The significance of glycosylation on the function of the calcium-sensing receptor (CaSR) dataset

This dataset is an outcome of a series of experiments conducted to investigate the significance of glycosylation on the function of the human calcium-sensing receptor (CaSR). The CaSR is a family C G protein-coupled receptor that plays an important role in regulating whole body extracellular calcium (Ca2+o) homeostasis.  The exact location of the binding sites for Ca2+o (and other cations) is still unknown, but experimental and in silico analysis has provided evidence that they may be in the extracellular domain and the transmembrane domain. Post translational modifications leads to the covalent attachment of a number of negatively charged sugars, which may provide a negatively charged surface for polyvalent cations to interact with.

There are 11 potential N-linked glycosylation sites in the CaSR’s extracellular domain.  A single conservative asparagine to glutatmine mutation was undertaken to disrupt individual N-linked glycosylation consensus sequences.  Alanine scanning mutations were also used to assess the significance of potential O-linked glycosylation sites (small clusters of Ser/The residues near the membrane anchor points of the exo-loops in the transmembrane domain).  These mutant receptors were then transiently expressed in HEK293 cells. The impact of each mutation on cell expression and function was assessed by the following techniques:

Western blotting: Hand cast SDS-PAGE gel and Membrane transfer was undertaken using the BioRad mini-protean system, Precast gradient gels (4 – 15%) were TGX precast gels from Biorad. Western blotting films were scanned in .JPG and .PNG format. Quantification of bands was undertaken using GNU Imaging Manipulation Program (GIMP) imaging software.

Cell surface expression ELISAs: Antibody based enzyme-linked immunosorbent assays (ELISAs) were used. The chromogenic substrate for horseradish peroxidase (HRP) was 3,3’,5,5’-Tetramethylbenzidine (TMB). Absorbance at 450 nM was read using an EnVision Multilabel Reader and exported using .XLS files. Data were represented and analysed using GraphPad Prism 5.

Microfluorimetry with fura-2 AM (a calcium-sensitive fluorophore): Fluorescent images were taken on a Zeiss Axiovert 200M microscope with a Carl Zeiss monochrome HSm digital camera using a 63x Zeiss long-distance objective. Excitation filters, emission filters and dichroic mirrors were all obtained from Zeiss. The fluorescent light source was provided by a Lambda Sutter DG-4 wavelength switcher. The ratiometric (340/380 nm) region-of-interest data were exported using Stallion software (.SLD files) and subsequently processed using GraphPad Prism 5.0.

The creation of the mutant cells and expression/function experiments were carried out by Dr Sarah Brennan and Karolina Windloch. Data analysis was undertaken by Dr Sarah Brennan. For further information, refer to the associated thesis.

本数据集为一系列实验的研究成果，旨在探究糖基化（glycosylation）对人类钙敏感受体（CaSR）功能的调控意义。CaSR属于C类G蛋白偶联受体（family C G protein-coupled receptor），在调节全身细胞外钙（Ca²⁺ₒ）稳态中发挥关键作用。目前，Ca²⁺ₒ（及其他阳离子）结合位点的确切位置仍未明确，但实验与计算机模拟（in silico）分析已证实，其结合位点可能位于细胞外结构域与跨膜结构域中。翻译后修饰会共价结合多种带负电的糖类，进而为多价阳离子与受体的相互作用提供带负电的结合表面。

CaSR的细胞外结构域中共存在11个潜在的N-连接糖基化位点。本研究通过单个保守的天冬酰胺→谷氨酰胺突变，以破坏单个N-连接糖基化共有序列；同时采用丙氨酸扫描突变技术，评估跨膜结构域外袢膜锚定位点附近丝氨酸/苏氨酸残基小簇所对应的潜在O-连接糖基化位点的功能重要性。随后将上述突变受体在人胚胎肾293（HEK293）细胞中进行瞬时表达。本研究通过以下技术手段评估各突变对受体细胞表达与功能的影响：

1. 蛋白质印迹（Western blotting）：采用伯乐（BioRad）微型蛋白电泳系统进行手工灌制十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）凝胶与膜转印实验，同时使用伯乐生产的TGX预制梯度凝胶（4%–15%）。蛋白质印迹胶片以JPG与PNG格式进行扫描，条带定量分析采用GNU图像处理程序（GIMP）完成。

2. 细胞表面表达酶联免疫吸附试验（ELISA）：本实验采用基于抗体的酶联免疫吸附试验。辣根过氧化物酶（HRP）的生色底物为3,3’,5,5’-四甲基联苯胺（TMB），以EnVision多标记读板仪读取450 nm处的吸光度值，结果以.XLS文件导出；数据通过GraphPad Prism 5软件进行可视化与统计分析。

3. 搭载钙敏感性荧光探针fura-2 AM的微荧光法：荧光成像在蔡司Axiovert 200M显微镜下完成，搭配蔡司单色HSm数码相机，使用63倍蔡司长工作距离物镜。激发滤光片、发射滤光片与二向色镜均购自蔡司，荧光光源由Lambda Sutter DG-4波长切换器提供。采用比率法（340/380 nm）采集的感兴趣区域数据通过Stallion软件导出为.SLD文件，后续使用GraphPad Prism 5.0进行数据处理。

突变细胞的构建与表达/功能实验由Sarah Brennan博士与Karolina Windloch完成，数据分析由Sarah Brennan博士负责。如需进一步了解相关信息，请参阅关联学位论文。