Qadri Abrar H, Prajapati Jyotsana, Faheem Iqball, Bhattacharjee Utsa, Padmanaban Hari Krishnan, Mulukala Sandeep Kn, Pasupulati Anil K
Department of Biochemistry, University of Hyderabad Hyderabad 500046, India.
Department of Microbiology and Cell Biology, Indian Institute of Science Bangalore 560012, India.
Int J Biochem Mol Biol. 2024 Apr 15;15(2):20-33. doi: 10.62347/UVSH8436. eCollection 2024.
Glomerular podocytes are specialized epithelial cells localized to the blood-urine interface of the kidney. Podocyte slit-diaphragm (SD), a size-and-charge-selective junction, is instrumental in blood ultrafiltration and the formation of protein-free urine. The SD consists of macromolecular complexes of several proteins, such as nephrin, podocin, and CD2-associated protein (CD2AP). CD2AP is an adapter protein and is considered to be crucial for the integrity of SD. Mutations in the SD proteins cause nephrotic syndrome (NS), characterized by proteinuria. SD proteins' structural features must be elucidated to understand the mechanism of proteinuria in NS. In this study, we expressed, purified, and biophysically characterized heterologously expressed human CD2AP.
Codon-optimized human CD2AP was expressed in Rosetta cells. The recombinant protein was induced with 1 mM IPTG and purified by Ni-NTA affinity chromatography. Analytical size-exclusion chromatography, blue native-PAGE, circular dichroism, and fluorescence spectroscopy were performed to decipher the oligomeric nature, secondary structural content, and tertiary packing of CD2AP.
Our analysis revealed that CD2AP adopts a predominantly disordered secondary structure despite exhibiting moderate tertiary packing, characterized by low helical and β-sheet content. CD2AP readily assembles into homo-oligomers, with octamers and tetramers constituting the primary population. Interestingly, the inherent flexibility of CD2AP's secondary structural elements appears resistant to thermal denaturation. Frameshift mutation (p.K579Efs*7) that leads to loss of the coiled-coil domain promotes aberrant oligomerization of CD2AP through SH3 domains.
We successfully expressed full-length human CD2AP in a heterologous system, wherein the secondary structure of CD2AP is predominantly disordered. CD2AP can form higher-order oligomers, and the significance of these oligomers and the impact of mutations in the context of size-selective permeability of SD needs further investigation.
肾小球足细胞是位于肾脏血液-尿液界面的特殊上皮细胞。足细胞裂孔隔膜（SD）是一种具有大小和电荷选择性的连接结构，在血液超滤和无蛋白尿液形成中起重要作用。SD由几种蛋白质的大分子复合物组成，如nephrin、podocin和CD2相关蛋白（CD2AP）。CD2AP是一种衔接蛋白，被认为对SD的完整性至关重要。SD蛋白的突变会导致以蛋白尿为特征的肾病综合征（NS）。必须阐明SD蛋白的结构特征，以了解NS中蛋白尿的机制。在本研究中，我们对异源表达的人CD2AP进行了表达、纯化及生物物理特性表征。
密码子优化的人CD2AP在Rosetta细胞中表达。重组蛋白用1 mM异丙基-β-D-硫代半乳糖苷（IPTG）诱导表达，并用镍-氮三乙酸（Ni-NTA）亲和层析法纯化。进行分析型尺寸排阻色谱、蓝色天然聚丙烯酰胺凝胶电泳、圆二色光谱和荧光光谱分析，以解析CD2AP的寡聚性质、二级结构含量和三级结构堆积。
我们的分析表明，尽管CD2AP表现出适度的三级结构堆积，但其二级结构主要为无序结构，螺旋和β折叠含量较低。CD2AP易于组装成同源寡聚体，八聚体和四聚体是主要群体。有趣的是，CD2AP二级结构元件的固有灵活性似乎使其对热变性具有抗性。导致卷曲螺旋结构域缺失的移码突变（p.K579Efs*7）通过Src同源3（SH3）结构域促进CD2AP的异常寡聚化。
我们在异源系统中成功表达了全长人CD2AP，其中CD2AP的二级结构主要为无序结构。CD2AP可形成高阶寡聚体，这些寡聚体的意义以及在SD大小选择性通透性背景下突变的影响需要进一步研究。
