Shipley J M, Grubb J H, Sly W S
Edward A. Doisy Department of Biochemistry and Molecular Biology, St. Louis University School of Medicine, Missouri 63104.
J Biol Chem. 1993 Jun 5;268(16):12193-8.
Phosphorylation of mannose residues on N-linked oligosaccharide side chains of lysosomal enzymes targets them to lysosomes. We used site-directed mutagenesis to observe the effect of eliminating selective glycosylation sites from human beta-glucuronidase on enzyme sorting. Expression studies allowed us to determine which of four potential sites were glycosylated, preferentially phosphorylated, and required for catalytic activity. All four sites of the human enzyme were glycosylated, whereas in the mouse and rat enzymes, only three of four sites are used. Sites 2 and 3 were preferentially phosphorylated. Elimination of sites 2 and 3 in combination markedly decreased sorting to lysosomes and increased enzyme secretion. Each of the four glycosylation sites could be eliminated individually without drastic reduction in catalytic activity. Activity was progressively lost as combinations of two, three, and four sites were eliminated. Wild-type enzyme produced in the presence of tunicamycin was also inactive, indicating that glycosylation is required for realization of enzyme activity. However, active enzyme could be deglycosylated with only minimal loss of activity. Mutant enzyme completely lacking glycosylation did not form tetramers. Partial restoration of tetramerization was achieved by the co-expression of normal rat enzyme, provided that the normal rat enzyme supplied at least two subunits to the tetramer.
溶酶体酶N - 连接寡糖侧链上的甘露糖残基磷酸化可将它们靶向至溶酶体。我们利用定点诱变来观察消除人β - 葡萄糖醛酸酶的选择性糖基化位点对酶分选的影响。表达研究使我们能够确定四个潜在位点中哪些被糖基化、优先磷酸化以及对催化活性是必需的。人酶的所有四个位点都被糖基化，而在小鼠和大鼠的酶中，四个位点中只有三个被利用。位点2和3优先被磷酸化。同时消除位点2和3显著降低了向溶酶体的分选并增加了酶的分泌。四个糖基化位点中的每一个都可以单独消除而不会使催化活性急剧降低。随着两个、三个和四个位点组合被消除，活性逐渐丧失。在衣霉素存在下产生的野生型酶也无活性，表明糖基化是实现酶活性所必需的。然而，活性酶可以去糖基化，且活性仅有最小程度的丧失。完全缺乏糖基化的突变酶不形成四聚体。通过共表达正常大鼠酶可部分恢复四聚化，前提是正常大鼠酶为四聚体提供至少两个亚基。
