PURIFICATION AND CHARACTERIZATION OF HUMAN LYSOSOMAL MEMBRANE-GLYCOPROTEINS
PURIFICATION AND CHARACTERIZATION OF HUMAN LYSOSOMAL MEMBRANE-GLYCOPROTEINS
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DOI：
10.1016/0003-9861(89)90597-3
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发表时间：
1989-01-01
影响因子：
3.9
通讯作者：
AUGUST, JT
中科院分区：
文献类型：
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作者：
MANE, SM;MARZELLA, L;AUGUST, JT
.apprx 的两种人类细胞溶酶体膜糖蛋白。 120 kDa、hLAMP-1 和 hLAMP-2 通过使用针对 U937 骨髓单核细胞白血病细胞或血液单核细胞制备的单克隆抗体进行鉴定。通过抗体亲和层析纯化这两种糖蛋白，并且发现每种糖蛋白都是人脾细胞的主要成分，代表.apprx。总洗涤剂可萃取蛋白质的 0.05%。两种分子均高度糖基化，合成为 40 至 45 kDa 的多肽，并通过添加 Asn 连接的寡糖进行共翻译修饰。 NH2-末端序列分析表明每个都是.apprx。与小鼠细胞相应的 mLAMP-1 或 mLAMP-2 50% 相同。对人血单核细胞、HL-60 和 U937 细胞的电子显微镜研究表明，这些糖蛋白的主要位置在细胞内、液泡和溶酶体结构中，但不在单核细胞的过氧化物酶阳性颗粒中。蛋白质在细胞器之间的运输通过它们在吞噬溶酶体膜中的显着积累来证明。在 U937 和 HL-60 细胞的质膜上也检测到了每种糖蛋白的一部分，但在多种其他组织培养细胞上未检测到。这种细胞表面表达可能与分化相关，因为在正常血液单核细胞的质膜中未检测到这些蛋白质，并且当用分化剂处理细胞时，它们在 U937 和 HL-60 细胞上的表达降低。在暴露于溶酶体试剂盐酸甲胺后，两种糖蛋白的细胞表面表达在血液单核细胞中显着增加，但在 U937 细胞中没有增加，表明这些细胞类型中 LAMP 相关膜流的差异。
Two human cell lysosomal membrane glycoproteins of .apprx. 120 kDa, hLAMP-1 and hLAMP-2, were identified by use of monoclonal antibodies prepared against U937 myelomonocytic leukemia cells or blood mononuclear cells. The two glycoproteins were purified by antibody affinity chromatography and each was found to be a major constituent of human spleen cells, representing .apprx. 0.05% of the total detergent-extractable protein. Both molecular were highly glycosylated, being synthesized as polypeptides of 40 to 45 kDa and cotranslationally modified by the addition of Asn-linked oligosaccharides. NH2-terminal sequence analysis indicated that each was .apprx. 50% identical to the corresponding mLAMP-1 or mLAMP-2 of mouse cells. Electron microscopic studies of human blood monocytes, HL-60, and U937 cells demonstrated that the principal location of these glycoproteins was intracellular, in vacuoles and lysosomal structures but not in the peroxidase-positive granules of monocytes. Transport of the proteins between organelles was evidenced by their marked accumulation in the membranes of phagolysosomes. A fraction of each glycoprotein was also detected on the plasma membranes of U937 and HL-60 cells but not on a variety of other tissue culture cells. This cell-surface expression may be differentiation related, since the proteins were not detected in the plasma membrane of normal blood monocytes and their expression on U937 and HL-60 cells was reduced when the cells were treated with differentiating agents. Cell-surface expression of both glycoproteins was markedly increased in blood monocytes but not in U937 cells after exposure to the lysosomotropic reagent methylamine HCl, indicating differences in LAMP-associated membrane flow in these cell types.