Structural determination of the sheath-forming polysaccharide of Sphaerotilus montanus using thiopeptidoglycan lyase which recognizes the 1,4 linkage between α-d-GalN and β-d-GlcA
Structural determination of the sheath-forming polysaccharide of Sphaerotilus montanus using thiopeptidoglycan lyase which recognizes the 1,4 linkage between α-d-GalN and β-d-GlcA
复制标题
使用硫肽聚糖裂解酶对 Sphaerotilus montanus 的鞘形成多糖进行结构测定，硫肽聚糖裂解酶可识别 α-d-GalN 和 β-d-GlcA 之间的 1,4 连接
DOI：
10.1016/j.ijbiomac.2021.05.001
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发表时间：
2021
影响因子：
8.2
通讯作者：
Takeda Minoru
中科院分区：
文献类型：
--
作者：
Kashiwabara Daisuke;Kondo Keiko;Usami Ryoji;Kan Daisuke;Kawamura Izuru;Kawasaki Yuta;Sato Michio;Nittami Tadashi;Suzuki Ichiro;Katahira Masato;Takeda Minoru
Sphaerotilusnatansis是一种在活性污泥中常见的丝状鞘形成细菌。它的鞘是由一种叫做硫肽聚糖的硫醇糖缀合物组装而成的。montanusATCC-BAA-2725是溪流生物膜的鞘形成成员，其鞘的形态与S.然而，它具有热敏感性，这使它区别于S。纳坦鞘。在这项研究中，化学成分和固态核磁共振分析表明，S。Montanussheath不含半胱氨酸，表明二硫键对于鞘的形成不是必需的。帖前通过N-乙酰化成功地溶解了montanussheath，允许溶液状态NMR分析以确定糖序列。鞘对由硫肽聚糖同化细菌Paenibacilluskoleovorans制备的硫肽聚糖裂解酶敏感。用4-氨基苯甲酸乙酯标记酶促底物的还原末端，然后进行HPLC。检测到两种衍生物，并确定了它们的结构。结果表明，鞘中没有肽段，其组装方式为：[→4）-β-d-GlcA-（1→4）-β-d-Glc-（1→3）-β-d-GalNAc-（1 → 4）-α-d-GalNAc-（1 → 4）-α-d-GalN-（1→]n（β-d-Glc和α-d-GalNAc分别以化学计量和亚化学计量的方式被3-O-乙酰化）。因此证实硫肽聚糖裂解酶切割α-d-GalN和β-d-GlcA之间的1，4键，而不管肽部分如何。此外，鞘的活体荧光染色表明，延长发生在尖端，与S。纳坦鞘。
Sphaerotilusnatansis a filamentous sheath-forming bacterium commonly found in activated sludge. Its sheath is assembled from a thiolic glycoconjugate called thiopeptidoglycan.S. montanusATCC-BAA-2725 is a sheath-forming member of stream biofilms, and its sheath is morphologically similar to that ofS. natans.However, it exhibits heat susceptibility, which distinguishes it from theS. natanssheath. In this study, chemical composition and solid-state NMR analyses suggest that theS. montanussheath is free of cysteine, indicating that disulfide linkage is not mandatory for sheath formation. TheS. montanussheath was successfully solubilized byN-acetylation, allowing solution-state NMR analysis to determine the sugar sequence. The sheath was susceptible to thiopeptidoglycan lyase prepared from the thiopeptidoglycan-assimilating bacterium,Paenibacilluskoleovorans. The reducing ends of the enzymatic digests were labeled with 4-aminobenzoic acid ethyl ester, followed by HPLC. Two derivatives were detected, and their structures were determined. We found that the sheath has no peptides and is assembled as follows: [→4)-β-d-GlcA-(1→4)-β-d-Glc-(1→3)-β-d-GalNAc-(1→4)-α-d-GalNAc-(1→4)-α-d-GalN-(1→]n(β-d-Glc and α-d-GalNAc are stoichiometrically and substoichiometrically 3-O-acetylated, respectively). Thiopeptidoglycan lyase was thus confirmed to cleave the 1,4 linkage between α-d-GalN and β-d-GlcA, regardless of the peptide moiety. Furthermore, vital fluorescent staining of the sheath demonstrated that elongation takes place at the tips, as with theS. natanssheath.