Fischer H D, Creek K E, Sly W S
J Biol Chem. 1982 Sep 10;257(17):9938-43.
We have purified phosphomannosyl-enzyme receptors from bovine liver on an affinity column composed of glycoproteins isolated from Dictyostelium discoideum secretions. Binding of human fibroblast beta-hexosaminidase B to receptors reconstituted into phosphatidylcholine liposomes was 1) specifically inhibited by mannose 6-phosphate, but not mannose 1-phosphate or glucose 6-phosphate, and 2) had properties similar to the previously reported binding of enzyme to receptors on cell surfaces and isolated membranes. In order to determine the structural features of the phosphomannosyl recognition marker required for receptor recognition, we covalently coupled purified receptor to an agarose gel bead support for affinity chromatography of phosphorylated, high mannose-type oligosaccharides isolated from fibroblast secretions radiolabeled with [2-3H]mannose. Neutral oligosaccharides and oligosaccharides containing one or two phosphates in phosphodiester linkage were not retained by the receptor column. By contrast, oligosaccharides bearing one phosphomonoester moiety were retarded on the column; those bearing two phosphomonoesters were bound to the column and were eluted with 10 mM mannose 6-phosphate. The binding of the oligosaccharides to the immobilized receptor correlates with their ability to be pinocytosed by fibroblasts and shows that the preferred recognition marker for the phosphomannosyl-enzyme receptor is a high mannose-type oligosaccharide chain bearing two uncovered phosphomannosyl groups.
我们在由从盘基网柄菌分泌物中分离的糖蛋白组成的亲和柱上，从牛肝中纯化了磷酸甘露糖基酶受体。人成纤维细胞β-己糖胺酶B与重构到磷脂酰胆碱脂质体中的受体的结合：1）被6-磷酸甘露糖特异性抑制，但不被1-磷酸甘露糖或6-磷酸葡萄糖抑制；2）具有与先前报道的酶与细胞表面和分离膜上受体的结合相似的特性。为了确定受体识别所需的磷酸甘露糖基识别标记的结构特征，我们将纯化的受体共价偶联到琼脂糖凝胶珠载体上，用于对从用[2-³H]甘露糖放射性标记的成纤维细胞分泌物中分离的磷酸化高甘露糖型寡糖进行亲和层析。中性寡糖和含有磷酸二酯键的一两个磷酸的寡糖不被受体柱保留。相比之下，带有一个磷酸单酯部分的寡糖在柱上被滞留；带有两个磷酸单酯的寡糖与柱结合，并用10 mM 6-磷酸甘露糖洗脱。寡糖与固定化受体的结合与其被成纤维细胞胞饮的能力相关，表明磷酸甘露糖基酶受体的首选识别标记是带有两个未被覆盖的磷酸甘露糖基的高甘露糖型寡糖链。
