Mast cell surfaceome characterization reveals CD98 heavy chain is critical for optimal cell function.
Mast cell surfaceome characterization reveals CD98 heavy chain is critical for optimal cell function.
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DOI：
10.1016/j.jaci.2021.07.014
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发表时间：
2022-03
期刊：
影响因子：
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通讯作者：
Piliponsky AM
中科院分区：
文献类型：
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作者：
Saha SS;Samanas NB;Miralda I;Shubin NJ;Niino K;Bhise G;Acharya M;Seo AJ;Camp N;Deutsch GH;James RG;Piliponsky AM
肥大细胞参与许多不同的病理状态，表明它们识别和响应各种刺激，因此需要丰富的细胞表面蛋白。然而，肥大细胞表面蛋白质组还没有得到全面的研究。我们的目标是进一步确定肥大细胞表面蛋白质组的特征，以更好地了解肥大细胞在健康和疾病中的作用。我们浓缩了在小鼠骨髓来源的肥大细胞(BMCMCs)中表达的糖基化表面蛋白，并用质谱分析对它们进行了鉴定。通过定量聚合酶链式反应和流式细胞仪分析，证实肥大细胞中存在新的表面蛋白。我们开发了一种簇状规则间隔短回文重复序列(CRISPR)/CRISPR相关蛋白9(Cas9)基因编辑方法来干扰BMCMCs中的相关基因。糖蛋白富集法鉴定了BMCMCs中的1270种蛋白质，其中378种定位于质膜。在质膜蛋白中，最常见的蛋白质类别是小GTP酶、受体和转运蛋白。一种这样的细胞表面蛋白是由Slc3a2基因编码的CD98重链(CD98hc)。SLC3a2基因阻断可显著降低CD98hc的表达、黏附和增殖。我们的研究表明，糖蛋白浓缩结合质谱仪可以用于识别肥大细胞中的新的表面分子。此外，我们还发现CD98hc在肥大细胞功能中起着重要作用。糖蛋白浓缩和质谱学使肥大细胞表面蛋白质组的全面表征成为可能。CD98hc的表达是肥大细胞最佳功能所必需的。
Mast cells are involved in many distinct pathological conditions, suggesting that they recognize and respond to various stimuli and thus require a rich repertoire of cell surface proteins. However, mast cell surface proteomes have not been comprehensively characterized. We aimed to further characterize the mast cell surface proteome to obtain a better understanding of how mast cells function in health and disease. We enriched for glycosylated surface proteins expressed in mouse bone marrow-derived cultured mast cells (BMCMCs) and identified them using mass spectrometry analysis. The presence of novel surface proteins in mast cells was validated by qPCR and flow cytometry analysis in BMCMCs and peritoneal mast cells (PMCs). We developed a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9) gene editing approach to disrupt genes of interest in BMCMCs. The glycoprotein enrichment approach resulted in the identification of 1270 proteins in BMCMCs, 378 of which were localized to the plasma membrane. The most common protein classes among plasma membrane proteins are small GTPases, receptors and transporters. One such cell surface protein is CD98 heavy chain (CD98hc) encoded by the Slc3a2 gene. Slc3a2 gene disruption resulted in a significant reduction in CD98hc expression, adhesion, and proliferation. Our study indicates that glycoprotein enrichment coupled with mass spectrometry can be used to identify novel surface molecules in mast cells. Moreover, we found that CD98hc plays an important role in mast cell function. Glycoprotein enrichment and mass spectrometry enabled a comprehensive characterization of the mast cell surface proteome. CD98hc expression is required for optimal mast cell function.