Background

Mast cells are involved in many distinct pathologic 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.

Objective

We aimed to further characterize the mast cell surface proteome to obtain a better understanding of how mast cells function in health and disease.

Methods

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 real-time quantitative PCR 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.

Results

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 were small GTPases, receptors, and transporters. One such cell surface protein was CD98 heavy chain (CD98hc), encoded by the Slc3a2 gene. Slc3a2 gene disruption resulted in a significant reduction in CD98hc expression, adhesion, and proliferation.

Conclusions

Glycoprotein enrichment coupled with mass spectrometry can be used to identify novel surface molecules in mast cells. Moreover, CD98hc plays an important role in mast cell function.

中文翻译：

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肥大细胞表面组特征揭示 CD98 重链对于最佳细胞功能至关重要

背景

肥大细胞参与许多不同的病理状况，表明它们识别各种刺激并对其作出反应，因此需要丰富的细胞表面蛋白库。然而，肥大细胞表面蛋白质组尚未得到全面表征。

客观的

我们旨在进一步表征肥大细胞表面蛋白质组，以更好地了解肥大细胞在健康和疾病中的功能。

方法

我们富集了在小鼠骨髓来源的培养肥大细胞 (BMCMC) 中表达的糖基化表面蛋白，并使用质谱分析对其进行了鉴定。通过 BMCMC 和腹膜肥大细胞 (PMC) 中的实时定量 PCR 和流式细胞术分析验证了肥大细胞中新型表面蛋白的存在。我们开发了一种成簇的规则间隔短回文重复序列 (CRISPR)/CRISPR 相关蛋白 9 (Cas9) 基因编辑方法来破坏 BMCMC 中感兴趣的基因。

结果

糖蛋白富集方法导致在 BMCMC 中鉴定出 1270 种蛋白质，其中 378 种位于质膜上。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基因破坏导致 CD98hc 表达、粘附和增殖显着减少。

结论

糖蛋白富集与质谱联用可用于鉴定肥大细胞中的新型表面分子。此外，CD98hc 在肥大细胞功能中起重要作用。