Published:20 December 2022,
Published Online:09 September 2022,
Received:18 April 2022,
Accepted:23 May 2022
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The sialidase-mediated desialylation plays an important role in the dynamic regulation of sialic acid (SA) on the cell surface and is involved in a variety of physiological and pathological processes. For one thing, the in vitro simulation and in-depth study of this important process will help us deeply understand many physiological processes related to SA, and for another, it will also lay a foundation for the construction of biomimetic materials involving enzymes. Here, we used glycopeptide nanofibers with a large exposure of SA on the surface as the starting structure and then replicated the sialidase-mediated desialylation process by adding sialidase to the solution. The results showed that the added sialidase indeed led to the cleavage of SA on the fiber surface and thus triggered the morphological transformation of glycopeptide fibers. We also regulated the kinetics of SA removal by changing the sialidase concentration, and the results showed that the rate of removal of sialic acid determined the evolution path of glycopeptide fibers. Slow removal of SA led to the gradual evolution of the initial double-helix glycopeptide fibers into micelles, and the high rate of SA excision allowed the initial double-helical glycopeptide fibers to eventually evolve into twisted nanoribbons. In addition, the application of this strategy in cell culture has also been demonstrated.
设计了一种使用唾液酸酶诱导糖肽纤维组装转化的方法,通过调节酶浓度,发现纤维的形态演化路径与唾液酸的切除速度有关. 这种转化在细胞培养中有潜在的应用.
Glycopeptides;
Self-assembly;
Sialidase;
Morphology transformation
唾液酸(sialic acid)处于众多脊椎动物细胞表面聚糖分子的末端,掩盖了底层聚糖链上的表位,赋予了细胞表面负电荷和亲水性[
由于糖缀合物结构本身的复杂性,均质且化学成分确定的糖缀合物很难通过生物来源获得.引入化学合成的糖聚合物、糖肽或糖脂[
在之前的工作中,我们利用合成的一系列由寡糖和寡肽2个组分组成的糖肽模型研究了寡糖和寡肽2个组分对于纤维缔合行为的影响,并发现最末端的唾液酸对于纤维的缔合行为有着至关重要的影响[
乙基-(3-二甲基氨基丙基)碳酰二亚胺盐酸盐、1-羟基苯并三唑、N,N-二异丙基乙胺、Boc-L-苯丙氨酸、L-苯丙氨酸甲酯盐酸盐、三氟乙酸等,购自百灵威科技有限公司;3'-唾液酸乳糖,购自Sigma-Aldrich公司;三氟甲磺酸钇,购自梯希爱(上海)化成工业发展有限公司;唾液酸酶(AdvanceBio Sialidase A),购自安捷伦科技有限公司,直接使用;超纯水经ULTRAPURE (TYPE1)WATER SYSTEM处理后使用.
核磁共振波谱(NMR):核磁共振碳谱和氢谱均在400 MHz核磁共振波谱仪(徳国Bruker)上测定得到.
基质辅助激光解吸电离-串联飞行时间质谱(Maldi-TOF):在5800 Maldi-TOF/TOF飞行时间质谱仪(美国AB SCIEX)上测定. 选用反式-2-(3-(4-叔丁基苯基)-2-甲基-2-亚丙烯基)丙二腈(DCTB)作为基质.
高效液相色谱(HPLC):在LC1100液相色谱仪(美国Agilent)上测定.
透射电镜(TEM):在HR2100透射电子显微镜(日本JEOL)或HT7800高反差透射电子显微镜(日本日立)上测定. 制样流程:首先将铜网进行亲水化处理,再将5 μL的样品滴于铜网,吸附1 min后吸去液体,随后取5 μL的醋酸铀溶液滴于铜网,吸附20 s后吸去液体,待铜网干燥后进行测试.
荧光发射光谱(FLS):在高分辨稳态瞬态荧光光谱测量系统(FLS1000,英国爱丁堡)上测定,激发波长为300 nm,测试样品体积为800 μL.
紫外光谱(UV-Vis):在紫外可见分光光度计(Lambda 750,美国Perkin-Elmer)上测定得到.
圆二色光谱(CD):在Chirascan 圆二色光谱仪(英国Applied Photophysics Ltd)上测试得到,光程为1 mm.
合成实验主要参照参考文献[
组装实验:取合成的3'SL-FFF糖肽分子5 mg,溶解在5 mL纯水中,超声至样品完全溶解,在室温下组装.
酶切形貌转变:取组装7天以上的糖肽组装液1 mL,预热至37 ℃,分别加入0.01、0.05、0.25 和0.5 U唾液酸酶,直接取样,通过HPLC分析酶切的反应效率,并进行相应的形貌表征.
细胞毒性测试:细胞的脱氢酶活性使用CCK-8试剂盒检测,由此来评估各种唾液酸乳糖糖肽的细胞毒性. 向孔板中接种RAW 264.7细胞悬液,每组进行3个平行实验,在恒温培养箱中预培养一段时间后,向各孔中加入梯度浓度的糖肽组装体(25~200 μg/mL),在与细胞共孵育24 h后加入10%的CCK-8试剂,继续孵育2 h,用酶标仪检测在450 nm处的吸光度.
细胞迁移(Transwell)实验:选用直径为5 μm的Transwell小室于24孔板中润湿5 min,在新的24孔板中加入750 μL的1640完全培养基,在上室加入75 μL的RAW 264.7细胞悬液和组装体,加酶组在培养3 h后加入,培养过夜后使用多聚甲醛固定、吉姆萨染液染色. 每个小室拍照随机选取3个视野,手动或ImageJ计数取平均值.
唾液酸乳糖作为一种来自母乳的寡糖,具有提高免疫力、抗感染和促进大脑发育等作用. 它在抑制病毒黏附[
Fig. 1 (a) Chemical structure of glycopeptide studied here. (b) HPLC analysis on the conversion from 3'SL-FFF to Lac-FFF. Inset: HPLC spectra of pure 3'SL-FFF (blue), 1:1 mixture of 3'SL-FFF and Lac-FFF (green) , pure Lac-FFF (yellow).
酶促自组装得到的组装体往往是热力学和动力学共同作用的结果,已有文献报道酶反应调节的酶促动力学会对自组装产生一定影响[
在此前的研究中我们发现,3'SL-FFF分子在水中组装成为双螺旋纤维的驱动力主要来自以下几个方面:寡糖部分之间的氢键相互作用和唾液酸部分贡献的静电排斥相互作用,寡肽部分之间的疏水相互作用,π-π相互作用以及氢键相互作用,其中端基的唾液酸部分对总体氢键(寡糖部分贡献的氢键+寡肽部分贡献的氢键)的贡献超过了30%,此外唾液酸部分提供的静电相互作用对于双螺旋纤维独特的PP II helix二级结构的形成以及双螺旋纤维离散的缔合行为都有着至关重要的影响. 这启发我们,如果能够利用唾液酸酶将双螺旋纤维表面的唾液酸切除,则纤维的自身形貌将很难继续维持. 如图
Fig. 2 TEM images of nanofibers assembled by (a) 3'SL-FFF after adding 0.01 U sialidase for (b) 12 h and (c) 24 h or adding 0.5 U sialidase for (e) 12 h and (f) 24 h. Representative AFM images of 3'SL-FFF nanofibers after adding (d) 0 U, (g) 0.01 U, (h) 0.5 U sialidase for 12 h.
为了在超分子水平上对转变过程有进一步的了解,进一步利用圆二色光谱和紫外-可见光光谱法对加入0.01和0.5 U唾液酸酶情况下组装体的转变过程进行了原位追踪. 紫外-可见光光谱中最大吸收峰值落在192 nm处,表明在水中聚集体的酰胺键排列成了螺旋结构[
Fig. 3 (a) Time-dependent UV-Vis absorbance at 192 nm with 3'SL-FFF at different concentrations of sialidase and the corresponding trend lines. (b) CD spectrum of 3'SL-FFF and Lac-FFF nanostructure obtained at different enzyme concentrations after 24 h. (c) Time-dependent CD intensity at 205 nm with 3'SL-FFF at different concentrations of sialidase. (d) Fluorescence emission spectra (excitation at 300 nm) of 3'SL-FFF and Lac-FFF nanostructure obtained at different enzyme concentrations after 24 h.
Fig. 4 Schematic presentation of sialidase-induced morphology transition.
进一步采用荧光发射光谱测试来探究糖肽在酶催化过程中有序排列的变化(
巨噬细胞的表面表达唾液酸结合性免疫球蛋白样凝集素(Siglec-1, CD169)[
首先确定得到的3'SL-FFF、Lac-FFF 2种组装体是否表现出对巨噬细胞的细胞毒性,分别设置了25、50、100、200 μg/mL的浓度梯度,将它们和巨噬细胞共孵育24 h后,如
Fig. 5 Cell viability of 3'SL-FFF and Lac-FFF.
为了验证转变前后组装体对巨噬细胞的黏附程度有一定程度的影响,设置了Transwell实验来进行验证(
Fig. 6 The effects of glyco-assemblies on the migration of RAW 264.7 cells detected by the transwell assay incubated with serum-free DMEM culture medium containing (a) 0.6% BSA or (b) 10% FBS; (d) nanofibrils assembled by 3'SL-FFF; (e) Lac-FFF prepared in advance; (f) Lac-FFF generated in situ; (c) Graphs of cell count statistics. Key: ***p<0.001.
综上所述,我们利用表面高“表达”唾液酸的糖肽纳米纤维作为载体,进行了唾液酸酶对纤维表面的唾液酸切除的动力学研究. 通过简单的控制加入的唾液酸酶的浓度,实现了不同的唾液酸切除速率,3'SL-FFF得到的组装体并不是单一产物,主要包含2种不同的转化路径. 随着酶浓度的增加,得到的产物与原来纤维的结构更加接近并且有序,这一点对酶促自组装过程中加入酶浓度的筛选具有一定意义. 最后初步探索了以巨噬细胞为模板,3'SL-FFF组装体的酶切转换作为辅助培养多细胞团基质的可能性.
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