The Shigella flexneri bacteriophage Sf6 tailspike protein (TSP)/endorhamnosidase is related to the bacteriophage P22 TSP and has a motif common to exo- and endoglycanases, and C-5 epimerases
The Shigella flexneri bacteriophage Sf6 tailspike protein (TSP)/endorhamnosidase is related to the bacteriophage P22 TSP and has a motif common to exo- and endoglycanases, and C-5 epimerases
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DOI：
10.1099/13500872-145-7-1649
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发表时间：
1999-07-01
期刊：
影响因子：
--
通讯作者：
Morona, R
中科院分区：
文献类型：
--
作者：
Chua, JEH;Manning, PA;Morona, R
温带噬菌体 Sf6 感染血清型 X 或 Y 的福氏志贺氏菌菌株，将它们分别转化为血清型 3a 或 3b。 Sf6 的尾刺蛋白 (TSP) 具有内切 1,3-α-L-鼠李糖苷酶（内切鼠李糖苷酶）活性，在噬菌体吸附到细胞表面的过程中导致脂多糖 O 抗原受体裂解。当用于Southern杂交时，P22基因g（编码P22 TSP）DNA探针与Sf6的限制性片段Pstl-7杂交。 Pstl-7 和相邻 Pstl-8 片段的 DNA 测序和分析揭示了一个 1872 bp（624 个氨基酸）的开放阅读框（ORF1），其氨基酸序列与噬菌体 P22 TSP N 端头结合域具有同源性。关键残基的高度保守表明该区域中的 Sf6 TSP 具有相似的二级和三级 N 端蛋白质结构以及相似的功能。此外，在残基 164 和 184 之间鉴定了一个氨基酸序列基序 (DFGX(3)DGX(6)AX(3)A)，该基序也被发现存在于各种原核和真核外切/内切聚糖酶、C-5 差向异构酶和噬菌体蛋白中。来自 T7 启动子的 ORF1 表达产生了 67 kDa 蛋白（通过 L-[S-35]蛋氨酸标记和 SDS-PAGE 检测），通过与完整的 Sh、福氏 Y 细胞一起孵育，对含有 ORF1 编码蛋白的热处理细胞质提取物进行分析，证明存在 O 抗原水解活性；因此 ORF1 编码 Sf6 TSP。 Sf6 TSP 对长链 Sh、福氏血清型 X 或 Y O 抗原表现出特异性和优先活性，其裂解导致寡糖片段的释放，通过荧光团辅助碳水化合物电泳 (FACE) 检测到，寡糖片段的大小与八糖一致。
The temperate bacteriophage Sf6 infects Shigella flexneri strains of serotype X or Y, converting them into serotypes 3a or 3b, respectively. The tailspike protein (TSP) of Sf6 possesses endo-1,3-alpha-L-rhamnosidase (endorhamnosidase) activity which results in cleavage of the lipopolysaccharide O-antigen receptor during the adsorption of the phage to the cell surface. When used in Southern hybridization, a P22 gene g (encoding P22 TSP) DNA probe hybridized with restriction fragment Pstl-7 of Sf6. DNA sequencing and analysis of Pstl-7 and the adjacent Pstl-8 fragment revealed an open reading frame (ORF1) of 1872 bp (624 amino acids) bearing amino acid sequence homology to the bacteriophage P22 TSP N-terminal head-binding domain. High conservation of key residues was suggestive of similar secondary and tertiary N-terminal protein structure and a similar function of the Sf6 TSP in this region. In addition, an amino acid sequence motif (DFGX(3)DGX(6)AX(3)A) was identified between residues 164 and 184 which was also found to exist in various prokaryotic and eukaryotic exo-/endoglycanases, C-5 epimerases and bacteriophage proteins. Expression of ORF1 from a T7 promoter produced a 67 kDa protein (detected by L-[S-35]methionine labelling and SDS-PAGE), Assay of heat-treated cytoplasmic extracts containing the ORF1-encoded protein by incubation with whole Sh, flexneri Y cells demonstrated that O-antigen hydrolysis activity was present; ORF1 therefore encodes Sf6 TSP. Sf6 TSP exhibited specific and preferential activity for long-chain Sh, flexneri serotype X or Y O-antigen,, cleavage of which resulted in the release of oligosaccharide fragments, consistent with octasaccharides in size, as detected by fluorophore-assisted carbohydrate electrophoresis (FACE).