McAlister M S, Davis B, Pfuhl M, Driscoll P C
Department of Biochemistry and Molecular Biology, University College London, UK.
Protein Eng. 1998 Oct;11(10):847-53. doi: 10.1093/protein/11.10.847.
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 the three-dimensional polypeptide fold is as yet unknown. Glycosylated CD5 domain 1 (CD5d1) has been obtained by expression by secretion from both Chinese hamster ovary (CHO) cells and Pichia pastoris. Recombinant CD5d1 expressed in this manner was shown to be correctly folded by binding to anti-CD5 L17F12/Leu1 monoclonal antibody. Preliminary nuclear magnetic resonance (NMR) spectra obtained for CD5d1 (residues 1-118) had spectral dispersion typical of a folded protein, but otherwise of such poor quality that NMR structural studies were not feasible. The analysis of glycoproteins by NMR is frustrated by sample heterogeneity and poor spectral quality associated with glycan resonance overlap and the potential for increased line-widths due to the large hydrodynamic volume. In order to pursue NMR structural studies of CD5d1 it was necessary to optimize the quality of NMR spectra of CD5d1. A range of constructs of varying length and carbohydrate content were expressed in CHO cells and in P. pastoris. In addition the P. pastoris CD5d1 proved susceptible to N-glycan cleavage with endoglycosidase H. The protein products were characterised using size exclusion chromatography, NMR measurement of translational self-diffusion coefficients and two-dimensional 1H nuclear Overhauser effect spectroscopy experiments. Removal of an eight residue O-glycosylated C-terminal peptide, in particular, resulted in significant improvements in the quality of the CD5d1 NMR data, while retaining native protein structure. Two-dimensional heteronuclear NMR spectroscopy of nitrogen-15 isotope labelled deglycosylated CD5d1 (residues 1-110) prepared from P. pastoris suggests that this protein product is now amenable to solution structure determination.
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，目前尚不清楚其三维多肽折叠结构。糖基化的CD5结构域1（CD5d1）已通过中国仓鼠卵巢（CHO）细胞和巴斯德毕赤酵母分泌表达获得。以这种方式表达的重组CD5d1通过与抗CD5 L17F12/Leu1单克隆抗体结合显示已正确折叠。为CD5d1（第1 - 118位氨基酸残基）获得的初步核磁共振（NMR）谱具有折叠蛋白典型的谱分散，但质量很差，以至于无法进行NMR结构研究。由于样品异质性以及与聚糖共振重叠相关的差的谱质量，以及由于大的流体动力学体积导致线宽增加的可能性，通过NMR分析糖蛋白受到阻碍。为了进行CD5d1的NMR结构研究，有必要优化CD5d1的NMR谱质量。在CHO细胞和巴斯德毕赤酵母中表达了一系列不同长度和碳水化合物含量的构建体。此外，巴斯德毕赤酵母表达的CD5d1被证明对内切糖苷酶H敏感，可进行N - 聚糖切割。使用尺寸排阻色谱、平移自扩散系数的NMR测量和二维1H核Overhauser效应光谱实验对蛋白质产物进行了表征。特别是去除一个八残基的O - 糖基化C末端肽，在保留天然蛋白质结构的同时，显著提高了CD5d1 NMR数据的质量。由巴斯德毕赤酵母制备的氮 - 15同位素标记的去糖基化CD5d1（第1 - 110位氨基酸残基）的二维异核NMR光谱表明，这种蛋白质产物现在适合进行溶液结构测定。
