Detection of site-specific glycosylation in proteins using flow cytometry.
Detection of site-specific glycosylation in proteins using flow cytometry.
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DOI：
10.1002/cyto.a.20773
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发表时间：
2009-10
期刊：
影响因子：
3.7
通讯作者：
Neelamegham, Sriram
中科院分区：
文献类型：
--
作者：
Jayakumar, Deepak;Marathe, Dhananjay D.;Neelamegham, Sriram
关键词：
我们测试了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位点特异性糖基化的可能性。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，修饰人白细胞糖蛋白PSGL-1（P-选择素糖蛋白配体-1）的N-末端部分，使其含有聚组氨酸标签，随后是蛋白水解切割位点。切割位点之前的氨基酸具有单个0-连接的糖基化位点。使用慢病毒递送方法，在两种哺乳动物细胞系CHO和HL-60的表面上表达称为PSGL-1（HT）的重组蛋白。结果表明，PSGL-1（HT）的N-末端部分可以从这些细胞释放的蛋白酶，所得的肽可以很容易地捕获和检测使用流式细胞仪珠测定。使用该策略，将肽免疫沉淀到带有针对聚组氨酸序列或人PSGL-1的mAb的珠上。使用识别唾液酸化Lewis-X表位的单克隆抗体HECA-452和CSLEX-1检测与释放的肽相关的碳水化合物表位。最后，从细胞中释放的肽可以使用镍螯合珠分离和富集。总的来说，这种将重组蛋白表达与流式细胞术相结合的方法可能有助于定量位点特异性糖基化的变化，用于基础科学和临床应用。
We tested the possibility that it is possible to 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.
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影响因子：
4.8
作者：
Marathe, Dhananjay D.;Chandrasekaran, E. V.;Neelamegham, Sriram
通讯作者：
Neelamegham, Sriram
影响因子：
7.8
作者：
Zhou, Q;Moore, K L;Smith, D F;Varki, A;McEver, R P;Cummings, R D
通讯作者：
Cummings, R D
影响因子：
20.3
作者：
Snapp, KR;Ding, H;Kansas, GS
通讯作者：
Kansas, GS
影响因子：
3.4
作者：
Xiao, ZH;Goldsmith, HL;Neelamegham, S
通讯作者：
Neelamegham, S
影响因子：
4.8
作者：
Liu, W;Ramachandran, V;McEver, RP
通讯作者：
McEver, RP