DNA-BINDING TO HUMAN-LEUKOCYTES - EVIDENCE FOR A RECEPTOR-MEDIATED ASSOCIATION, INTERNALIZATION, AND DEGRADATION OF DNA
DNA-BINDING TO HUMAN-LEUKOCYTES - EVIDENCE FOR A RECEPTOR-MEDIATED ASSOCIATION, INTERNALIZATION, AND DEGRADATION OF DNA
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DOI：
10.1172/jci112226
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发表时间：
1985-12-01
影响因子：
15.9
通讯作者：
MERRITT, MM
中科院分区：
文献类型：
--
作者：
BENNETT, RM;GABOR, GT;MERRITT, MM
以前的研究表明，白色血细胞的外膜上有DNA。在这项研究中，我们着手确定外源DNA是否以与配体受体结合相容的方式与细胞结合。将纯化的白色血细胞群、中性粒细胞（多形核白细胞，PMN）、粘附单核细胞（ADMC）、玫瑰花结淋巴细胞（E+细胞）和非玫瑰花结淋巴细胞（E-细胞）与放射性标记的λ噬菌体DNA以递增浓度孵育。[~ 3 H]DNA的结合是一个饱和的过程，过冷的DNA和预先用胰蛋白酶消化细胞会抑制[~ 3 H]DNA的结合。纯化的细胞膜制备物的速率区带密度离心证实DNA与细胞外表面结合。所有四种细胞类型的解离常数为. apprx。10-9 M，从0.81 × 10 - 4到0.81 × 10 - 4。103至2.6 ×103个λ噬菌体DNA分子与每个细胞结合，这取决于细胞类型。结合不被RNA、聚脱氧腺苷酸-聚脱氧胸苷酸（poly[d（A）. d（T）]）或单核苷酸。将十二烷基硫酸钠（SDS）聚丙烯酰胺凝胶电泳（PAGE）从PMN、ADMC、E+和E-细胞中分离的蛋白质在硝酸纤维素片上进行电泳印迹;生物素标记的DNA探针表明，在分子量为30，000的位置迁移了一种DNA结合分子。同位素和免疫荧光研究表明，DNA被内化并降解为寡核苷酸，这一过程被放线菌酮抑制。这些结果支持这样的观点，即在白色血细胞上存在DNA的共同结合位点，该结合的化学计量与配体受体关系相容，并且这是负责外源DNA的内吞作用和降解的明显受体。
Previous studies have indicated that white blood cells possess DNA on their outer membranes. In this study we set out to determine whether exogenous DNA bound to cells in a fashion compatible with a ligand receptor union. Purified populations of white blood cells; neutrophils (polymorphonuclear leukocytes, PMN), adherent mononculear cells (ADMC), rosetting lymphocytes (E+ cells), and nonrosetting lymphocytes (E-cells) were incubated with radiolabeled lambda phage DNA in increasing concentrations. Binding of [3H]DNA was saturable process and was inhibited by excess cold DNA and prior trypsinization of the cells. Rate zonal density centrifugation of purified cell membrane preparations confirmed that DNA was binding to the outer cell surface. The dissociation constant for all four cell types was .apprx. 10-9 M, and from 0.81 .times. 103 to 2.6 .times. 103 molecules of lambda phage DNA bound to each cell depending upon cell type. Binding was not competitively inhibited by RNA, polydeoxyadenylic acid-polydeoxythymidylic acid (poly[d(A) .cntdot. d(T)]), or mononucleotides. Sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE)-separated proteins from PMN, ADMC, E+, and E-cells were electrophoretically blotted onto nitrocellulose sheets; a probe of biotin-labeled DNA indicated a single species of DNA-binding molecule migrating in a position consistent with a molecular weight of 30,000. Isotopic and immunofluorescent studies indicate that DNA is internalized and degraded to oligonucleotides; this process is inhibited by cycloheximide. These results support the notion that there is a common binding site for DNA on white blood cells, that the stoichiometry of the association is compatible with a ligand receptor relationship, and that this is apparent receptor is responsible for the endocytosis and degradation of exogenous DNA.