Chitin-Based Organic Networks: An Integral Part of Cell Wall Biosilica in the Diatom Thalassiosira pseudonana
Chitin-Based Organic Networks: An Integral Part of Cell Wall Biosilica in the Diatom Thalassiosira pseudonana
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DOI：
10.1002/anie.200905028
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发表时间：
2009-01-01
影响因子：
16.6
通讯作者：
van Pee, Karl-Heinz
中科院分区：
文献类型：
--
作者：
Brunner, Eike;Richthammer, Patrick;van Pee, Karl-Heinz
硅藻[1]细胞壁是天然杂化材料的杰出例子，并表现出非常有趣的机械和光学特性。[2]它们的结构和组成继续激发各种仿生合成方法。[3]硅藻是二氧化硅生物矿化研究的首选模式生物。[4]它们的分层结构细胞壁含有无定形二氧化硅以及特殊的生物分子。在过去的十年中，已经确定了三种不同类型的生物分子：1）silaffins，高度后修饰的肽/蛋白质;[5] 2）长链多胺（LCPA）;[6]和3）高度酸性的silacidins。[7]两性离子型硅杂环化合物自组装成超分子聚集体。对于LCPA [8]观察到相同的情况，只要存在适当选择的抗衡离子如正磷酸盐或焦磷酸盐，或带负电荷的肽如硅酸肽。无论是聚集的silaffins以及长链多胺诱导快速二氧化硅沉淀在体外从含草酸的解决方案。为了鉴定这些分子，将生物二氧化硅溶解在HF或NH4F中。然后发现Silaffins、LCPA和silacidins溶解在提取溶液中。硅藻物种Thalassiosira damanana是这一领域的模式生物;[4]其基因组已被完全测序。[9]最近的离子磨损扫描电子显微镜[10]以及原子力显微镜研究[11]对T. Escherichana揭示了在生长的细胞壁内存在丝状纳米级和微米级结构，这些结构显然含有中心模板有机结构（"线性蛋白质"）。[10]甲壳素（聚-N-乙酰-d-葡萄糖胺）存在于许多钙基生物矿物中。[12]它被假定为形成不溶性支架或隔室，其中几丁质相关的生物分子控制钙生物矿化事件。然而，到目前为止，甲壳素还没有被确定在硅藻细胞壁的生物二氧化硅形成。另一方面，一些硅藻物种如海链藻（Thalassiosira sp.）从高度结晶的β-几丁质合成外部纤维。[13]有趣的是，最近的T.这表明几丁质在细胞壁生物合成中可能起作用。[14]此外，在固体核磁共振光谱分析中观察到多糖如几丁质的特征信号。紫云英细胞壁。[15]本工作的目的是阐明几丁质在T.特别是关于嵌入或紧密结合到生物二氧化硅中的"内部"几丁质的存在。扫描电镜（SEM）图像显示T.图1中显示了Escherichia的细胞壁。使用基于十二烷基硫酸钠（SDS）和乙二胺四乙酸（EDTA）处理的既定方法提取细胞壁（见实验部分）。培养物在相同条件下生长，但使用流式离心机或过滤器收获（见实验部分）。过滤后的样品含有大量众所周知的外部甲壳素纤维（图1，顶部）。这一观察结果通过13 C固态NMR光谱得到证实（图2）。过滤样品的光谱主要由结晶β-甲壳素特征化学位移处的强窄共振[16]。在用Calcofluor白色染色后，可以观察到材料的强烈荧光，Calcofluor是一种优先结合β-1，4-结合多糖的荧光染料。这证实了存在大量的外部的、染料可接近的甲壳素（参见支持信息）。相反，外部的甲壳素是从用流动离心机收集的样品中除去的。
Diatom [1] cell walls are outstanding examples of natural hybrid materials and exhibit extraordinarily interesting mechanical and optical properties.[2] Their structure and composition continue to inspire a variety of biomimetic synthesis approaches.[3] Diatoms are preferred model organisms in silica biomineralization studies.[4] Their hierarchically structured cell walls contain amorphous silica as well as special biomolecules. Over the past decade, three different classes of such biomolecules have been identified: 1) the silaffins, highly post-translationally modified peptides/proteins;[5] 2) long-chain polyamines (LCPAs);[6] and 3) the highly acidic silacidins.[7] The zwitterionic silaffins self-assemble into supramolecular aggregates. The same was observed for LCPAs [8] provided a properly chosen counterion such as orthophosphate or pyrophosphate, or a negatively charged peptide such as silacidin is present. Both the aggregated silaffins as well as the long-chain polyamines induce rapid silica precipitation in vitro from silicic acid containing solutions. To identify these molecules, biosilica was dissolved in HF or NH4F. Silaffins, LCPAs, and silacidins were then found to be dissolved in the extraction solutions. The diatom species Thalassiosira pseudonana is an established model organism in this area;[4] its genome has been completely sequenced.[9] Recent ion-abrasion scanning electron microscopic [10] as well as atomic force microscopic studies [11] on cell wall formation in T. pseudonana revealed the presence of filamentous nano-and microscale structures within the growing cell wall which apparently contain central templating organic structures (“linear proteins”).[10] Chitin (poly-N-acetyl-d-glucosamine) occurs in numerous calciumbased biominerals.[12] It is assumed to form insoluble scaffolds or compartments, wherein chitin-associated biomolecules control calcium biomineralization events. So far, however, chitin has not been identified in biosilica formation in diatom cell walls. On the other hand, several diatom species such as Thalassiosira sp. synthesize external fibers from highly crystalline β-chitin.[13] Interestingly, recent gene expression studies of T. pseudonana indicate a possible role of chitin in cell wall biosynthesis.[14] Furthermore, signals characteristic for polysaccharides such as chitin were observed in solid-state NMR spectroscopic analyses of T. pseudonana cell walls.[15] The aim of the present work is the elucidation of the possible role of chitin in T. pseudonana cell walls, in particular with respect to the presence of “internal” chitin embedded in or tightly bound to the biosilica. SEM images of T. pseudonana cell walls are shown in Figure 1. The cell walls were extracted using the established method based on treatment with sodium dodecylsulfate (SDS) and ethylenediamine tetraacetic acid (EDTA)(see the Experimental Section). Cultures were grown under identical conditions but harvested either with the use of a flow centrifuge or a filter (see Experimental Section). The filtered samples contain large amounts of the well-known external chitin fibers (Figure1, top). This observation is confirmed by 13C solid-state NMR spectroscopy (Figure 2). The spectrum of the filtered sample is dominated by intense, narrow resonances at chemical shifts characteristic of crystalline β-chitin [16]. An intense fluorescence of the material can be observed after staining with Calcofluor White, a fluorescence dye that preferentially binds to β-1, 4-bound polysaccharides. This confirms the presence of high amounts of external, dye-accessible chitin (see the Supporting Information).In contrast, external chitin is removed from samples harvested with a flow centrifuge …