MOLECULAR-CLONING AND FUNCTIONAL EXPRESSION OF BACTERIOPHAGE-PK1E-ENCODED ENDONEURAMINIDASE ENDO NE
MOLECULAR-CLONING AND FUNCTIONAL EXPRESSION OF BACTERIOPHAGE-PK1E-ENCODED ENDONEURAMINIDASE ENDO NE
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DOI：
10.1111/j.1365-2958.1995.tb02409.x
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发表时间：
1995-05-01
影响因子：
3.6
通讯作者：
FROSCH, M
中科院分区：
文献类型：
--
作者：
GERARDYSCHAHN, R;BETHE, A;FROSCH, M
均聚β-2，8-连接唾液酸（PSA）已被发现作为脓毒症和脑膜炎引起的细菌病原体的荚膜成分，并在真核细胞上作为神经细胞粘附分子（NCAM）的翻译后修饰。该多糖被噬菌体编码的内切-N-乙酰神经氨酸酶E（Endo NE）特异性识别和降解。因此，Endo NE已成为研究细菌致病机制和真核生物形态发生的有价值的工具。在这份报告中，我们描述的分子克隆的Endo NE和表达的功能活性的重组酶。克隆的DNA序列（2436 bp）编码811个氨基酸的多肽，其5'端含有完全保守的神经氨酸酶基序。该酶在大肠杆菌中表达，SDS-PAGE电泳显示为约74 kDa的单一条带，其669个氨基酸残基的中心结构域与最近克隆的Endo NF的同源性约为90%，重组酶对细菌的裂解和PSA降解的催化作用均被针对完整噬菌体颗粒的多克隆抗血清有效地抑制，C-末端区域似乎对酶功能至关重要，因为同源结构域外的32个氨基酸的截短完全消除了Endo NE活性。我们的数据还表明，38 kDa的蛋白质，以前被认为是一个亚基的内NE全酶，是一个单独的基因位点的产品，是不必要的体外解聚酶活性。
Homopolymeric beta-2,8-linked sialic acid (PSA) has been found as a capsular component of sepsis- and meningitis-causing bacterial pathogens, and on eukaryotic cells as a post-translational modification of the neural cell adhesion molecule (NCAM). The polysaccharide is specifically recognized and degraded by a phage-encoded enzyme, the endo-N-acetylneuraminidase E (Endo NE). Endo NE therefore has become a valuable tool in the study of bacterial pathogenesis and eukaryotic morphogenesis. In this report we describe the molecular cloning of Endo NE and the expression of a functionally active recombinant enzyme. The cloned DNA sequence (2436 bp) encodes a polypeptide of 811 amino acids, which at the 5' end contains a totally conserved neuraminidase motif. Expressed in Escherichia coli, the enzyme migrates as a single band of approximately 74 kDa in SDS-PAGE, A central domain of 669 amino acid residues is about 90% homologous to the recently cloned Endo NF, Both phage-induced lysis of bacteria and the catalysis of PSA degradation by the recombinant enzyme are efficiently inhibited by a polyclonal antiserum raised against the intact phage particle, The C-terminal region seems to he essential to enzymatic functions, as truncation of 32 amino acids outside the homology domain completely abolishes Endo NE activity. Our data also indicate that the 38 kDa protein, previously assumed to be a subunit of the Endo NE holoenzyme, is the product of a separate gene locus and is not necessary for in vitro depolymerase activity.