Bezouska K, Nepovím A, Horváth O, Pospísil M, Hamann J, Feizi T
Department of Biochemistry, Faculty of Science, Charles University Prague, Czech Republic.
Biochem Biophys Res Commun. 1995 Mar 8;208(1):68-74. doi: 10.1006/bbrc.1995.1306.
CD69 is a signal transducing molecule of hematopoietic cells. Previous molecular cloning of CD69 has revealed a type II transmembrane orientation and the presence of an extracellular domain related to the Ca(2+)-dependent (C-type) animal lectins. As the predicted amino acid sequence for the lectin-like domain is highly divergent from those of other C-type lectin-like proteins - a feature shared with NKR-P1 of natural killer cells - CD69 and NKR-P1 are among proteins assigned to a separate group, group V. To initiate ligand identification studies, we have prepared soluble forms of CD69 protein by bacterial expression of its extracellular portion. We show that cysteine 68 located in the short membrane-proximal neck region of CD69 which adjoins the C-terminal lectin-like domain is a critical element for dimerization. We have evidence that the soluble dimeric CD69 has a tight association with calcium, a feature shared with NKR-P1, and that it is a carbohydrate-binding protein with N-acetyl-D-glucosamine and N-acetyl-D-galactosamine as the best inhibitors: 4-8 x 10(-5) M giving 50% inhibition of binding to N-acetyl-D-glucosamine neoglycoprotein. Thus, the tight association with calcium and high affinities for carbohydrate binding appear to be features of at least two members of the C-type lectin group V.
CD69是造血细胞的一种信号转导分子。先前对CD69的分子克隆揭示了其II型跨膜方向以及存在一个与钙依赖性（C型）动物凝集素相关的细胞外结构域。由于凝集素样结构域的预测氨基酸序列与其他C型凝集素样蛋白的序列高度不同——这一特征与自然杀伤细胞的NKR-P1相同——CD69和NKR-P1属于被归为一个单独组（V组）的蛋白质。为了启动配体鉴定研究，我们通过细菌表达CD69蛋白的细胞外部分制备了可溶性形式的CD69蛋白。我们发现，位于CD69短的膜近端颈部区域（毗邻C末端凝集素样结构域）的半胱氨酸68是二聚化的关键元件。我们有证据表明，可溶性二聚体CD69与钙紧密结合，这一特征与NKR-P1相同，并且它是一种碳水化合物结合蛋白，N-乙酰-D-葡萄糖胺和N-乙酰-D-半乳糖胺是最佳抑制剂：4 - 8×10⁻⁵ M时对与N-乙酰-D-葡萄糖胺新糖蛋白的结合产生50%的抑制。因此，与钙的紧密结合和对碳水化合物结合的高亲和力似乎是C型凝集素V组至少两个成员的特征。
