Graduate School of Life Science and Frontier Research Center for Post-Genomic Science and Technology, Hokkaido University, N21, W11, Kita-ku, Sapporo 001-0021, Japan.
Biochemistry. 2010 Mar 23;49(11):2604-14. doi: 10.1021/bi100094g.
Recombinant glycosyltransferases are potential biocatalysts for the construction of a compound library of oligosaccharides, glycosphingolipids, glycopeptides, and various artificial glycoconjugates on the basis of combined chemical and enzymatic synthetic procedures. The structurally defined glycan-related compound library is a key resource both in the basic studies of their functional roles in various biological processes and in the discovery research of new diagnostic biomarkers and therapeutic reagents. Therefore, it is clear that the immobilization of extremely unstable membrane-bound glycosyltransferases on some suitable supporting materials should enhance the operational stability and activity of recombinant enzymes and makes facile separation of products and recycling use of enzymes possible. Until now, however, it seems that no standardized protocol preventing a significant loss of enzyme activity is available due to the lack of a general method of site-selective anchoring between glycosyltransferases and scaffold materials through a stable covalent bond. Here we communicate a versatile and efficient method for the immobilization of recombinant glycosyltransferases onto commercially available solid supports by means of transpeptidase reaction by Staphylococcus aureus sortase A. This protocol allowed for the first time highly specific conjugation at the designated C-terminal signal peptide moiety of recombinant human beta1,4-galactosyltransferase or recombinant Helicobacter pylori alpha1,3-fucosyltransferase with simple aliphatic amino groups displayed on the surface of solid materials. Site-specifically immobilized enzymes exhibited the desired sugar transfer activity, an improved stability, and a practical reusability required for rapid and large-scale synthesis of glycoconjugates. Considering that most mammalian enzymes responsible for the posttranslational modifications, including the protein kinase family, as well as glycosyltransferases are unstable and highly oriented membrane proteins, the merit of our strategy based on "site-specific" transpeptidation is evident because the reaction proceeds only at an engineered C-terminus without any conformational influence around the active sites of both enzymes as well as heptad repeats of rHFucT required to maintain native secondary and quaternary structures during the dimerization on cell surfaces.
基于化学和酶促合成程序的组合，重组糖基转移酶是构建寡糖、糖脂、糖肽和各种人工糖缀合物化合物库的潜在生物催化剂。结构定义的糖相关化合物库是基础研究其在各种生物过程中的功能作用以及发现新的诊断生物标志物和治疗试剂的关键资源。因此，很明显，将极其不稳定的膜结合糖基转移酶固定在一些合适的支撑材料上，应该可以提高重组酶的操作稳定性和活性，并使产物的分离和酶的循环使用成为可能。然而，到目前为止，由于缺乏通过稳定的共价键在糖基转移酶和支架材料之间进行位点选择性锚定的通用方法，似乎没有防止酶活性显著损失的标准化方案。在这里，我们通过金黄色葡萄球菌 sortase A 的转肽酶反应，传达了一种将重组糖基转移酶固定在商业可得的固体载体上的通用且有效的方法。该方案首次允许在重组人β1,4-半乳糖基转移酶或重组幽门螺杆菌α1,3-岩藻糖基转移酶的指定 C 末端信号肽部分与简单脂肪族氨基上进行高度特异性的缀合，这些氨基在固体材料表面显示。定点固定的酶表现出所需的糖转移活性、提高的稳定性和用于糖缀合物快速大规模合成所需的实际可重复使用性。考虑到大多数负责翻译后修饰的哺乳动物酶，包括蛋白激酶家族以及糖基转移酶，都是不稳定的和高度定向的膜蛋白，我们基于“定点”转肽的策略的优点是显而易见的，因为该反应仅在工程化的 C 末端进行，而不会对酶的活性位点以及在细胞表面上二聚化时维持天然二级和四级结构所需的 rHFucT 的七肽重复产生任何构象影响。
