Wang Yunling, Norgård Maria, Andersson Göran
Division of Pathology, Department of Laboratory Medicine, Karolinska Institutet, Karolinska University Hospital, S-141 86 Huddinge, Sweden.
Arch Biochem Biophys. 2005 Mar 1;435(1):147-56. doi: 10.1016/j.abb.2004.11.029.
Purple acid phosphatase (PAP), also known as tartrate-resistant acid phosphatase or uteroferrin, contains two potential consensus N-glycosylation sites at Asn(97) and Asn(128). In this study, endogenous rat bone PAP was found to possess similar N-glycan structures as rat recombinant PAP heterologously expressed in baculovirus-infected Sf9 insect cells. PAP from Sf9 cells was shown to contain two N-linked oligosaccharides, whereas PAP expressed by mammalian CHO-K1 cells was less extensively glycosylated. The extent of N-glycosylation affected the catalytic properties of the enzyme, as N97Q and N128Q mutants, containing a single oligosaccharide chain, exhibited a lower substrate affinity and catalytic activity compared to those of the fully glycosylated PAP in the native, monomeric state. The differences in substrate affinity and catalytic activity were abolished and partially restored, respectively, by proteolytic cleavage in the loop domain, indicating that the extent of N-glycosylation influences the interaction of the repressive loop domain with catalytically important residues.
紫色酸性磷酸酶（PAP），也称为抗酒石酸酸性磷酸酶或子宫铁蛋白，在天冬酰胺（97）和天冬酰胺（128）处含有两个潜在的共有N-糖基化位点。在本研究中，发现内源性大鼠骨PAP具有与在杆状病毒感染的Sf9昆虫细胞中异源表达的大鼠重组PAP相似的N-聚糖结构。来自Sf9细胞的PAP显示含有两个N-连接的寡糖，而由哺乳动物CHO-K1细胞表达的PAP糖基化程度较低。N-糖基化的程度影响了该酶的催化特性，因为含有单个寡糖链的N97Q和N128Q突变体与天然单体状态下完全糖基化的PAP相比，表现出较低的底物亲和力和催化活性。通过在环结构域中的蛋白水解切割分别消除并部分恢复了底物亲和力和催化活性的差异，表明N-糖基化的程度影响了抑制性环结构域与催化重要残基的相互作用。
