Ye Xuanqing, Wang Jiamin, Qiao Zilin, Yang Di, Wang Jiao, Abudureyimu Ayimuguli, Yang Kun, Feng Yuping, Ma Zhongren, Liu Zhenbin
Gansu Tech Innovation Center of Animal Cell, Biomedical Research Center, Northwest Minzu University, Lanzhou 730030, China.
Mol Omics. 2021 Feb 1;17(1):121-129. doi: 10.1039/d0mo00055h. Epub 2020 Nov 17.
MDCK cells are a key reagent in modern vaccine production. As MDCK cells are normally adherent, creation of suspension cells for vaccine production using genetic engineering approaches is highly desirable. However, little is known regarding the mechanisms and effectors underlying MDCK cell adhesion. In this study, we performed a comparative analysis of whole protein levels between MDCK adhesion and suspension cells using an iTRAQ-based (isobaric tags for relative and absolute quantitation) proteomics approach. We found that expression of several proteins involved in cell adhesion exhibit reduced expression in suspension cells, including at the mRNA level. Proteins whose expression was reduced in suspension cells include cadherin 1 (CDH1), catenin beta-1 (CTNNB1), and catenin alpha-1 (CTNNA1), which are involved in intercellular adhesion; junction plakoglobin (JUP), desmoplakin (DSP), and desmoglein 3 (DSG3), which are desmosome components; and transglutaminase 2 (TGM2) and alpha-actinin-1 (ACTN1), which regulate the adhesion between cells and the extracellular matrix. A functional verification experiment showed that inhibition of E-cadherin significantly reduced intercellular adhesion of MDCK cells. E-Cadherin did not significantly affect the proliferation of MDCK cells and the replication of influenza virus. These findings reveal possible mechanisms underlying adhesion of MDCK cells and will guide the creation of MDCK suspension cells by genetic engineering.
MDCK细胞是现代疫苗生产中的关键试剂。由于MDCK细胞通常是贴壁的，因此使用基因工程方法创建用于疫苗生产的悬浮细胞非常可取。然而，关于MDCK细胞粘附的潜在机制和效应分子知之甚少。在本研究中，我们使用基于iTRAQ（相对和绝对定量的等压标签）的蛋白质组学方法，对MDCK贴壁细胞和悬浮细胞之间的全蛋白水平进行了比较分析。我们发现，几种参与细胞粘附的蛋白质的表达在悬浮细胞中降低，包括在mRNA水平。在悬浮细胞中表达降低的蛋白质包括参与细胞间粘附的钙粘蛋白1（CDH1）、β-连环蛋白1（CTNNB1）和α-连环蛋白1（CTNNA1）；桥粒斑珠蛋白（JUP）、桥粒芯蛋白（DSP）和桥粒芯糖蛋白3（DSG3），它们是桥粒的组成成分；以及转谷氨酰胺酶2（TGM2）和α-辅肌动蛋白-1（ACTN1），它们调节细胞与细胞外基质之间的粘附。功能验证实验表明，抑制E-钙粘蛋白可显著降低MDCK细胞的细胞间粘附。E-钙粘蛋白对MDCK细胞的增殖和流感病毒的复制没有显著影响。这些发现揭示了MDCK细胞粘附的潜在机制，并将指导通过基因工程创建MDCK悬浮细胞。
