Identification of a novel, dendritic cell-associated molecule, dectin-1, by subtractive cDNA cloning
Identification of a novel, dendritic cell-associated molecule, dectin-1, by subtractive cDNA cloning
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DOI：
10.1074/jbc.m909512199
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发表时间：
2000-06-30
影响因子：
4.8
通讯作者：
Takashima, A
中科院分区：
文献类型：
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作者：
Ariizumi, K;Shen, GL;Takashima, A
树突状细胞（DC）是抗原呈递细胞的特殊亚群，其特点是具有激活免疫幼稚T细胞的强大能力。通过将小鼠表皮源性DC细胞系XS52表达的mrna与巨噬细胞系J774表达的mrna相减，我们鉴定出了该DC细胞系选择性表达的5个新基因。其中一个基因编码了一个由244个氨基酸组成的II型膜整合多肽，在cooh末端含有一个假定的碳水化合物识别结构域基序。这种分子被称为“dectin-1”，在XS52 DC细胞系中mRNA和蛋白水平都大量表达，而在非DC细胞系（包括J774巨噬细胞系）中则不表达。Dectin-1 mRNA主要在脾脏和胸腺（通过Northern blotting）以及皮肤DC细胞，即朗格汉斯细胞（通过逆转录-聚合酶链反应）中检测到。亲和纯化的抗dectin-1抗体在XS52 DC细胞株和dectin-1转染的COS-1细胞的膜中鉴定出一个43 kda的糖蛋白。含有dectin-1细胞外结构域的his标记重组蛋白在抗cd3单克隆抗体存在的亚理想浓度下，与T细胞表面特异性结合，促进T细胞增殖。这些体外结果表明DC上的dectin-1可能与T细胞上尚未定义的配体结合，从而传递T细胞共刺激信号。这些结果不仅证明了减法cDNA克隆对鉴定DC表达的独特基因的有效性，也为研究dectin-1的生理功能提供了框架。
Dendritic cells (DC) are special subsets of antigen presenting cells characterized by their potent capacity to activate immunologically naive T cells. By subtracting the mRNAs expressed by the mouse epidermus-derived DC line XS52 with the mRNAs expressed by the J774 macrophage line, we identified five novel genes that were expressed selectively by this DC line. One of these genes encoded a type II membrane-integrated polypeptide of 244 amino acids containing a putative carbohydrate recognition domain motif at the COOH-terminal end. This molecule, termed "dectin-1," was expressed abundantly at both mRNA and protein levels by the XS52 DC line, but not by non-DC lines (including the J774 macrophage line). Dectin-1 mRNA was detected predominantly in spleen and thymus (by Northern blotting) and in skin-resident DC, i.e. Langerhans cells (by reverse transcription-polymerase chain reaction). Affinity-purified antibody against dectin-1 identified a 43-kDa glycoprotein in membrane fractions isolated from the XS52 DC line and from the dectin-1 cDNA-transfected COS-1 cells. His-tagged recombinant proteins containing the extracellular domains of dectin-1 showed marked and specific binding to the surface of T cells and promoted their proliferation in the presence of anti-CD3 monoclonal antibody at suboptimal concentrations. These in vitro results suggest that dectin-1 on DC may bind to as yet undefined ligand(s) on T cells, thereby delivering T cell co-stimulatory signals. Not only do these results document the efficacy of subtractive cDNA cloning for the identification of unique genes expressed by DC, they also provide a framework for studying the physiological function of dectin-1.