Chemical and Biological Engineering, State University of New York, Buffalo, New York 14260, USA.
Cytometry A. 2009 Oct;75(10):866-73. doi: 10.1002/cyto.a.20773.
We tested the possibility that we may express unique peptide probes on cell surfaces, and detect site-specific glycosylation on these peptides using flow cytometry. Such development can enhance the application of flow cytometry to detect and quantify post-translational modifications in proteins. To this end, the N-terminal section of the human leukocyte glycoprotein PSGL-1 (P-selectin glycoprotein ligand-1) was modified to contain a poly-histidine tag followed by a proteolytic cleavage site. Amino acids preceding the cleavage site have a single O-linked glycosylation site. The recombinant protein called PSGL-1 (HT) was expressed on the surface of two mammalian cell lines, CHO and HL-60, using a lentiviral delivery approach. Results demonstrate that the N-terminal portion of PSGL-1 (HT) can be released from these cells by protease, and the resulting peptide can be readily captured and detected using cytometry-bead assays. Using this strategy, the peptide was immunoprecipitated onto beads bearing mAbs against either the poly-histidine sequence or the human PSGL-1. The carbohydrate epitope associated with the released peptide was detected using HECA-452 and CSLEX-1, monoclonal antibodies that recognize the sialyl Lewis-X epitope. Finally, the peptide released from cells could be separated and enriched using nickel chelate beads. Overall, such an approach that combines recombinant protein expression with flow cytometry may be useful to quantify changes in site-specific glycosylation for basic science and clinical applications.
我们测试了一种可能性，即在细胞表面表达独特的肽探针，并使用流式细胞术检测这些肽上的特定糖基化位点。这种发展可以增强流式细胞术在检测和定量蛋白质翻译后修饰中的应用。为此，我们修饰了人白细胞糖蛋白 PSGL-1（P 选择素糖蛋白配体-1）的 N 端部分，使其包含一个多组氨酸标签和一个蛋白水解切割位点。在切割位点之前的氨基酸只有一个 O-连接的糖基化位点。通过慢病毒递送方法，将称为 PSGL-1（HT）的重组蛋白表达在 CHO 和 HL-60 两种哺乳动物细胞系的表面。结果表明，PSGL-1（HT）的 N 端部分可以被蛋白酶从这些细胞中释放出来，并且可以使用细胞术珠分析轻易地捕获和检测到释放的肽。使用这种策略，肽可以被免疫沉淀到带有针对多组氨酸序列或人 PSGL-1 的 mAb 的珠子上。使用识别唾液酸化 Lewis-X 表位的单克隆抗体 HECA-452 和 CSLEX-1 检测与释放肽相关的碳水化合物表位。最后，细胞释放的肽可以使用镍螯合珠进行分离和富集。总之，这种将重组蛋白表达与流式细胞术相结合的方法可能对基础科学和临床应用中特定糖基化位点的定量变化很有用。
