Isolation and characterization of complement receptor type 1 (CR1) storage vesicles from human neutrophils using antibodies to the cytoplasmic tail of CR1
Isolation and characterization of complement receptor type 1 (CR1) storage vesicles from human neutrophils using antibodies to the cytoplasmic tail of CR1
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DOI：
10.1182/blood.v89.12.4555
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发表时间：
1997-06-15
期刊：
影响因子：
20.3
通讯作者：
Berger, M
中科院分区：
文献类型：
--
作者：
Kumar, A;Wetzler, E;Berger, M
中性粒细胞（PMN）活化与从细胞内库易位的几种膜蛋白的表面表达增加有关，间接证据表明，静息PMN中补体受体1型（CR 1）的细胞内储存库不同于传统颗粒，并且可能是其中也储存白蛋白的分泌囊泡，但尚不清楚该隔室是同质的还是异质的，为了分离和表征含CR 1的囊泡，我们使用了针对CR 1胞质尾区中独特序列的抗体。亲和纯化的IgG被用来吸附CR 1存储囊泡从轻膜部分（γ-带）的氮空泡的休息中性粒细胞。免疫吸附剂可以定量地去除含CR 1的囊泡，而非免疫IgG的对照吸附剂没有显示出CR 1的特异性结合。特异性分离的囊泡的免疫印迹也显示白蛋白、衰变加速因子、Fc γ RIII和CR 3的富集;而HLA I类在这些囊泡中不可检测。用Triton X-100处理后特异性分离的囊泡的酶测定表明，这些囊泡含有大部分细胞的潜在碱性磷酸酶。还鉴定了含有白蛋白但不含CR 1且不与抗CR 1吸附剂结合的囊泡的额外群体。免疫电子显微镜显示，特别是分离的囊泡的平均直径为0.086至0.1 μ m和染色阳性的CR 1和白蛋白，这些结果表明，CR 1存储囊泡可以分离与抗体对CR 1的胞质尾，并显示这些囊泡也含有白蛋白以及糖基磷脂酰肌醇锚定蛋白。这些结果是最兼容的假设，即CR 1含有囊泡已经出现了内吞检索的蛋白质，已在质膜上。(C)1997年，美国血液学会。
Neutrophil (PMN) activation is associated with increased surface expression of several membrane proteins that are translocated from intracellular pools, Indirect evidence suggests that the intracellular storage pools of complement receptor type 1 (CR1) in resting PMN are distinct from traditional granules and may be the secretory vesicles in which albumin is also stored, but it is not known if this compartment is homogeneous or heterogeneous, To isolate and characterize the CR1-containing vesicles, we used antibodies against unique sequences in the cytoplasmic tail of CR1. Affinity-purified IgG was used to adsorb CR1 storage vesicles from the light membrane fraction (gamma-band) of nitrogen cavitates of resting PMN. The immunoadsorbent could quantitatively remove the CR1-containing vesicles, whereas control adsorbents with nonimmune IgG showed no specific binding of CR1. Immunoblots of specifically isolated vesicles also showed enrichment of albumin, decay-accelerating factor, Fc gamma RIII, and CR3; whereas HLA class I was not detectable in these vesicles. Enzyme assay of specifically isolated vesicles after treatment with Triton X-100 showed that these vesicles contained most of the cells' latent alkaline phosphatase. An additional population of vesicles containing albumin, but not CR1, and that did not bind to anti-CR1 adsorbent was also identified. Immunoelectron microscopy showed that the specifically isolated vesicles had mean diameters of 0.086 to 0.1 mu m and stained positive for CR1 and albumin, These results indicate that CR1 storage vesicles can be isolated with antibodies against the cytoplasmic tail of CR1 and show that these vesicles also contain albumin as well as glycosyl-phosphatidyl inositol-anchored proteins. These results are most compatible with the hypothesis that CR1-containing vesicles have arisen by endocytic retrieval of proteins that had been on the plasma membrane. (C) 1997 by The American Society of Hematology.