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。

1. 缓冲器和试剂的制备
<ol><li>
3 mM 甲醇钠溶液的制备
	<ol>
<li>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。</li>
	</ol>
</li>
	<li>
三盐酸缓冲液的制备
	<ol>
<li>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。</li>
		<li>将氢氧化钠 (1 米, 在水中) 或 HCl (20%, 在水中) 添加到搅拌液中, 同时观察 ph 值和 ph 值, 并将 ph 值调整到8.0。</li>
		<li>增加需要的水量, 以达到100毫升的最终体积。使用0.22 µm 无菌过滤系统过滤解决方案。 注: 100 毫升三盐酸缓冲将包含150毫米氯化钠, 10 毫米三盐酸, 和5毫米 EDTA (pH 8.0)。该溶液可贮存在4° c 以下几周。</li>
	</ol>
</li>
	<li>
抑肽酶溶液的制备
	<ol>
<li>在1毫升水中溶解10毫克抑肽酶 (1.54 毫米)。分将抑肽酶溶液转化为 10 x 1.5 毫升塑料管 (每根100µL)。存放在-20 ° c 几个星期。</li>
	</ol>
</li>
	<li>
leupeptin 溶液的制备
	<ol>
<li>在2毫升水中溶解10毫克 leupeptin (10 毫米)。分 leupeptin 溶液 10 x 1.5 毫升塑料管。存放在-20 ° c 几个星期。</li>
	</ol>
</li>
	<li>
磺氟 (PMSF) 溶液的制备
	<ol>
<li>在2毫升乙醇 (100 mM) 中溶解34.8 毫克 PMSF。分 10 x 1.5 mL 塑料管中的 PMSF 溶液。存放在-20 ° c 几个星期。</li>
	</ol>
</li>
	<li>
12二氨基-45-methyldioxybenzol 二盐酸盐 (DMB) 溶液的制备
	<ol>
<li>结合15.62 毫克 DMB, 352 µL β-基, 和48µL 亚硫酸氢钠溶液 (39%), 并添加9.6 毫升水 (6.9 毫米 DMB, 500 毫米β-基, 和0.19% 亚硫酸氢钠)。分的 DMB 解决方案在 40 x 1.5 毫升塑料管 (每250µL)。商店在-20 ° c 保护了几个星期。</li>
	</ol>
</li>
	<li>
1 M 乙酸酸 (TFA) 溶液的制备
	<ol>
<li>在15毫升塑料管 (1 米) 中加入770.3 µL 100% TFA 至9.23 毫升水。存放在4° c 几个星期。</li>
	</ol>
</li>
	<li>
120毫米 TFA 溶液的制备
	<ol>
<li>在15毫升塑料管中加入92.4 µL TFA (100%) 至9.91 毫升水 (120 mM)。存放在4° c 几个星期。</li>
	</ol>
</li>
	<li>
400毫米氢氧化钠 (氢氧化钠) 溶液的制备
	<ol>
<li>在15毫升的塑料管中溶解10毫升水 (400 毫米) 中的160毫克氢氧化钠。存放在4° c 几个星期。</li>
	</ol>
</li>
	<li>
西部印迹裂解液的制备
	<ol>
<li>溶解 5.84 g 氯化钠, 0.1 g 三 (羟甲基)-aminomethan (三), 0.1 g CaCl2, 0.95 g 氯化镁2, 和 1 g Triton-X100 在100毫升水和调整 pH 值到7.8。贮存在4° c。在使用裂解缓冲液之前, 加入100µL 的蛋白酶抑制剂 (抑肽酶、leupeptin 和 PMSF)。</li>
	</ol>
</li>
	<li>
十二烷基硫酸钠聚丙烯酰胺凝胶电泳 (SDS-页) 缓冲剂的研制
	<ol>
<li>在100毫升水中溶解0.3 克三, 1.5 克甘氨酸, 0.1 克 SDS, 并将 pH 值调整到7.3。存储在 RT。</li>
	</ol>
</li>
	<li>
免疫印迹缓冲液的制备
	<ol>
<li>在90毫升水中溶解0.3 克三, 1.1 克甘氨酸, 加入10毫升乙醇。存储在 RT。</li>
	</ol>
</li>
	<li>
堵塞溶液的制备
	<ol>
<li>在25毫升磷酸缓冲盐水 (PBS) 中溶解1克无脂牛奶的能量。总是准备新鲜。</li>
	</ol>
</li>
</ol>2. ManNProp 的合成及相关的N-酰基修饰 Mannosamines
<ol><li>将431.2 毫克甘露盐酸盐溶于10毫升3毫米的甲醇钠溶液中, 在50毫升的玻璃瓶中加入搅拌棒。</li>
	<li>在冰上冷却混合物到0° c。对搅拌溶液, 缓慢添加滴210µL 丙氯 (2.4 摩尔), 或248µL butanoyl 氯 (2.4 摩尔) 分别合成 ManNProp 或 ManNBut。孵育搅拌混合物在0° c 为 4 h。</li>
	<li>将溶液转化为50毫升的塑料管。用薄针戳 4-8 孔到塑料管的盖子。使用液氮快速冷冻溶液, 随后 lyophilize (冷冻干燥) 48 小时或直至完全干燥。</li>
	<li>混合350克硅凝胶60与750毫升乙酸乙酯/甲醇/水 (15:2: 1) (这是一个暂停)。在使用前, 用硅胶60悬浮液填充玻璃柱 (直径35毫米和70厘米), 再用100毫升的乙酸乙酯/甲醇/水 (15:2: 1) 洗涤准备好的柱子。</li>
	<li>在10毫升乙酸乙酯/甲醇/水 (15:2: 1) 中溶解2.3 步 (5 克干制品) 中的干制品, 并将其用吸管放到硅胶柱上。收集500毫升后的4毫升分数 (ManNProp)。注: ManNBut 将在700毫升后大约洗。</li>
	<li>将洗馏分转化为15毫升塑料管。用薄针戳 3-6 孔到塑料管的盖子。用液氮快速冷冻溶液, 随后 lyophilize, 直到完全干燥。 注: 冻干产品可在室温下保存数月。</li>
	<li>通过质谱法验证产品的纯度 (见9节)。 注: 另外, 纯度也可以通过1H 核磁共振 (NMR) 进行验证。</li>
</ol>3. 细胞培养
<ol><li>在罗斯威尔公园纪念研究所 (RPMI) 培养基中含有10% 胎牛血清, 100 U 青霉素, 100 毫克链霉素, 2 毫米 l-谷氨酰胺, 含有5毫米 ManNAc, 5 毫米 ManNProp, 或5毫米 ManNBut, 在37° c 和 5% CO2。 注意: 以培养基培养的无 ManNAc 或类似物的细胞用作对照。</li>
	<li>
在应用 mannosamines 之前, 通过孵化10分钟的胰蛋白酶/EDTA (0.25%, 0.02%, PBS) 和计数使用 Neubauer 室或细胞计数器, 分离的凯利神经母细胞瘤。根据盘子/盘子的大小, 在定义的数字上播种细胞。
	<ol>
<li>为了测定唾液酸单糖, 在500µL 培养基中, 种子 1 x 105细胞变成48井组织培养板。为分析唾液酸, 种子 1 x 106细胞在3毫升培养基上6厘米直径细胞培养皿。孵育在37° c 和 5% CO2。每24小时更换一次介质。 注: MGE 的效果随着治疗总时间的增加而增大。为高新陈代谢的效率治疗细胞为 5-7 天。</li>
	</ol>
</li>
	<li>
治疗后, 醒酒培养基。用 PBS 洗盘子/盘子。分离的细胞, 孵化他们10分钟的胰蛋白酶/EDTA (0.25%, 0.02%, 在 PBS)。通过在分离细胞中加入相同体积的细胞培养基来中和胰蛋白酶。将分离的细胞转化为15毫升的塑料管。离心样品3分钟在 500 x g 和丢弃上清液。
	<ol>
<li>添加5毫升 PBS 和离心细胞 (见步骤 3.3)。重复洗涤程序两次。 注: 无上清的水洗细胞颗粒可在-20 ° c 贮存数天。如果唾液酸的表达将被阐明继续10节。</li>
	</ol>
</li>
</ol>4. 高效液相色谱分析的细胞裂解
<ol><li>
HPLC 裂解液的制备
	<ol>
<li>结合5毫升三盐酸缓冲液, 5 µL 抑肽酶溶液, 20 µL leupeptin 溶液, 50 µL PMSF 溶液。 注: 5 毫升 HPLC 裂解缓冲液将含有150毫米氯化钠, 10 毫米三盐酸, 5 毫米 EDTA, 1.54 µM 抑肽酶, 40 µM Leupeptin, 1 毫米 PMSF (pH 8.0)。在细胞裂解之前, 这个缓冲液应该一直准备新鲜。</li>
	</ol>
</li>
	<li>将所收集的细胞小球 (步骤 3.3) 重新悬浮在500µL ice-cold HPLC 裂解缓冲液中, 并在三十年代的中高振幅下用超声波处理器针三次 ultra-sonicate 样品。冰上的样品冷却至少1分钟之间的周期。</li>
</ol>5. 膜馏分的分离
<ol><li>离心裂解细胞样品2小时在 2万 x g 和4° c。将上清液 (大约480µL) 分离, 代表胞浆蛋白, 在1.5 毫升塑料管中。确定这些分数的蛋白质浓度使用,例如, 布拉德福德或二辛可宁酸 (免疫分析) 化验。 注: 蛋白质浓度 (约1毫克/毫升) 的胞浆组分后, 可与受测的唾液酸种类的数量有关。颗粒代表膜分数, 在步骤6.1 中使用。</li>
</ol>6. 酸性水解
注意: 在细胞膜上的寡糖和多糖被水解成单糖。此外, 可能的O-在单糖中的修改也被水解。这是必要的定量 HPLC 分析, 因为绝大多数的唾液酸的膜分数是自然合并在糖, 可能会承担o-乙酰或o-lactolyl 修改。
<ol><li>添加150µL 1 M TFA-溶液的分离膜分数 (颗粒从5节)。在80° c 下以 200-600 rpm 的震动为样本孵育4小时。</li>
	<li>离心样品大约30分钟在 2万 x g 和21° c 使用0.5 毫升滤管与 3 kDa 排除膜, 直到上部阶段容量小于20µL。 注意: 这一步对处理更高的分子碎片很重要。</li>
	<li>将包括唾液酸类在内的小分子的低相转化为2毫升塑料管。用薄针戳 2-4 孔到塑料管的盖子。快速冷冻样品使用液氮, 然后 lyophilize 他们过夜。 注: 干燥后的样品可在 RT 中存放数天. 更换无孔盖, 以延长贮存时间。</li>
</ol>7. 荧光标签
<ol><li>在10µL 120 mM TFA 溶液中重新挂干样品, 并加入50µL DMB 溶液。将样品转移到深色的1.5 毫升管子中, 以保护它们免受紫外线照射。</li>
	<li>对于标准, 溶解1µL Neu5Ac (60 ng/毫升, 在水中) 或1µL Neu5Gc (60 ng/毫升, 在水中) 在10µL 120 毫米 TFA 解决方案在黑暗1.5 毫升管。添加50µL DMB 解决方案。</li>
	<li>孵育细胞膜样品和标准为 1.5 h 在56° c 保护免受光。在孵化后, 加入4µL 400 毫米氢氧化钠溶液, 以停止对每个样品的标记反应。</li>
</ol>8. HPLC 分析
注: 在 C18 RP 柱 (110 Å、3µm 粒度、4.6 x 150 mm)、荧光检测器和分数收集器的 HPLC 系统上对标签样品进行分析。所用溶剂为甲醇、乙腈和水。如果 HPLC 系统缺乏内部气, 请务必在测量前加气所有的溶剂。
<ol><li>将该柱的温度设置为40° c, 并分别配置 373 nm 激发和 448 nm 的荧光探测器。注入10µL 样品体积和分离探针为50分钟在0.5 毫升/分钟流速与甲醇/乙腈/水 (6:8:86) 作为淋洗。对于质谱分析, 收集感兴趣的峰值。 注: Neu5Gc 洗后6-8 分钟, Neu5Ac 后 9-12 分钟, Neu5Prop 后 17-23 分钟, 和 Neu5But 38-44 分钟。分馏样品可以被存放为几个 h 在4° c 保护免受光。</li>
	<li>在测量每样样品后, 用甲醇/乙腈/水 (6:25:69), 用甲醇/乙腈/水 (7.5: 1.0), 在1.0 毫升/分钟流速 (≈1.5 柱卷) 上, 将该柱洗净7.5 分钟, 并 re-equilibrate 系统为6:8 分钟。</li>
	<li>对于定量分析, 使用各自的 HPLC 系统22的操作软件, 计算出感兴趣的唾液酸峰值曲线下的面积。 注: 获得的数据可与 Neu5Ac 或 Neu5Gc 标准以及胞浆组分中测定的蛋白质浓度有关 (见5.1 节)。</li>
</ol>9. 唾液酸单糖的质谱分析
注: 利用液相色谱 (LC) 电喷雾质谱 (ESI), 可进一步分析溶液中的活性峰, 以验证非自然唾液酸的含量。
<ol><li>在质谱分析中, 将采集到的20µL 样品注入到 LC/质量选择性检测系统中, 以79.9% 甲醇、20% 异丙醇和0.1% 甲酸为淋洗, 0.5 毫升/分钟流量, 4 kV 毛细管电压和350° c 毛细管温度22,23。</li>
	<li>在各自的 LC/调峰系统的评价软件中, 选择总离子色谱中的峰值。查看已解决峰值的质量频谱。显示300和700之间的正质量/电荷比。 注: DMB 标记的唾液酸导致增加的分子质量116.2 克/摩尔。DMB 标记的唾液酸种类有以下分子质量: DMB-Neu5Ac = 424.2 克/摩尔, DMB-Neu5Gc = 441.8 克/摩尔, DMB-Neu5Prop = 436.2 克/摩尔, DMB-Neu5But = 453.2 克/摩尔. 普通加为乙腈、钠或异丙醇。</li>
</ol>10. 唾液酸在凯利神经母细胞瘤中的免疫印迹分析
<ol><li>
细胞裂解的制备
	<ol>
<li>在步骤3.3 和旋涡中加入1毫升的西部印迹裂解缓冲液。在冰上孵育30分钟。漩涡每5分钟。离心样品1.5 小时在 2万 x g 和4° c。从裂解细胞中提取含有蛋白质的上清液。</li>
		<li>测定蛋白质组分的蛋白质浓度,如: 如、布拉德福德或免疫分析。在裂解缓冲液中将样品稀释到1.5 µg/µL 的蛋白质浓度。</li>
		<li>为 SDS 页准备样品, 将10µL Laemmli 样本缓冲器添加到90µL 蛋白分数 (1.5 µg/µL), 并将样品煮沸为 5 min24。</li>
	</ol>
</li>
	<li>
免疫
	<ol>
<li>使用 8% SDS-丙烯酰胺凝胶, 20-30 µL 袋和0.75 或1毫米厚度。在室温下运行25毫安的凝胶2小时。</li>
		<li>小心地从凝胶中取下玻璃盖。切出并丢弃含有装载袋的凝胶的上部。将凝胶放在硝化棉膜上 (0.2 µm), 以前浸泡在西部印迹缓冲液中。根据系统的推荐将蛋白质转移到硝化纤维上 (例如,在4° c 时, 为 1 h 250 毫安)。</li>
		<li>从印迹系统中除去硝化纤维素膜。染色的印迹与胭脂红红色, 以可视化的蛋白质。将硝化纤维素膜转移到塑料室中, 并加入10毫升堵塞溶液。在 RT 或夜间在4° c 孵育1小时。</li>
		<li>醒酒在 PBS 中加入了阻断溶液并添加单克隆 anti-polySia 735 抗体 (1 µg/毫升)。在 RT 或隔夜在4° c 下孵育膜1小时。孵育后, 用 PBS 至少清洗三次膜5分钟。</li>
		<li>添加多克隆 anti-mouse IgG 二级抗体 (1 µg/毫升), 再加上辣根过氧化物酶 (HRP) 或 PBS 中的荧光标签。在 RT 上孵育1小时的膜。孵育后, 用 PBS 至少清洗三次膜5分钟。</li>
		<li>将膜转移到适当的成像仪的盘上。根据制造商的说明检测信号。</li>
	</ol>
</li>
</ol>

作者没有什么可透露的。

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?...

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。

<table><tbody><tr><td>Cells</td><td>Sigma-Aldrich</td><td>92110411</td><td></td></tr><tr><td>RPMI medium</td><td>Sigma-Aldrich</td><td>R8758</td><td></td></tr><tr><td>75 ml tissue culture flasks</td><td>Greiner</td><td>690175</td><td></td></tr><tr><td>48-well plates</td><td>Corning</td><td>3548</td><td></td></tr><tr><td>FCS</td><td>PAA</td><td>A15-102</td><td></td></tr><tr><td>Pen/Strep</td><td>Gibco</td><td>15140-122</td><td></td></tr><tr><td>Trypsine</td><td>Gibco</td><td>15400-054</td><td></td></tr><tr><td>EDTA</td><td>Roth</td><td>X986.1</td><td></td></tr><tr><td>Tris</td><td>Serva</td><td>37190.03</td><td></td></tr><tr><td>SDS</td><td>Roth</td><td>2326.2</td><td></td></tr><tr><td>SDS-PAGE equipment (tanks, glassware etc., machine</td><td>VWR</td><td>SDS Gel/Blot</td><td></td></tr><tr><td>Acrylamide</td><td>Roth</td><td>3019.1</td><td></td></tr><tr><td>Protein ladder</td><td>Fisher Scientific</td><td>267620</td><td></td></tr><tr><td>Nitrocellulose</td><td>GE Healthcare</td><td>10600002</td><td></td></tr><tr><td>Ponceau red</td><td>Roth</td><td>5938.2</td><td></td></tr><tr><td>Milk powder</td><td>Roth</td><td>T145.3</td><td></td></tr><tr><td>ECL</td><td>Millipore</td><td>WBLUF 0500</td><td></td></tr><tr><td>0.5 ml Centrifugal Filter Unit with 3 kDa membrane</td><td>Merck-Millipore</td><td>UFC500324</td><td></td></tr><tr><td>15 mL centrifuge tubes</td><td>Sigma-Aldrich (Corning)</td><td>CLS430791-500EA</td><td></td></tr><tr><td>2-Mercaptoethanol</td><td>Sigma-Aldrich</td><td>M6250-10ML</td><td></td></tr><tr><td>2-Propanol</td><td>Sigma-Aldrich</td><td>34965-1L</td><td>HPLC gradient grade</td></tr><tr><td>4,5-Methylenedioxy-1,2-phenylenediamine dihydrochloride</td><td>Sigma-Aldrich</td><td>D4784-50MG</td><td></td></tr><tr><td>48 well, flat bottom tissue culture plate</td><td>Sigma-Aldrich (Corning)</td><td>CLS3548-100EA</td><td></td></tr><tr><td>50 mL centrifuge tubes</td><td>Sigma-Aldrich (Corning)</td><td>CLS430829-500EA</td><td></td></tr><tr><td>Acetonitrile</td><td>Sigma-Aldrich</td><td>34967-1L</td><td>HPLC gradient grade</td></tr><tr><td>Aprotinin from bovine lung</td><td>Sigma-Aldrich</td><td>A1153-10MG</td><td>lyophilized powder, 3-8 TIU/mg solid</td></tr><tr><td>Butyryl chloride</td><td>Sigma-Aldrich</td><td>109614-250G</td><td></td></tr><tr><td>C18 RP column</td><td>Phenomenex</td><td>00F-4435-E0</td><td>110 &amp;Aring;, 3 &amp;micro;m particle size, 4.6 x 150 mm</td></tr><tr><td>D-Mannosamine hydrochloride</td><td>Sigma-Aldrich</td><td>M4670-1G</td><td></td></tr><tr><td>Dulbecco`s Phosphate Buffered Salt Solution</td><td>PAN Biotech</td><td>P04-36500</td><td></td></tr><tr><td>Ethylenediaminetetraacetic acid</td><td>Sigma-Aldrich</td><td>E9884-100G</td><td></td></tr><tr><td>Formic acid</td><td>Sigma-Aldrich</td><td>56302-50ML-GL</td><td></td></tr><tr><td>Hydrochloric acid solution</td><td>Sigma-Aldrich</td><td>H1758-100ML</td><td>36.5-38.0%, in water</td></tr><tr><td>Leupeptin</td><td>Sigma-Aldrich</td><td>L2884-10MG</td><td></td></tr><tr><td>Methanol</td><td>Carl-Roth</td><td>T169.2</td><td>HPLC gradient grade</td></tr><tr><td>N-Acetyl-D-mannosamine</td><td>Sigma-Aldrich</td><td>A8176-250MG</td><td></td></tr><tr><td>N-Acetylneuraminic acid</td><td>Sigma-Aldrich</td><td>A0812-25MG</td><td></td></tr><tr><td>N-Glycolylneuraminic acid</td><td>Sigma-Aldrich</td><td>G9793-10MG</td><td></td></tr><tr><td>Phenylmethanesulfonyl fluoride</td><td>Sigma-Aldrich</td><td>P7626-250MG</td><td></td></tr><tr><td>Propionyl chloride</td><td>Sigma-Aldrich</td><td>P51559-500G</td><td></td></tr><tr><td>Safe-Lock Tubes, 1.5 mL, amber (light protection)</td><td>Eppendorf</td><td>30120191</td><td></td></tr><tr><td>Safe-Lock Tubes, 1.5 mL, colorless</td><td>Eppendorf</td><td>30120086</td><td></td></tr><tr><td>Sodium bisulfite solution</td><td>Sigma-Aldrich</td><td>13438-1L-R-D</td><td>40%, in water</td></tr><tr><td>Sodium chloride</td><td>Sigma-Aldrich</td><td>746398-500G-D</td><td></td></tr><tr><td>Sodium hydroxide</td><td>Sigma-Aldrich</td><td>795429-500G-D</td><td></td></tr><tr><td>Sodium hydroxide solution</td><td>Sigma-Aldrich</td><td>319511-500ML</td><td>1.0 M, in water</td></tr><tr><td>Sodium methoxide</td><td>Sigma-Aldrich</td><td>164992-5G</td><td></td></tr><tr><td>Trifluoroacetic acid</td><td>Sigma-Aldrich</td><td>T6508-100ML-D</td><td></td></tr><tr><td>Tris hydrochloride</td><td>Sigma-Aldrich</td><td>T5941-100G</td><td></td></tr><tr><td>Trypsin 0.25 %/EDTA 0.02 % in PBS</td><td>PAN Biotech</td><td>P10-019100</td><td></td></tr><tr><td>Water</td><td>Carl-Roth</td><td>T905.1</td><td>HPLC gradient grade</td></tr><tr><td>Silica Gel 60</td><td>Carl-Roth</td><td>9779.1</td><td></td></tr><tr><td>HPLC</td><td>Agilent</td><td></td><td></td></tr><tr><td>ESI-MSD-System</td><td>Agilent</td><td></td><td></td></tr></tbody></table>

metabolic glycoengineering of sialic acid using n-acyl-modified mannosamines

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。

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...

Watch this Scientific Journal Video about Metabolic Glycoengineering of Sialic Acid Using N-acyl-modified Mannosamines at JoVE.com

Metabolic Glycoengineering of Sialic Acid Using N-acyl-modified Mannosamines

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。

利用N酰基修饰 Mannosamines 对唾液酸的代谢 Glycoengineering

Sialic acid (Sia) is a highly important constituent of glycoconjugates, such as N- and O-glycans or glycolipids. Due to its position at the non-reducing ...

metabolic-glycoengineering-sialic-acid-using-n-acyl-modified

Research

JoVE Journal

Bioengineering

Metabolic Glycoengineering of Sialic Acid Using N-acyl-modified Mannosamines

12.6K 次观看.  LMU München. 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。

视频: Metabolic Glycoengineering of Sialic Acid Using N-acyl-modified Mannosamines

High-throughput Synthesis of Carbohydrates and Functionalization of Polyanhydride Nanoparticles

Transdisciplinary approaches involving areas such as material design, nanotechnology, chemistry, and immunology have to be utilized to rationally design efficacious vaccines carriers. Nanoparticle-based platforms can prolong the persistence of vaccine antigens, which could improve vaccine immunogenicity1. Several biodegradable polymers have been studied as vaccine delivery vehicles1; in particular, polyanhydride particles have demonstrated the ability to provide sustained release of stable protein antigens and to activate antigen presenting cells and modulate immune responses2-12.
The molecular design of these vaccine carriers needs to integrate the rational selection of polymer properties as well as the incorporation of appropriate targeting agents. High throughput automated fabrication of targeting ligands and functionalized particles is a powerful tool that will enhance the ability to study a wide range of properties and will lead to the design of reproducible vaccine delivery devices.
The addition of targeting ligands capable of being recognized by specific receptors on immune cells has been shown to modulate and tailor immune responses10,11,13 C-type lectin receptors (CLRs) are pattern recognition receptors (PRRs) that recognize carbohydrates present on the surface of pathogens. The stimulation of immune cells via CLRs allows for enhanced internalization of antigen and subsequent presentation for further T cell activation14,15. Therefore, carbohydrate molecules play an important role in the study of immune responses; however, the use of these biomolecules often suffers from the lack of availability of structurally well-defined and pure carbohydrates. An automation platform based on iterative solution-phase reactions can enable rapid and controlled synthesis of these synthetically challenging molecules using significantly lower building block quantities than traditional solid-phase methods16,17.
Herein we report a protocol for the automated solution-phase synthesis of oligosaccharides such as mannose-based targeting ligands with fluorous solid-phase extraction for intermediate purification. After development of automated methods to make the carbohydrate-based targeting agent, we describe methods for their attachment on the surface of polyanhydride nanoparticles employing an automated robotic set up operated by LabVIEW as previously described10. Surface functionalization with carbohydrates has shown efficacy in targeting CLRs10,11 and increasing the throughput of the fabrication method to unearth the complexities associated with a multi-parametric system will be of great value (Figure 1a).

In this article, a high throughput method is presented for the synthesis of oligosaccharides and their attachment to the surface of polyanhydride nanoparticles for further use in targeting specific receptors on antigen presenting cells.

碳水化合物和聚酸酐纳米粒子功能化的高通量合成

Transdisciplinary approaches involving areas such as material design, nanotechnology, chemistry, and immunology have to be utilized to rationally design ...

科研

生物工程

Profiling Anti-Neu5Gc IgG in Human Sera with a Sialoglycan Microarray Assay

Cells are covered with a cloak of carbohydrate chains (glycans) that is commonly altered in cancer and that includes variations in sialic acid (Sia) expression. These are acidic sugars that have a 9-carbon backbone and that cap vertebrate glycans on cell surfaces. Two of the major Sia forms in mammals are N-acetylneuraminic acid (Neu5Ac) and its hydroxylated form, N-glycolylneuraminic acid (Neu5Gc). Humans cannot produce endogenous Neu5Gc due to the inactivation of the gene encoding cytidine 5&#39;monophosphate-Neu5Ac (CMP-Neu5Ac) hydroxylase (CMAH). Foreign Neu5Gc is acquired by human cells through the dietary consumption of red meat and dairy and subsequently appears on diverse glycans on the cell surface, accumulating mostly on carcinomas. Consequently, humans have circulating anti-Neu5Gc antibodies that play diverse roles in cancer and other chronic inflammation-mediated diseases and that are becoming potential diagnostic and therapeutic targets. Here, we describe a high-throughput sialoglycan microarray assay to assess such anti-Neu5Gc antibodies in the human sera. Neu5Gc-containing glycans and their matched pairs of controls (Neu5Ac-containing glycans), each with a core primary amine, are covalently linked to epoxy-coated glass slides. We exemplify the printing of 56 slides in a 16-well format using a specific nano-printer capable of generating up to 896 arrays per print. Each slide can be used to screen 16 different human sera samples for the evaluation of anti-Neu5Gc antibody specificity, intensity, and diversity. The protocol describes the complexity of this robust tool and provides a basic guideline for those aiming to investigate the response to Neu5Gc dietary carbohydrate antigen in diverse clinical samples in an array format.

A sialoglycan microarray assay can be used to evaluate anti-Neu5Gc antibodies in human sera, making it a potential high-throughput diagnostic assay for cancer and other chronic inflammation-mediated human diseases.

使用唾液酸多糖微阵列测定在人血清中分析抗Neu5Gc IgG

Cells are covered with a cloak of carbohydrate chains (glycans) that is commonly altered in cancer and that includes variations in sialic acid (Sia) ...

行为学

Chemo-enzymatic Synthesis of N-glycans for Array Development and HIV Antibody Profiling

We present a highly efficient way for the rapid preparation of a wide range of N-linked oligosaccharides (estimated to exceed 20,000 structures) that are commonly found on human glycoproteins. To achieve the desired structural diversity, the strategy began with the chemo-enzymatic synthesis of three kinds of oligosaccharyl fluoride modules, followed by their stepwise &#945;-selective glycosylations at the 3-O and 6-O positions of the mannose residue of the common core trisaccharide having a crucial &#946;-mannoside linkage. We further attached the N-glycans to the surface of an aluminum oxide-coated glass (ACG) slide to create a covalent mixed array for the analysis of hetero-ligand interaction with an HIV antibody. In particular, the binding behavior of a newly isolated HIV-1 broadly neutralizing antibody (bNAb), PG9, to the mixture of closely spaced Man5GlcNAc2 (Man5) and 2,6-di-sialylated bi-antennary complex type N-glycan (SCT) on an ACG array, opens a new avenue to guide the effective immunogen design for HIV vaccine development. In addition, our ACG array embodies a powerful tool to study other HIV antibodies for hetero-ligand binding behavior.

Chemo-enzymatic Synthesis of N-glycans for Array Development and HIV Antibody Profiling

A modular approach to the synthesis of N-glycans for attachment to an aluminum oxide-coated glass slide (ACG slide) as a glycan microarray has been developed and its use for the profiling of an HIV broadly neutralizing antibody has been demonstrated.

用于阵列发育和 HIV 抗体分析的糖的化学酶法合成

We present a highly efficient way for the rapid preparation of a wide range of N-linked oligosaccharides (estimated to exceed 20,000 structures) that are ...

化学

Regioselective O-Glycosylation of Nucleosides via the Temporary 2',3'-Diol Protection by a Boronic Ester for the Synthesis of Disaccharide Nucleosides

Disaccharide nucleosides, which consist of disaccharide and nucleobase moieties, have been known as a valuable group of natural products having multifarious bioactivities. Although chemical O-glycosylation is a commonly beneficial strategy to synthesize disaccharide nucleosides, the preparation of substrates such as glycosyl donors and acceptors requires&#160;tedious protecting&#160;group manipulations and a purification at each synthetic step. Meanwhile, several research groups have reported that boronic and borinic esters serve as a protecting or activating group of carbohydrate derivatives to achieve the regio- and/or stereoselective acylation, alkylation, silylation, and glycosylation. In this article, we demonstrate the procedure for the regioselective O-glycosylation of unprotected ribonucleosides utilizing boronic acid. The esterification of 2&#39;,3&#39;-diol of ribonucleosides with boronic acid makes the temporary protection of diol, and, following O-glycosylation with a glycosyl donor in the presence of p-toluenesulfenyl chloride and silver triflate, permits the regioselective reaction of the 5&#39;-hydroxyl group to afford the disaccharide nucleosides. This method could be applied to various nucleosides, such as guanosine, adenosine, cytidine, uridine, 5-metyluridine, and 5-fluorouridine. This article and the accompanying video represent&#160;useful (visual) information for the O-glycosylation of unprotected nucleosides and their analogs for the synthesis of not only disaccharide nucleosides, but also a variety of biologically relevant derivatives.

Regioselective O-Glycosylation of Nucleosides via the Temporary 2',3'-Diol Protection by a Boronic Ester for the Synthesis of Disaccharide Nucleosides

Here, we present protocols for the synthesis of disaccharide nucleosides by the regioselective O-glycosylation of ribonucleosides via a temporary protection of their 2&#39;,3&#39;-diol moieties utilizing a cyclic boronic ester. This method applies to several unprotected nucleosides such as adenosine, guanosine, cytidine, uridine, 5-methyluridine, and 5-fluorouridine to give corresponding disaccharide nucleosides.

苷 2 ', 3 ' -二醇酯对双糖苷合成的选择性O-糖基化

Disaccharide nucleosides, which consist of disaccharide and nucleobase moieties, have been known as a valuable group of natural products having ...

Nonspecific Proteolysis-Based Glycomics Strategy for Bacterial Glycoproteins

Protein glycosylation is one of the most common and complex post-translational modifications. Many techniques have been developed to characterize the specific roles of glycans, the relationship between their structures and their impact on the functions of proteins. A common method for glycan analysis is to employ exoglycosidase cleavage to release N-linked glycans from glycoproteins or glycopeptides using Peptide-N-Glycosidase F (PNGase F). However, the glycan-protein linkages in bacteria are different and there is no enzyme available to release glycans from bacterial glycoproteins. In addition, free glycans have also been described in mammalian cells, bacteria, yeast, plants, and fish. In this article, we present a method that can characterize the N-linked glycosylation system in Campylobacter jejuni by detecting asparagine (Asn)-linked and free glycans that are not attached to their target proteins. In this method, total proteins from C. jejuni were digested by Pronase E with a higher enzyme to protein ratio (2:1&#8722;3:1) and a longer incubation time (48&#8722;72 h). The resulted Asn-glycans and free glycans were then purified using porous graphitic carbon cartridges, permethylated, and analyzed by mass spectrometry.

This study presents a method for glycomics analysis of glycoproteins by a combination of pronase E digestion, permethylation, and mass spectrometry analysis. This method is capable of analyzing all types of N-linked glycans, including bacterial N-glycans.

Protein glycosylation is one of the most common and complex post-translational modifications. Many techniques have been developed to characterize the ...

生物化学

Targeting Cysteine Thiols for in Vitro Site-specific Glycosylation of Recombinant Proteins

Stromal interaction molecule-1 (STIM1) is a type-I transmembrane protein located on the endoplasmic reticulum (ER) and plasma membranes (PM). ER-resident STIM1 regulates the activity of PM Orai1 channels in a process known as store operated calcium (Ca2+) entry which is the principal Ca2+ signaling process that drives the immune response. STIM1 undergoes post-translational N-glycosylation at two luminal Asn sites within the Ca2+ sensing domain of the molecule. However, the biochemical, biophysical, and structure biological effects of N-glycosylated STIM1 were poorly understood until recently due to an inability to readily obtain high levels of homogeneous N-glycosylated protein. Here, we describe the implementation of an in vitro chemical approach which attaches glucose moieties to specific protein sites applicable to understanding the underlying effects of N-glycosylation on protein structure and mechanism. Using solution nuclear magnetic resonance spectroscopy we assess both efficiency of the modification as well as the structural consequences of the glucose attachment with a single sample. This approach can readily be adapted to study the myriad glycosylated proteins found in nature.

Targeting Cysteine Thiols for in Vitro Site-specific Glycosylation of Recombinant Proteins

Biochemical and structural analyses of glycosylated proteins require relatively large amounts of homogeneous samples. Here, we present an efficient chemical method for site-specific glycosylation of recombinant proteins purified from bacteria by targeting reactive Cys thiols.

靶向半胱氨酸硫用于体外重组蛋白的特异糖基化

Stromal interaction molecule-1 (STIM1) is a type-I transmembrane protein located on the endoplasmic reticulum (ER) and plasma membranes (PM). ER-resident ...

免疫与感染

Rapid Antibody Glycoengineering in Chinese Hamster Ovary Cells

Recombinant monoclonal antibodies bind specific molecular targets and, subsequently, induce an immune response or inhibit the binding of other ligands. However, monoclonal antibody functionality and half-life may be reduced by the type and distribution of host-specific glycosylation. Attempts to produce superior antibodies have inspired the development of genetically modified producer cells that synthesize glyco-optimized antibodies. Glycoengineering typically requires the generation of a stable knockout or knockin cell line using methods such as clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9. Monoclonal antibodies produced by engineered cells are then characterized using mass spectrometric methods to determine if the desired glycoprofile has been obtained. This strategy is time-consuming, technically challenging, and requires specialists. Therefore, an alternative strategy that utilizes streamlined protocols for genetic glycoengineering and glycan detection may assist endeavors toward optimal antibodies. In this proof-of-concept study, an IgG-producing Chinese hamster ovary cell served as an ideal host to optimize glycoengineering. Short interfering RNA targeting the Fut8 gene was delivered to Chinese hamster ovary cells, and the resulting changes in FUT8 protein expression were quantified. The results indicate that knockdown by this method was efficient, leading to a ~60% reduction in FUT8. Complementary analysis of the antibody glycoprofile was performed using a rapid yet highly sensitive technique: capillary gel electrophoresis and laser-induced fluorescence detection. All knockdown experiments showed an increase in afucosylated glycans; however, the greatest shift achieved in this study was ~20%. This protocol simplifies glycoengineering efforts by harnessing in silico design tools, commercially synthesized gene targeting reagents, and rapid quantification assays that do not require extensive prior experience. As such, the time efficiencies offered by this protocol may assist investigations into new gene targets.

The glycosylation pattern of an antibody determines its clinical performance, thus industrial and academic efforts to control glycosylation persist. Since typical glycoengineering campaigns are time- and labor-intensive, the generation of a rapid protocol to characterize the impact of glycosylation genes using transient silencing would prove useful.

仓鼠卵巢细胞的快速抗体糖工程

Recombinant monoclonal antibodies bind specific molecular targets and, subsequently, induce an immune response or inhibit the binding of other ligands. ...

使用唾液酸酶处理从分离的 PBMC 中去除唾液酸化单核细胞衍生的 DC

Generation of Monocyte-Derived Dendritic Cells with Differing Sialylated Phenotypes

Sialic acids are negatively charged monosaccharides typically found at the termini of cell surface glycans. Due to their hydrophilicity and biophysical characteristics, they are involved in numerous biological processes, such as modulation of the immune response, recognition of self and non-self antigens, carbohydrate-protein interactions, etc. The cellular content of sialic acid is regulated by sialidase, which catalyzes the removal of sialic acid residues. Several studies have shown that sialo-glycans are critical in monitoring immune surveillance by engaging with cis and trans inhibitory Siglec receptors on immune cells. Likewise, glyco-immune checkpoints in cancer are becoming crucial targets for developing immunotherapies. Additionally, dendritic cells (DCs) are envisioned as an important component in immunotherapies, especially in cancer research, due to their unique role as professional antigen-presenting cells (APC) and their capacity to trigger adaptive immune responses and generate immunologic memory. Nevertheless, the function of DCs is dependent on their full maturation. Immature DCs have an opposing function to mature DCs and a high sialic acid content, which further dampens their maturation level. This downregulates the ability of immature DCs to activate T-cells, leading to a compromised immune response. Consequently, removing sialic acid from the cell surface of human DCs induces their maturation, thus increasing the expression of MHC molecules and antigen presentation. In addition, it can restore the expression of co-stimulatory molecules and IL-12, resulting in DCs having a higher ability to polarize T-cells toward a Th1 phenotype and specifically activate cytotoxic T-cells to kill tumor cells. Therefore, sialic acid has emerged as a key modulator of DCs and is being used as a novel target to advance their therapeutic use. This study provides a unique approach to treat in vitro monocyte-derived DCs with sialidase, aimed at generating DC populations with different cell surface sialic acid phenotypes and tailored maturation and co-stimulatory profiles.

作者聚焦:使用基于唾液酶的细胞表面酶处理树突状细胞成熟 

A unique, comprehensive protocol to generate de-sialylated human monocyte-derived dendritic cells (mo-DCs) from isolated peripheral blood mononuclear cells (PBMCs) using a sialidase treatment is presented. Further, methods to assess the phenotypic and functional characterization of mo-DCs and evaluate how sialidase treatment improves the maturation level of mo-DCs are described.

具有不同唾液酸化表型的单核细胞来源的树突状细胞的生成

Sialic acids are negatively charged monosaccharides typically found at the termini of cell surface glycans. Due to their hydrophilicity and biophysical ...

利用N酰基修饰 Mannosamines 对唾液酸的代谢 Glycoengineering

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Nonspecific Proteolysis-Based Glycomics Strategy for Bacterial Glycoproteins

靶向半胱氨酸硫用于体外重组蛋白的特异糖基化

仓鼠卵巢细胞的快速抗体糖工程

具有不同唾液酸化表型的单核细胞来源的树突状细胞的生成

具有不同唾液酸化表型的单核细胞来源的树突状细胞的生成

具有不同唾液酸化表型的单核细胞来源的树突状细胞的生成