McAlister M S, Brown M H, Willis A C, Rudd P M, Harvey D J, Aplin R, Shotton D M, Dwek R A, Barclay A N, Driscoll P C
Department of Biochemistry and Molecular Biology, University College London, UK.
Eur J Biochem. 1998 Oct 1;257(1):131-41. doi: 10.1046/j.1432-1327.1998.2570131.x.
CD5 is a type-I transmembrane glycoprotein found on thymocytes, T-cells and a subset of B-cells. The extracellular region consists of three domains belonging to the scavenger receptor cysteine-rich (SRCR) superfamily, for which no three-dimensional structure has been obtained. Recombinant soluble CD5 domain 1 (CD5d1), the N-terminal SRCR domain, has been expressed in both chinese hamster ovary (CHO) cells and Pichia pastoris. CD5d1 was shown to be correctly folded by binding to the CD5 monoclonal antibody Leul. Circular dichroism and NMR analyses indicate that CD5d1 has a high beta-sheet content. CD5d1 from both CHO cells and P. pastoris have very similar properties. The disulphide bonding pattern was determined and is consistent with that found for the group-A SRCR domain of type-1 macrophage scavenger receptor and MARCO, the macrophage receptor with collagenous structure. Observations have been made of the role of glycosylation of CD5. P. pastoris expression provides large quantities of correctly folded recombinant CD5d1 for multidimensional NMR and for X-ray crystallographic studies. The whole extracellular region of CD5, expressed as a chimaera with rat CD4 domains 3 and 4 (cCD5d1-3-CD4d3+4), was studied by electron microscopy and carbohydrate analysis to gain an overview of the structure of the extracellular portion of intact CD5. Carbohydrate analysis identified N-linked glycans on CD5 domains 1 and 2, and sialylated O-linked glycans on the linker peptide between domains 1 and 2. Electron microscopy and carbohydrate analysis together suggest that the extracellular region of CD5 forms a rod-like structure with domain 1 distal from the cell surface and separated from domains 2 and 3 by an O-glycosylated peptide linker region.
CD5是一种I型跨膜糖蛋白，存在于胸腺细胞、T细胞和一部分B细胞上。其细胞外区域由三个属于富含半胱氨酸清道夫受体（SRCR）超家族的结构域组成，目前尚未获得其三维结构。重组可溶性CD5结构域1（CD5d1），即N端SRCR结构域，已在中国仓鼠卵巢（CHO）细胞和巴斯德毕赤酵母中表达。通过与CD5单克隆抗体Leul结合，表明CD5d1折叠正确。圆二色性和核磁共振分析表明，CD5d1具有较高的β-折叠含量。来自CHO细胞和巴斯德毕赤酵母的CD5d1具有非常相似的特性。确定了二硫键连接模式，该模式与1型巨噬细胞清道夫受体和具有胶原结构的巨噬细胞受体MARCO的A组SRCR结构域一致。已经观察到CD5糖基化的作用。巴斯德毕赤酵母表达为多维核磁共振和X射线晶体学研究提供了大量正确折叠的重组CD5d1。通过电子显微镜和碳水化合物分析研究了与大鼠CD4结构域3和4形成嵌合体的CD5整个细胞外区域（cCD5d1-3-CD4d3+4），以全面了解完整CD5细胞外部分的结构。碳水化合物分析确定了CD5结构域1和2上的N-连接聚糖，以及结构域1和2之间连接肽上的唾液酸化O-连接聚糖。电子显微镜和碳水化合物分析共同表明，CD5的细胞外区域形成棒状结构，结构域1远离细胞表面，并通过O-糖基化肽连接区域与结构域2和3分开。
