Unexpected tolerance of glycosylation by UDP-GalNAc:polypeptide alpha-N-acetylgalactosaminyltransferase revealed by electron capture dissociation mass spectrometry: carbohydrate as potential protective groups.
Unexpected tolerance of glycosylation by UDP-GalNAc:polypeptide alpha-N-acetylgalactosaminyltransferase revealed by electron capture dissociation mass spectrometry: carbohydrate as potential protective groups.
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电子捕获解离质谱法揭示了 UDP-GalNAc：多肽 α-N-乙酰半乳糖胺基转移酶对糖基化的意外耐受：碳水化合物作为潜在的保护基团。
作者：
Yayoi Yoshimura;Takahiko Matsushita;N. Fujitani;Yasuhiro Takegawa;Haruhiko Fujihira;K. Naruchi;Xiao;Naomi Manri;Takeshi Sakamoto;Kentaro Kato;H. Hinou;S. Nishimura
UDP-GalNAc：多肽α-N-乙酰半乳糖胺转移酶（ppGalNAcTs，EC 2.4.1.41），是通过引入α-GalNAc残基在粘蛋白合成中用O-聚糖引发翻译后修饰的关键酶家族，在结构上由催化结构域和凝集素结构域组成。已知在常见的粘蛋白糖蛋白中多个Ser/Thr残基被指定为潜在的O-糖基化位点，并且该酶家族的20多种不同的同种型有助于产生密集的O-糖基化粘蛋白糖蛋白。然而，似乎ppGalNAcTs的凝集素结构域的功能作用仍不清楚。我们认为，电子捕获解离质谱法（ECD-MS），一个有前途的方法，在糖肽的肽连接高度选择性的碎片，以产生独特的c和z系列的离子，应该允许精确的结构表征，以揭示粘蛋白肽的O-糖基化的机制ppGalNAcTs。在本研究中，证明了由电喷雾源、隔离前体离子的线性离子阱、ECD装置和TOF质谱仪组成的系统是识别优先O-糖基化位点而不分解碳水化合物部分的好工具。应当注意，用于ECD的电子在1.75至9.75 eV的范围内加速，这取决于感兴趣的糖肽的结构。我们首次揭示了在具有α-Man、α-Fuc和β-Gal残基以及α-GalNAc残基的各种非天然糖肽中，通过ppGalNAcT 2在潜在糖基化位点处的α-GalNAc残基的额外安装顺利进行。结果可能表明，ppGalNAcT 2没有区分完全预取代的糖残基的功能基团，d-，l-构型，甚至α-，β-立体化学的异头碳原子的配置方面时，相对较短的合成肽受体底物。ppGalNAcT 2的意想不到的特性促使我们通过用选择性可去除的糖保护Thr/Ser残基的一些羟基来挑战α-GalNAc残基在所需位置的定点安装，特别是作为“碳水化合物作为保护基团”的新概念，朝着生物学上重要的粘蛋白糖肽的系统化学和酶促合成的目标。
UDP-GalNAc:polypeptide alpha-N-acetylgalactosaminyltransferases (ppGalNAcTs, EC 2.4.1.41), a family of key enzymes that initiate posttranslational modification with O-glycans in mucin synthesis by introduction of alpha-GalNAc residues, are structurally composed of a catalytic domain and a lectin domain. It has been known that multiple Ser/Thr residues are assigned in common mucin glycoproteins as potential O-glycosylation sites and more than 20 distinct isoforms of this enzyme family contribute to produce densely O-glycosylated mucin glycoproteins. However, it seems that the functional role of the lectin domain of ppGalNAcTs remains unclear. We considered that electron capture dissociation mass spectrometry (ECD-MS), a promising method for highly selective fragmentation at peptide linkages of glycopeptides to generate unique c and z series of ions, should allow for precise structural characterization to uncover the mechanism in O-glycosylation of mucin peptides by ppGalNAcTs. In the present study, it was demonstrated that a system composed of an electrospray source, a linear RFQ ion trap that isolates precursor ions, the ECD device, and a TOF mass spectrometer is a nice tool to identify the preferential O-glycosylation sites without any decomposition of the carbohydrate moiety. It should be noted that electrons used for ECD are accelerated within a range from 1.75 to 9.75 eV depending on the structures of glycopeptides of interest. We revealed for the first time that additional installation of a alpha-GalNAc residue at potential glycosylation sites by ppGalNAcT2 proceeds smoothly in various unnatural glycopeptides having alpha-Man, alpha-Fuc, and beta-Gal residues as well as alpha-GalNAc residues. The results may suggest that ppGalNAcT2 did not differentiate totally presubstituted sugar residues in terms of configuration of functional groups, d-, l-configuration, and even alpha-, beta-stereochemistry at an anomeric carbon atom when relatively short synthetic peptides were employed for the acceptor substrates. Unexpected characteristics of ppGalNAcT2 motivated us to challenge site-directed installation of alpha-GalNAc residues at desired position(s) by protecting some hydroxyl groups of Thr/Ser residues with selectively removable sugars, notably a novel concept as "carbohydrate as protective groups", toward a goal of the systematic chemical and enzymatic synthesis of biologically important mucin glycopeptides.
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影响因子：
3.8
作者：
Sung, H. H.;Castro, I.;Gonzalez, M-J
通讯作者：
Gonzalez, M-J
影响因子：
14.8
作者：
Ingale, Sampat;AWolfert, Margreet;Boons, Geert-Jan
通讯作者：
Boons, Geert-Jan
影响因子：
4.3
作者：
Kinarsky, L;Suryanarayanan, G;Sherman, S
通讯作者：
Sherman, S
影响因子：
15
作者：
Coltart, DM;Royyuru, AK;Live, DH
通讯作者：
Live, DH
影响因子：
21.8
作者：
Boltje, Thomas J.;Buskas, Therese;Boons, Geert-Jan
通讯作者：
Boons, Geert-Jan