The periodontal pathogen Tannerella forsythia utilizes host sialic acids as a nutrient source. To also make O-acetylated sialyl residues susceptible to the action of its sialidase and sialic acid uptake system, Tannerella produces NanS, an O-acetylesterase with two putative catalytic domains. Here, we analyzed NanS by homology modeling, predicted a catalytic serine–histidine–aspartate triad for each catalytic domain and performed individual domain inactivation by single alanine exchanges of the triad nucleophiles S32 and S311. Subsequent functional analyses revealed that both domains possess sialyl-O-acetylesterase activity, but differ in their regioselectivity with respect to position O9 and O7 of sialic acid. The 7-O-acetylesterase activity inherent to the C-terminal domain of NanS is unique among sialyl-O-acetylesterases and fills the current gap in tools targeting 7-O-acetylation. Application of the O7-specific variant NanS-S32A allowed us to evidence the presence of cellular 7,9-di-O-acetylated sialoglycans by monitoring the gain in 9-O-acetylation upon selective removal of acetyl groups from O7. Moreover, we established de-7,9-O-acetylation by wild-type NanS as an easy and efficient method to validate the specific binding of three viral lectins commonly used for the recognition of (7),9-O-acetylated sialoglycans. Their binding critically depends on an acetyl group in O9, yet de-7,9-O-acetylation proved advantageous over de-9-O-acetylation as the additional removal of the 7-O-acetyl group eliminated ligand formation by 7,9-ester migration. Together, our data show that NanS gained dual functionality through recruitment of two esterase modules with complementary activities. This enables Tannerella to scavenge 7,9-di-O-acetylated sialyl residues and provides a novel, O7-specific tool for studying sialic acid O-acetylation.

中文翻译：

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Tannerella forsythia 的唾液酸-O-乙酰酯酶 NanS 包含两个催化模块，对唾液酸的 O7 和 O9 具有不同的区域特异性

牙周病原体Tannerella forsythia利用宿主唾液酸作为营养来源。为了也使O-乙酰化唾液酸残基对其唾液酸酶和唾液酸摄取系统的作用敏感，Tannerella 产生了 NanS，这是一种具有两个推定催化结构域的O-乙酰酯酶。在这里，我们通过同源模型分析了 NanS，预测了每个催化结构域的催化丝氨酸-组氨酸-天冬氨酸三联体，并通过三联体亲核试剂 S32 和 S311 的单个丙氨酸交换进行了单个结构域的失活。随后的功能分析表明，这两个结构域都具有唾液酸-O-乙酰酯酶活性，但它们对唾液酸的 O9 和 O7 位的区域选择性不同。7- ONanS 的 C 末端结构域所固有的 -乙酰酯酶活性在唾液酸-O -乙酰酯酶中是独一无二的，并填补了目前针对 7 - O -乙酰化的工具的空白。O7 特异性变体 NanS-S32A 的应用使我们能够通过监测从 O7 中选择性去除乙酰基后9- O-乙酰化的增益来证明细胞 7,9-二-O-乙酰化唾液聚糖的存在。此外，我们通过野生型 NanS 建立了去 7,9- O-乙酰化作为一种​​简单有效的方法来验证三种病毒凝集素的特异性结合，这些凝集素通常用于识别 (7),9- O-乙酰化唾液聚糖。它们的结合主要取决于 O9 中的乙酰基，但 de-7,9-O-乙酰化证明优于去-9- O-乙酰化，因为额外去除7 - O-乙酰基消除了由7,9-酯迁移形成的配体。总之，我们的数据表明，NanS 通过招募具有互补活性的两个酯酶模块获得了双重功能。这使 Tannerella 能够清除 7,9-二-O-乙酰化的唾液酸残基，并为研究唾液酸O-乙酰化提供了一种新的 O7 特异性工具。