Exo-enzymatic addition of diazirine-modified sialic acid to cell surfaces enables photocrosslinking of glycoproteins
Exo-enzymatic addition of diazirine-modified sialic acid to cell surfaces enables photocrosslinking of glycoproteins
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通过酶外酶法将二氮丙啶修饰的唾液酸添加到细胞表面，从而实现糖蛋白的光交联
DOI：
10.1101/2021.10.07.463072
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发表时间：
2021
期刊：
影响因子：
--
通讯作者：
Jennifer J. Kohler
中科院分区：
文献类型：
--
作者：
Nageswari Yarravarapu;Rohit Sai Reddy Konada;Narek Darabedian;Nichole J. Pedowitz;Soumya N Krishnamurthy;Matthew R. Pratt;Jennifer J. Kohler
聚糖结合通常介导细胞外大分子识别事件。这些结合相互作用的准确表征可能是困难的，因为在纯化和分析步骤期间发生解离和混乱。已经寻求使用光交联方法来原位共价捕获聚糖依赖性相互作用，然而使用代谢聚糖工程方法来掺入光交联糖类似物仅限于某些细胞类型。在这里，我们报告了一种酶外标记方法，添加二氮丙啶修饰的唾液酸（SiaDAz）的细胞表面糖缀合物。该方法涉及化学酶促合成二氮丙啶修饰的CMP-唾液酸（CMP-SiaDAz），然后通过唾液酸转移酶催化将SiaDAz添加到去唾液酸化的细胞表面。细胞表面SiaDAz-化与多种细胞类型相容，并且通过Daudi B细胞中存在的内源性细胞外唾液酸转移酶活性促进。这种用于细胞外添加α2-6连接的SiaDAz的方法能够实现CD 22的UV诱导交联，证明了共价捕获聚糖介导的结合相互作用的实用性。
Glycan binding often mediates extracellular macromolecular recognition events. Accurate characterization of these binding interactions can be difficult because of dissociation and scrambling that occur during purification and analysis steps. Use of photocrosslinking methods has been pursued to covalently capture glycan-dependent interactions in situ however use of metabolic glycan engineering methods to incorporate photocrosslinking sugar analogs is limited to certain cell types. Here we report an exo-enzymatic labeling method to add a diazirine-modified sialic acid (SiaDAz) to cell surface glycoconjugates. The method involves chemoenzymatic synthesis of diazirine-modified CMP-sialic acid (CMP-SiaDAz), followed by sialyltransferase-catalyzed addition of SiaDAz to desialylated cell surfaces. Cell surface SiaDAz-ylation is compatible with multiple cell types and is facilitated by endogenous extracellular sialyltransferase activity present in Daudi B cells. This method for extracellular addition of α2-6-linked SiaDAz enables UV-induced crosslinking of CD22, demonstrating the utility for covalent capture of glycan-mediated binding interactions.
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