Wang W C, Lee N, Aoki D, Fukuda M N, Fukuda M
La Jolla Cancer Research Foundation, Cancer Research Center, California 92037.
J Biol Chem. 1991 Dec 5;266(34):23185-90.
The poly-N-acetyllactosamines on neutrophils and monocytes have been shown to serve as ligands for various selectins present on endothelial cells and platelets. We have previously shown that only a limited number of glycoproteins contain poly-N-acetyllactosamine and found that lysosomal membrane glycoproteins (lamps) are the major glycoproteins carrying poly-N-acetyllactosamine. In order to understand the reason why only certain glycoproteins can be modified by poly-N-acetyllactosamine, we have utilized 21 degrees C incubation conditions, which were previously shown to cause the accumulation of glycoproteins at the trans-Golgi. HL-60 cells were labeled with [3H]galactose at 21 or 37 degrees C for 6 or 24 h, and lamp-1 and lamp-2 were immunoprecipitated. Upon examination by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, each lamp from HL-60 cells incubated at 21 degrees C exhibited a much broader, slower migrating band than that isolated from the cells incubated at 37 degrees C. The number of N-glycans containing poly-N-acetyllactosamine, estimated by their binding to tomato lectin column, increased approximately 30-50% after incubation at 21 degrees C than incubation at 37 degrees C. The analysis of oligosaccharides released by endo-beta-galactosidase digestion demonstrates that the amount of side chains containing three or more N-acetyllactosamine repeats increased about 100% after incubation at 21 degrees C, and methylation analysis confirmed these results. The same analysis and the results obtained by ion-exchange chromatography also provided evidence that the N-glycans of lamps are sialylated at 21 degrees C as much as at 37 degrees C. Pulse-chase experiments using [35S]methionine labeling indicated that the time necessary for processing of lamps is much longer at 21 degrees C than at 37 degrees C. These results therefore indicate that incubation at 21 degrees C causes the lamps to reside longer within the Golgi complex, and such longer residence allows lamps to acquire more polylactosaminoglycan. These results also suggest that the time necessary for moving through the Golgi complex is a critical factor for poly-N-acetyllactosamine formation.
中性粒细胞和单核细胞上的多聚N-乙酰乳糖胺已被证明可作为内皮细胞和血小板上各种选择素的配体。我们之前已表明只有有限数量的糖蛋白含有多聚N-乙酰乳糖胺，并发现溶酶体膜糖蛋白（LAMPs）是携带多聚N-乙酰乳糖胺的主要糖蛋白。为了理解为何只有某些糖蛋白能被多聚N-乙酰乳糖胺修饰，我们采用了21℃的孵育条件，此前已表明该条件会导致糖蛋白在反式高尔基体中积累。HL-60细胞在21℃或37℃下用[³H]半乳糖标记6或24小时，然后免疫沉淀LAMP-1和LAMP-2。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳检测，在21℃孵育的HL-60细胞中的每种LAMP都呈现出比在37℃孵育的细胞中分离出的条带更宽、迁移更慢的条带。通过与番茄凝集素柱结合估计，含有多聚N-乙酰乳糖胺的N-聚糖数量在21℃孵育后比在37℃孵育后增加了约30 - 50%。对经内切β-半乳糖苷酶消化释放的寡糖的分析表明，含有三个或更多N-乙酰乳糖胺重复序列的侧链数量在21℃孵育后增加了约100%，甲基化分析证实了这些结果。相同的分析以及离子交换色谱获得的结果也提供了证据，表明LAMPs的N-聚糖在21℃和37℃下的唾液酸化程度相同。使用[³⁵S]甲硫氨酸标记的脉冲追踪实验表明，在21℃下加工LAMPs所需的时间比在
