The C-terminal cysteine-rich region dictates specific catalytic properties in chimeras of the ectonucleotidases NTPDase1 and NTPDase2.
The C-terminal cysteine-rich region dictates specific catalytic properties in chimeras of the ectonucleotidases NTPDase1 and NTPDase2.
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DOI：
10.1046/j.1432-1033.2001.01896.x
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发表时间：
2001
期刊：
影响因子：
--
通讯作者：
P. Heine;N. Braun;Jean Sévigny;Simon C. Robson;Jörg Servos;Herbert Zimmermann
中科院分区：
文献类型：
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作者：
P. Heine;N. Braun;Jean Sévigny;Simon C. Robson;Jörg Servos;Herbert Zimmermann
外核苷三磷酸二磷酸水解酶（e - ntpases）包括一个新的外核苷酶家族，在胞外核苷酸的水解中起重要作用。相关的NTPDase1（外链apyrase）和NTPDase2（外链atpase）具有共同的膜结构，在N端和c端都有跨膜结构域，一个广泛的胞外环，具有五个“apyrase保守区域”（ACR1至ACR5），以及一个富含半胱氨酸的c端区域。CHO细胞中表达的NTPDase1对ATP和ADP的水解作用相同，而NTPDase2对ATP的水解作用高于ADP。此外，重组NTPDase1将ATP水解为AMP，仅形成少量的游离ADP。相反，当ATP被NTPDase2水解时，ADP是主要的游离产物。为了确定这些催化性能差异的分子结构域，构建了嵌合cdna，其中NTPDase1的n端序列长度增加，被NTPDase2的相应序列取代，反之亦然。转换点包含在ACR1到ACR5之间。嵌合cdna在CHO细胞中表达，并通过免疫细胞化学验证其表面表达。采用高效液相色谱法测定ATP和ADP的水解率以及ADP和AMP的产物生成。ACR3和ACR5之间的氨基酸残基，特别是ACR4和ACR5之间富含半胱氨酸的区域，赋予了嵌合酶对应于各自野生型酶的表型。蛋白质结构而不是保守的acr可能是决定相关野生型酶之间催化性能差异的主要相关因素。
Ecto-nucleoside triphosphate diphosphohydrolases (E-NTPDases) comprise a novel family of ectonucleotidases that are important in the hydrolysis of extracellular nucleotides. The related NTPDase1 (ecto-apyrase) and NTPDase2 (ecto-ATPase) share a common membrane topography with a transmembrane domain at both the N- and C-terminus, an extensive extracellular loop with five 'apyrase conserved regions' (ACR1 to ACR5), and a cysteine-rich C-terminal region. Whereas NTPDase1 expressed in CHO cells hydrolyzes ATP and ADP equivalently, NTPDase2 has a high preference for the hydrolysis of ATP over ADP. In addition recombinant NTPDase1 hydrolyzes ATP to AMP with the formation of only minor amounts of free ADP. In contrast, ADP appears as the major free product when ATP is hydrolyzed by NTPDase2. In order to determine molecular domains responsible for these differences in catalytic properties, chimeric cDNAs were constructed in which N-terminal sequences of increasing length of NTPDase1 were substituted by the corresponding sequences of NTPDase2 and vice versa. The turnover points were contained within ACR1 to ACR5. Chimeric cDNAs were expressed in CHO cells and surface expression was verified by immunocytochemistry. ATP and ADP hydrolysis rates and ADP and AMP product formation were determined using HPLC. Amino-acid residues between ACR3 and ACR5 and in particular the cysteine-rich region between ACR4 and ACR5 conferred a phenotype to the chimeric enzymes that corresponded to the respective wild-type enzyme. Protein structure rather than the conserved ACRs may be of major relevance for determining differences in the catalytic properties between the related wild-type enzymes.