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首页> 外文期刊>Biochemistry >Highly Oriented Recombinant Glycosyltransferases: Site-Specific Immobilization of Unstable Membrane Proteins by Using Staphylococcus aureus Sortase A
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Highly Oriented Recombinant Glycosyltransferases: Site-Specific Immobilization of Unstable Membrane Proteins by Using Staphylococcus aureus Sortase A

机译：高度定向的重组糖基转移酶：通过使用金黄色葡萄球菌分选酶A的特定位置固定化不稳定的膜蛋白。

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摘要

Recombinant glycosyltransferases are potential biocatalysts for the construction of a compoundnlibrary of oligosaccharides, glycosphingolipids, glycopeptides, and various artificial glycoconjugates on thenbasis of combined chemical and enzymatic synthetic procedures. The structurally defined glycan-relatedncompound library is a key resource both in the basic studies of their functional roles in various biologicalnprocesses and in the discovery research of new diagnostic biomarkers and therapeutic reagents. Therefore, it isnclear that the immobilization of extremely unstable membrane-bound glycosyltransferases on some suitablensupportingmaterials should enhance the operational stability and activity of recombinant enzymes andmakesnfacile separation of products and recycling use of enzymes possible. Until now, however, it seems that nonstandardized protocol preventing a significant loss of enzyme activity is available due to the lack of a generalnmethod of site-selective anchoring between glycosyltransferases and scaffold materials through a stablencovalent bond. Here we communicate a versatile and efficient method for the immobilization of recombinantnglycosyltransferases onto commercially available solid supports by means of transpeptidase reaction bynStaphylococcus aureus sortase A. This protocol allowed for the first time highly specific conjugation at thendesignated C-terminal signal peptide moiety of recombinant human β1,4-galactosyltranseferase or recombi-nnant Helicobacter pylori R1,3-fucosyltransferase with simple aliphatic amino groups displayed on the surfacenof solid materials. Site-specifically immobilized enzymes exhibited the desired sugar transfer activity, annimproved stability, and a practical reusability required for rapid and large-scale synthesis of glycoconjugates.nConsidering thatmostmammalian enzymes responsible for the posttranslationalmodifications, including thenprotein kinase family, as well as glycosyltransferases are unstable and highly oriented membrane proteins, thenmerit of our strategy based on “site-specific” transpeptidation is evident because the reaction proceeds only atnan engineered C-terminus without any conformational influence around the active sites of both enzymes asnwell as heptad repeats of rHFucT required tomaintain native secondary and quaternary structures during thendimerization on cell surfaces.
机译：重组糖基转移酶是潜在的生物催化剂，可用于在结合化学和酶促合成程序的基础上构建寡糖，糖鞘脂，糖肽和各种人工糖缀合物的复合文库。结构上定义的与聚糖相关的化合物库是其在各种生物过程中的功能作用的基础研究以及在新的诊断性生物标记物和治疗剂的发现研究中的重要资源。因此，尚不清楚极不稳定的膜结合糖基转移酶在某些合适的支持材料上的固定应增强重组酶的操作稳定性和活性，并使产物的分离和酶的回收利用成为可能。然而，到目前为止，由于缺乏糖基转移酶和支架材料之间通过稳定的共价键固定位点的一般方法，似乎可以使用防止酶活性显着降低的非标准化方案。在这里，我们交流一种通用且有效的方法，用于通过金黄色葡萄球菌分选酶A的转肽酶反应将重组糖基糖基转移酶固定在可商购的固体支持物上。该方案首次允许在重组人β1的指定C端信号肽部分进行高特异性缀合。 ，4-半乳糖基转铁酶或重组幽门螺杆菌R1,3-岩藻糖基转移酶在固体物质表面具有简单的脂肪族氨基。定点固定的酶表现出所需的糖转移活性，提高的稳定性和快速，大规模合成糖缀合物所需的实用可重复使用性。n考虑到负责翻译后修饰的大多数哺乳动物酶（包括蛋白激酶家族以及糖基转移酶）不稳定且膜蛋白高度定向，因此我们基于“位点特异性”转肽的策略的优点是显而易见的，因为该反应仅在工程改造的C末端进行，而在两种酶的活性位点附近均无构象影响，而rHFucT的七肽重复序列则需要保持天然第二级然后在细胞表面二聚过程中的四级结构。

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  • 来源
    《Biochemistry》 |2010年第11期|p.2604-2614|共11页
  • 作者

    Takaomi Ito Reiko Sadamoto Kentaro Naruchi Hiroko Togame Hiroshi Takemoto Hirosato Kondo and Shin-Ichiro Nishimura;

  • 作者单位

    ‡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,§Discovery Research Laboratories, Pharmaceutical Research Division,Shionogi and Company, Ltd., N21, W11, Kita-ku, Sapporo 001-0021, Japan,) Ochanomizu University, 2-1-1, Otsuka,Bunkyo-ku, Tokyo 112-8610, Japan, and ^Discovery Research Laboratories, Shionogi and Company, Ltd., 12-4,Sagisu 5-chome, Fukushima-ku, Osaka 541-0045, Japan;

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