LYSOSOME-ASSOCIATED MEMBRANE-PROTEINS - CHARACTERIZATION OF LAMP-1 OF MACROPHAGE-P388 AND MOUSE EMBRYO 3T3 CULTURED-CELLS
LYSOSOME-ASSOCIATED MEMBRANE-PROTEINS - CHARACTERIZATION OF LAMP-1 OF MACROPHAGE-P388 AND MOUSE EMBRYO 3T3 CULTURED-CELLS
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DOI：
10.1016/0003-9861(85)90727-1
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发表时间：
1985-01-01
影响因子：
3.9
通讯作者：
AUGUST, JT
中科院分区：
文献类型：
--
作者：
CHEN, JW;PAN, W;AUGUST, JT
溶酶体相关膜蛋白 (LAMP)-1 是小鼠胚胎 3T3 细胞的主要糖蛋白，与溶酶体膜特异性相关，在 P388 巨噬细胞中被鉴定，并与 NIH 3T3 细胞的同源糖蛋白进行比较。使用抗 LAMP-1 单克隆抗体的免疫荧光显微镜显示，抗原分布在整个 P388 细胞中，包括褶皱边缘或伪足，与吖啶橙积累的模式相同。使用 1D4B 单克隆抗体通过亲和层析从 P388 细胞中纯化 LAMP-1，产生均质糖蛋白，占去污剂提取的总细胞蛋白的 0.1%。 P388 LAMP-1 的表观质量为 130,000 至 150,000，而 3T3 糖蛋白的表观质量为 105,000 至 115,000。胰蛋白酶肽分析表明两种纯化的糖蛋白高度同源。通过用[35S]蛋氨酸进行脉冲追踪标记来分析多种细胞系中的蛋白质合成；在每种情况下，LAMP-1都是作为表观MW为92,000的前体合成的，然后转化为平均MW从110,000到140,000不同的异质成熟形式。通过对糖蛋白的生物合成和寡糖组成的研究，检验了这些明显的质量差异的基础。通过用糖苷内切酶 H 处理[35S]甲硫氨酸脉冲标记分子的免疫沉淀物，从 HaNIH 和 P388 细胞中获得 45,000 Da [道尔顿]的核心多肽。用莫能菌素处理的细胞含有 80,000 至 85,000 Da 的异质分子。神经氨酸酶处理显着降低了成熟LAMP-1的等电异质性，而对分子的表观MW或不同细胞系之间的差异几乎没有影响。糖胺聚糖合成的β-D-木糖苷抑制对LAMP-1的表观质量影响很小。
Lysosome-associated membrane protein (LAMP)-1, a major glycoprotein of mouse embryo 3T3 cells and specifically associated with the lysosomal membrane, was identified in P388 macrophage cells and compared with the homologous glycoprotein of NIH 3T3 cells. Immunofluorescence microscopy with anti-LAMP-1 monoclonal antibodies shows that the antigen was distributed throughout P388 cells including the ruffled edges or pseudopodia, identical to the pattern of acridine orange accumulation. LAMP-1 was purified from P388 cells by affinity chromotography with 1D4B monoclonal antibody, yielding a homogenous glycoprotein comprising 0.1% of the total detergent-extracted cell protein. The apparent mass of P388 LAMP-1 was 130,000 to 150,000 compared to the 3T3 glycoprotein of 105,000 to 115,000. Analysis of tryptic peptides indicated that the 2 purified glycoproteins were highly homologous. Protein synthesis was analyzed in a variety of cell lines by pulse-chase labeling with [35S]methionine; in every case, LAMP-1 was synthesized as a precursor of apparent MW 92,000, and then converted to heterogeneous mature forms differing in average MW from 110,000 to 140,000. The basis for these apparent differences in mass was examined by studies of the biosynthesis and oligosaccharide composition of the glycoprotein. Core polypeptides of 45,000 Da [dalton] were obtained from both HaNIH and P388 cells by treating immunoprecipitates of [35S]methionine pulse-labeled molecules with endoglycosidase H. Cells treated with monensin contained heterogeneous molecules of 80,000 to 85,000 Da. Isoelectric heterogeneity of mature LAMP-1 was markedly reduced by treatment with neuraminidase whereas there was little effect on the apparent MW of the molecules or the differences between the various cell lines. .beta.-D-Xyloside inhibition of glycosaminoglycan synthesis had little effect on the apparent mass of LAMP-1.