Comprehensive analysis of transglutaminase substrate preference by cDNA display coupled with next-generation sequencing and bioinformatics.
Comprehensive analysis of transglutaminase substrate preference by cDNA display coupled with next-generation sequencing and bioinformatics.
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DOI：
10.1038/s41598-022-17494-4
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发表时间：
2022-08-09
影响因子：
4.6
通讯作者：
Nakano, Hideo
中科院分区：
文献类型：
--
作者：
Damnjanovic, Jasmina;Odake, Nana;Fan, Jicheng;Camagna, Maurizio;Jia, Beixi;Kojima, Takaaki;Nemoto, Naoto;Hitomi, Kiyotaka;Nakano, Hideo
cDNA展示是一种基于蛋白质及其相应mRNA/cDNA之间共价键的体外展示技术，广泛用于基于蛋白质和肽的结合亲和力以高通量方式从大型文库（1012）中选择蛋白质和肽。在这里，我们开发了一个使用cDNA展示和下一代测序（NGS）的平台，用于快速和全面的转氨酶2（TG 2）底物分析，TG 2是一种交联蛋白质中谷氨酰胺和赖氨酸残基的酶。在筛选和选择在反应性谷氨酰胺处随机化的对照肽文库之后，筛选来自反应性谷氨酰胺的在位置-1、+1、+2和+3处随机化的展示肽的组合文库，随后进行NGS和生物信息学分析，这表明TG 2对在位置-1处具有谷氨酰胺的肽的强烈偏好（Gln-Gln基序）和+3位的异亮氨酸或缬氨酸。高度富集的肽确实含有所示的序列，并显示出比先前通过噬菌体展示选择的肽更高的作为TG 2底物的反应性，因此代表了用于TG 2研究的新型候选肽探针。此外，所获得的关于底物谱的信息可用于鉴定潜在的TG 2蛋白靶标。该平台将进一步用于其他TG同工酶的底物分析，以及用于更大生物分子的选择和进化。
cDNA display is an in vitro display technology based on a covalent linkage between a protein and its corresponding mRNA/cDNA, widely used for the selection of proteins and peptides from large libraries (1012) in a high throughput manner, based on their binding affinity. Here, we developed a platform using cDNA display and next-generation sequencing (NGS) for rapid and comprehensive substrate profiling of transglutaminase 2 (TG2), an enzyme crosslinking glutamine and lysine residues in proteins. After screening and selection of the control peptide library randomized at the reactive glutamine, a combinatorial library of displayed peptides randomized at positions − 1, + 1, + 2, and + 3 from the reactive glutamine was screened followed by NGS and bioinformatic analysis, which indicated a strong preference of TG2 towards peptides with glutamine at position − 1 (Gln-Gln motif), and isoleucine or valine at position + 3. The highly enriched peptides indeed contained the indicated sequence and showed a higher reactivity as TG2 substrates than the peptide previously selected by phage display, thus representing the novel candidate peptide probes for TG2 research. Furthermore, the obtained information on substrate profiling can be used to identify potential TG2 protein targets. This platform will be further used for the substrate profiling of other TG isozymes, as well as for the selection and evolution of larger biomolecules.
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DOI：
10.1016/j.bbamcr.2011.09.012
发表时间：
2012-02
期刊：
Biochimica et biophysica acta
影响因子：
--
作者：
Gundemir S;Colak G;Tucholski J;Johnson GV
通讯作者：
Johnson GV
影响因子：
3.5
作者：
Nemoto, N;MiyamotoSato, E;Yanagawa, H
通讯作者：
Yanagawa, H
影响因子：
2.7
作者：
Baggio, R;Burgstaller, P;Wagner, RW
通讯作者：
Wagner, RW
DOI：
10.1073/pnas.0702251104
发表时间：
2007-09-04
影响因子：
11.1
作者：
Ju, Wujian;Valencia, C. Alexander;Liu, Rihe
通讯作者：
Liu, Rihe
影响因子：
4.4
作者：
Iversen, Rasmus;du Pre, M. Fleur;Sollid, Ludvig M.
通讯作者：
Sollid, Ludvig M.