Pontow S E, Blum J S, Stahl P D
Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
J Biol Chem. 1996 Nov 29;271(48):30736-40. doi: 10.1074/jbc.271.48.30736.
The macrophage mannose receptor specifically recognizes proteins and particles bearing mannose terminal oligosaccharide chains. In the present study, we examined the ability of newly synthesized receptor to bind ligand. Human monocyte-derived macrophages were pulse-labeled with [35S]Met and prepared for affinity chromatography on mannose-Sepharose. Mannose receptor in the flow-through and eluted fractions was detected by fluorography following immunoprecipitation and gel electrophoresis. Labeled mannose receptor was found exclusively in the nonbinding fraction until 10 min of chase. Following a 60-min chase, 67-86% of newly synthesized receptor was precipitated from the bound column fraction. The half-time for development of receptor binding activity was determined to be 35-40 min compared with a 45-min half-time for development of endoglycosidase H resistance. Mannose receptor synthesized by cells incubated in brefeldin A required more than 120 min to acquire endoglycosidase H resistance and maximal binding activity. Inhibitors of N-linked oligosaccharide processing or of O-glycosylation had no effect on the development of mannose receptor binding activity. Monensin prevented terminal sialylation of oligosaccharide side chains but did not inhibit receptor activation. Inclusion of aluminum fluoride in the chase media reversibly inhibited development of endoglycosidase H resistance and mannose-binding activity. We conclude that the mannose receptor undergoes delayed activation following synthesis and suggest that the activating event(s) occur following exit of the receptor from the endoplasmic reticulum and prior to its entry into the trans-Golgi.
巨噬细胞甘露糖受体可特异性识别带有甘露糖末端寡糖链的蛋白质和颗粒。在本研究中，我们检测了新合成的受体结合配体的能力。用人单核细胞衍生的巨噬细胞进行[35S]甲硫氨酸脉冲标记，并制备用于甘露糖-琼脂糖亲和层析。通过免疫沉淀和凝胶电泳后的荧光自显影检测流过部分和洗脱部分中的甘露糖受体。在追踪10分钟之前，标记的甘露糖受体仅在非结合部分被发现。追踪60分钟后，67-86%新合成的受体从结合柱部分沉淀出来。受体结合活性发展的半衰期确定为35-40分钟，而内切糖苷酶H抗性发展的半衰期为45分钟。在布雷菲德菌素A中孵育的细胞合成的甘露糖受体需要超过120分钟才能获得内切糖苷酶H抗性和最大结合活性。N-连接寡糖加工或O-糖基化的抑制剂对甘露糖受体结合活性的发展没有影响。莫能菌素可阻止寡糖侧链的末端唾液酸化，但不抑制受体激活。在追踪培养基中加入氟化铝可可逆地抑制内切糖苷酶H抗性和甘露糖结合活性的发展。我们得出结论，甘露糖受体在合成后经历延迟激活，并表明激活事件发生在受体从内质网排出后、进入反式高尔基体之前。
