利用N酰基修饰 Mannosamines 对唾液酸的代谢 Glycoengineering
1Max von Pettenkofer-Institut & Genzentrum, Virologie, Nationales Referenzzentrum für Retroviren, Medizinische Fakultät,LMU München, 2Institut für Laboratoriumsmedizin, klinische Chemie und Pathobiochemie,Charité – Universitätsmedizin Berlin, 3Institut für Physiologische Chemie,Martin-Luther-Universität Halle-Wittenberg
Summary
唾液酸是一种典型的单糖单位, 见于糖。它涉及过多的分子和细胞相互作用。在这里, 我们提出了一个方法来修改细胞表面唾液酸表达使用代谢 glycoengineering 与N-acetylmannosamine 衍生物。
Abstract
唾液酸是糖的重要组成部分, 如N和O-糖或脂。由于它的位置在还原总站的寡糖和多糖, 以及其独特的化学特性, 唾液酸参与了多种不同的受体配体相互作用。通过改变唾液酸在细胞表面的表达, 将影响唾液酸依赖性的相互作用。这有助于研究唾液酸依赖性的相互作用, 并有可能以有益的方式影响某些疾病。通过代谢 glycoengineering (MGE), 可以调节唾液酸在细胞表面的表达。在这里, 细胞, 组织, 甚至整个动物都是用 C2-modified 衍生物的N-acetylmannosamine (ManNAc) 来处理的。这些氨基糖充当唾液酸前体分子, 因此被代谢到相应的唾液酸种类和表达的糖。应用这种方法对各种生物过程产生了有趣的影响。例如, 它可以大大减少唾液酸 (polySia) 在处理神经元细胞中的表达, 从而影响神经元的生长和分化。在这里, 我们展示了两个最常见的 C2-modified n-acylmannosamine 衍生物的化学合成, n-propionylmannosamine (ManNProp) 以及n-butanoylmannosamine (ManNBut), 并进一步显示这些非氨基糖可用于细胞培养实验。用高效液相色谱 (HPLC) 对改性唾液酸的表达进行了定量分析, 并通过质谱法进行了进一步的分析。利用唾液酸抗体对唾液酸表达的影响进行了免疫印迹。
Introduction
唾液酸是一种单糖, 通常可以在糖的还原总站找到, 如N-和O-糖或脂。在所有单糖中, 唾液酸具有独特的化学特性。它有一个 9 C 原子骨干, 一个羧基在 C-1 位置, 是 deprotonated, 从而负电荷在生理条件下, 和一个氨基功能的 C-5 位置。虽然超过50自然发生的唾液酸变种已被描述为迄今1, 在人类中发现的主要形式的唾液酸是N-乙酰酸 (Neu5Ac)。其他哺乳动物也表达了更高的数量的N-glycolylneuraminic 酸 (Neu5Gc)23。
由于其暴露在糖的位置, 唾液酸参与了过多的受体-配体相互作用,例如,流感病毒对宿主细胞的血依赖性绑定4。具有重要生物学功能的唾液酸表位, 尤其在胚胎发生和神经系统中, 是唾液酸。唾液酸是一种高达200α28连接的唾液酸的聚合物。唾液酸的主要蛋白载体是神经细胞黏附分子 (分子)。唾液酸的表达调节了分子的粘附特性, 即唾液酸的表达减少了粘连, 增加了与神经系统的可塑性5。
(聚) 唾液酸的表达变化最终会影响多种不同的生物相互作用。这可以用来研究已知的唾液酸相关过程的分子水平, 以揭示新的糖相互作用, 或探索可能的治疗方法。有不同的方法可利用在细胞表面上唾液酸的表示可以被调整, 例如治疗与唾液酸具体糖苷 (sialidases), 抑制酵素参与在唾液酸生物合成中6 ,7,8, 或击倒或更改唾液酸生物合成的关键酶的表达式9。
另一种用于调节唾液酸表达的方法是 MGE (也称为代谢性寡糖工程, 教育部)。在这里, 细胞, 组织, 甚至动物都是用非衍生物的 ManNAc 来进行 C2-amino 修饰。作为前体分子的唾液酸, 在细胞摄取后, 这些 ManNAc 类似物是单向代谢到非唾液酸和可以表达的 sialylated 糖。使用含有脂肪 C2-modifications 的 ManNAc 衍生物 (如 ManNProp 或 ManNBut) 处理的细胞, 在其 propionylneuraminic10中合并n-Neu5Prop 酸 (butanoylneuraminic) 或n-Neu5But 酸 (糖),11. 通过引入 ManNAc C2-position 的功能组, 非的唾液酸可以通过施陶丁格结扎或叠氮化 alkine 加与荧光染料相结合, 从而在单元格表面上可视化12。
这些非唾液酸的表达对许多生物过程有着耐人寻味的影响, 包括病原体感染、肿瘤细胞黏附和迁移、一般细胞黏附以及血管化和分化 (供审查请参见: Wratil et al.13). 有趣的是, MGE 与N-酰基修饰的 mannosamines 也可以用来干扰唾液酸的表达。唾液酸是由两种不同的 polysialyltransferases (ST8SiaII 和 ST8SiaIV) 产生的。已经证明, polysialyltransferase ST8SiaII 是由非自然的唾液酸前体抑制的, 如 ManNProp 或 ManNBut14,15。此外, 它已经证明在人类神经母细胞瘤 ManNProp 或 ManNBut 应用也减少 sialylation 总15。
MGE 与N-酰基修饰 mannosamines 是一种易于应用的方法, 已成功使用, 不仅在哺乳动物和细菌细胞培养, 而且在整个动物的不同物种, 如线虫线虫16,斑马鱼17, 或鼠标18,19,20,21。特别是含脂肪修饰的 ManNAc 衍生物, 包括 ManNProp 和 ManNBut, 都是地细胞毒, 即使在 millimolar 浓度的培养基或血浆中也是如此。此外, 它们相对容易合成。
在这里, 我们提供了如何使用 MGE 与N-乙酰修饰 mannosamines 的详细信息。首先, 解释了这一领域中最广泛使用的两种 ManNAc 衍生物的化学合成, ManNProp 和 ManNBut。接下来, 我们将展示如何在体内实验中应用 MGE。以 ManNAc 衍生物为例, 选择了神经母细胞线凯利在治疗后唾液表位的表达减少。采用 HPLC 法对细胞表面的非唾液酸进行定量分析, 并通过质谱法进一步分析。
Protocol
1. 缓冲器和试剂的制备 3 mM 甲醇钠溶液的制备 在50毫升甲醇 (3 mM) 中, 在100毫升的玻璃瓶中溶解8.1 毫克钠醇, 搅拌棒。室温 (RT) 贮存数周。 三盐酸缓冲液的制备 结合8.766 克氯化钠, 157 毫克三盐酸, 146 毫克 EDTA 在100毫升玻璃瓶与搅拌棒和溶解在80毫升水。 将氢氧化钠 (1 米, 在水中) 或 HCl (20%, 在水中) 添加到搅拌液中, 同时观?…
Representative Results
在图 2中描述了荧光标记 Neu5Ac 和 Neu5Gc 标准的 HPLC 谱。使用本文所描述的方法, DMB 标记的 Neu5Gc 通常 elutes 在 7-9 分钟洗脱时间之间, 和 DMB-Neu5Ac 之间 10-12 分钟。色谱中的几个小峰通常出现在 2-6 分钟之间。这些峰值代表未 DMB 和反应中间体25。 图 3显示了单元格裂解…
Discussion
如果化学合成的 ManNAc 衍生物, ManNProp 和 ManNBut 通过质谱分析, 只有正确的质量峰值的两个标本应确定。因此, 可以假定产品的纯度超过99%。在裂解细胞的膜片段中检测到少量的 Neu5Gc, 通常不存在于人类细胞29。这最有可能发生通过一个打捞途径, 招募 Neu5Gc 从胎儿牛血清 sialoglycoconjugates 在媒体30。用唾液酸的天然前驱物进行生物合成, ManNAc, 大大降低了 Neu5Gc 表位?…
Declarações
The authors have nothing to disclose.
Acknowledgements
我们感谢阮先生校对手稿和进行富有成效的讨论。此外, 我们感谢 j. Dernedde 和阮先生帮助我们准备视频拍摄。大多数视频镜头都是在 r. 罗腾堡实验室拍摄的。我们还感谢马克斯普朗克研究所的胶体和接口, 并为我们提供免费进入他们的质谱仪设施。RH 得到了 DFG (ProMoAge) 的支持。
Materials
| Cells | Sigma-Aldrich | 92110411 | |
| RPMI medium | Sigma-Aldrich | R8758 | |
| 75 ml tissue culture flasks | Greiner | 690175 | |
| 48-well plates | Corning | 3548 | |
| FCS | PAA | A15-102 | |
| Pen/Strep | Gibco | 15140-122 | |
| Trypsine | Gibco | 15400-054 | |
| EDTA | Roth | X986.1 | |
| Tris | Serva | 37190.03 | |
| SDS | Roth | 2326.2 | |
| SDS-PAGE equipment (tanks, glassware etc., machine | VWR | SDS Gel/Blot | |
| Acrylamide | Roth | 3019.1 | |
| Protein ladder | Fisher Scientific | 267620 | |
| Nitrocellulose | GE Healthcare | 10600002 | |
| Ponceau red | Roth | 5938.2 | |
| Milk powder | Roth | T145.3 | |
| ECL | Millipore | WBLUF 0500 | |
| 0.5 ml Centrifugal Filter Unit with 3 kDa membrane | Merck-Millipore | UFC500324 | |
| 15 mL centrifuge tubes | Sigma-Aldrich (Corning) | CLS430791-500EA | |
| 2-Mercaptoethanol | Sigma-Aldrich | M6250-10ML | |
| 2-Propanol | Sigma-Aldrich | 34965-1L | HPLC gradient grade |
| 4,5-Methylenedioxy-1,2-phenylenediamine dihydrochloride | Sigma-Aldrich | D4784-50MG | |
| 48 well, flat bottom tissue culture plate | Sigma-Aldrich (Corning) | CLS3548-100EA | |
| 50 mL centrifuge tubes | Sigma-Aldrich (Corning) | CLS430829-500EA | |
| Acetonitrile | Sigma-Aldrich | 34967-1L | HPLC gradient grade |
| Aprotinin from bovine lung | Sigma-Aldrich | A1153-10MG | lyophilized powder, 3-8 TIU/mg solid |
| Butyryl chloride | Sigma-Aldrich | 109614-250G | |
| C18 RP column | Phenomenex | 00F-4435-E0 | 110 Å, 3 µm particle size, 4.6 x 150 mm |
| D-Mannosamine hydrochloride | Sigma-Aldrich | M4670-1G | |
| Dulbecco`s Phosphate Buffered Salt Solution | PAN Biotech | P04-36500 | |
| Ethylenediaminetetraacetic acid | Sigma-Aldrich | E9884-100G | |
| Formic acid | Sigma-Aldrich | 56302-50ML-GL | |
| Hydrochloric acid solution | Sigma-Aldrich | H1758-100ML | 36.5-38.0%, in water |
| Leupeptin | Sigma-Aldrich | L2884-10MG | |
| Methanol | Carl-Roth | T169.2 | HPLC gradient grade |
| N-Acetyl-D-mannosamine | Sigma-Aldrich | A8176-250MG | |
| N-Acetylneuraminic acid | Sigma-Aldrich | A0812-25MG | |
| N-Glycolylneuraminic acid | Sigma-Aldrich | G9793-10MG | |
| Phenylmethanesulfonyl fluoride | Sigma-Aldrich | P7626-250MG | |
| Propionyl chloride | Sigma-Aldrich | P51559-500G | |
| Safe-Lock Tubes, 1.5 mL, amber (light protection) | Eppendorf | 30120191 | |
| Safe-Lock Tubes, 1.5 mL, colorless | Eppendorf | 30120086 | |
| Sodium bisulfite solution | Sigma-Aldrich | 13438-1L-R-D | 40%, in water |
| Sodium chloride | Sigma-Aldrich | 746398-500G-D | |
| Sodium hydroxide | Sigma-Aldrich | 795429-500G-D | |
| Sodium hydroxide solution | Sigma-Aldrich | 319511-500ML | 1.0 M, in water |
| Sodium methoxide | Sigma-Aldrich | 164992-5G | |
| Trifluoroacetic acid | Sigma-Aldrich | T6508-100ML-D | |
| Tris hydrochloride | Sigma-Aldrich | T5941-100G | |
| Trypsin 0.25 %/EDTA 0.02 % in PBS | PAN Biotech | P10-019100 | |
| Water | Carl-Roth | T905.1 | HPLC gradient grade |
| Silica Gel 60 | Carl-Roth | 9779.1 | |
| HPLC | Shimadzu |
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Citar este artigo
Wratil, P. R., Horstkorte, R. Metabolic Glycoengineering of Sialic Acid Using N-acyl-modified Mannosamines. J. Vis. Exp. (129), e55746, doi:10.3791/55746 (2017).