突变特征的β-半乳糖Asp332——> Asn和Arg148——>爵士,和多态性,Ser532 - - >通用电气,在一个GM1 gangliosidosis。
文章的细节
-
引用
复制到剪贴板 -
张,Bagshaw R, Hilson W,嗳哟Y, Hinek,克拉克JT,卡拉汉JW
突变特征的β-半乳糖Asp332——> Asn和Arg148——>爵士,和多态性,Ser532 - - >通用电气,在一个GM1 gangliosidosis。
j . 2000 348年6月15日;Pt 3:621-32。
- PubMed ID
-
10839995 (在PubMed]
- 文摘
-
我们有识别和特征三个错义突变患者1 G型gangliosidosis (M1),即一个替换的G在核苷酸位置1044 (G1044——>;在一个等位基因外显子10),将Asp(332)转化为天冬酰胺和一个突变(C492 - - >外显子4,导致氨基酸的变化参数(148)——> Ser)和多态性(A1644 - - >外显子15克,导致改变爵士(532年)——> G)等位基因。这个病人有不到1%的活动和最小可检测水平的免疫反应性的残余β-半乳糖苷酶蛋白在成纤维细胞。占上述研究结果,进行了一系列表达式和immunolocalization研究来评估每一个突变的影响。瞬态COS-1细胞过度的互补编码Asp (332) Asn, Arg(148)爵士和爵士(532)g变异产生丰富,大量的前体β-半乳糖苷酶活动的0,84和81% cDNA克隆野生型苷(GP8)。vector-driven水平以来的表达式是更少的中国仓鼠卵巢(CHO)细胞比COS-1细胞,我们知道,进行外源β-半乳糖溶酶体处理这些细胞中表达时,瞬时表达研究进行的参数(148)爵士和爵士(532)g,产生酶的活性形式。在这种情况下,Arg(148)爵士和爵士(532)g产品引发了分别为11%和86%的控制活动。这些结果并不意外,因为Arg (148) Ser突变主要构象变化引入的蛋白质,我们预期这将是退化的内质网(ER),而多态性将产生接近于正常的活动。检查Asp (332) Asn突变的影响催化活性,我们孤立曹永久克隆转染与Asp (332) Asn Asp (332) Glu结构,由substrate-analogue-affinity色谱纯化的酶,确定动力学参数。V (max)值的突变重组酶明显减少(不到0.9%的控制),和K (m)值与相应的野生型酶相比持平孤立的在同一时间。 Both the Arg(148)Ser beta-galactosidase in CHO cells and Asp(332)Asn beta-galactosidases (in COS-1 and CHO cells) produced abundant immunoreaction in the perinuclear area, consistent with localization in the ER. A low amount was detected in lysosomes. Incubation of patient fibroblasts in the presence of leupeptin, which reduces the rate of degradation of lysosomal beta-galactosidase by thiol proteases, had no effect on residual enzyme activity, and immunostaining was again detected largely in the perinuclear area (localized to the ER) with much lower amounts in the lysosomes. In summary, the Arg(148)Ser mutation has no effect on catalytic activity, whereas the Asp(332)Asn mutation seriously reduces catalytic activity, suggesting that Asp(332) might play a role in the active site. Immunofluorescence studies indicate the expressed mutant proteins with Arg(148)Ser and Asp(332)Asn mutations are held up in the ER, where they are probably degraded, resulting in only minimum amounts of the enzyme becoming localized in the lysosomes. These results are completely consistent with findings in the cultured fibroblasts. Our results imply that most of the missense mutations described in G(M1) gangliosidosis to date have little effect on catalytic activity, but do affect protein conformation such that the resulting protein cannot be transported out of the ER and fails to arrive in the lysosome. This accounts for the minimal amounts of enzyme protein and activity seen in most G(M1) gangliosidosis patient fibroblasts.