characteristic of microsomes. The aberrant intracellular localization and the reduced cellular content of 4-sulphatase polypeptide was consistent with the observed structural modification leading to retention and degradation of the protein within an early vacuolar compartment.
硫酸酯酶家族的酶具有序列相似区域，但关于硫酸酯酶的结构 - 功能关系以及高度保守氨基酸的作用，人们了解得相对较少。已证明4 - 硫酸酯酶第91 - 95位氨基酸序列CTPSR在所有已测序的硫酸酯酶中高度保守。由于4 - 硫酸酯酶第91位的半胱氨酸可能在其活性位点、底物结合位点或关键结构域的一部分中发挥作用，并且在VI型黏多糖贮积症（MPS VI）患者中该残基没有自然发生的突变，因此选择对其进行突变分析。产生了两个改变4 - 硫酸酯酶第91位氨基酸的突变，即C91S和C91T，并在中国仓鼠卵巢细胞中进行表达。对来自C91S细胞系的蛋白质进行生化分析表明，未检测到4 - 硫酸酯酶活性，但4 - 硫酸酯酶多肽水平相对正常（为野生型对照蛋白水平的180%）。使用一组十种单克隆抗体进行表位检测表明，C91S多肽与野生型4 - 硫酸酯酶具有相似的免疫反应性，这表明C91S替代并未在蛋白质中诱导主要的结构变化。在任何测试的MPS VI患者中均未观察到与正常水平的4 - 硫酸酯酶蛋白相关的催化活性降低，并且所有患者都显示出用同一组抗体检测到4 - 硫酸酯酶蛋白的结构修饰[布鲁克斯、麦考特、吉布森、阿什顿、舒特和霍普伍德（1991年）《美国人类遗传学杂志》48卷，710 - 719页]。酶活性丧失但未检测到蛋白质构象变化表明，半胱氨酸91可能是催化过程中的关键残基。相比之下，对来自C91T细胞系的蛋白质分析显示，催化无活性的4 - 硫酸酯酶多肽水平较低（为野生型对照蛋白水平的0.37%），其表位缺失或被掩盖，这表明蛋白质结构或构象发生了改变。对C91T细胞系进行亚细胞分级分离研究表明，在微粒体特征性细胞器中4 - 硫酸酯酶多肽含量比例较高。4 - 硫酸酯酶多肽异常的细胞内定位和细胞含量降低与观察到的结构修饰一致，导致该蛋白质在早期液泡区室中滞留并降解。
