Biochemical characterization of human glutamate carboxypeptidase III
Biochemical characterization of human glutamate carboxypeptidase III
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DOI：
10.1111/j.1471-4159.2006.04341.x
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发表时间：
2007-05-01
影响因子：
4.7
通讯作者：
Konvalinka, Jan
中科院分区：
文献类型：
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作者：
Hlouchova, Klara;Barinka, Cyril;Konvalinka, Jan
人谷氨酸羧肽酶II(GCPII)是一种跨膜金属肽酶，主要存在于脑、小肠和前列腺中。在大脑中，它会分解N-乙酰-L-天冬氨酸，释放出游离的谷氨酸。在中风和其他神经退行性变的模型中，抑制GCPII已被证明具有神经保护作用。在前列腺癌中，它被称为前列腺特异性膜抗原，一种癌症标记物。最近，人谷氨酸羧基肽酶III(GCPIII)被克隆出来，它是一个氨基酸同源性为67%的GCPII同源物。虽然GCPII被认为是一个重要的药物靶点，但目前还没有关于人类GCPIII的生化研究。在这里，我们报告了重组人GCPIII的克隆、表达和鉴定。我们发现GCPIII缺乏二肽基肽酶IV的活性，它的活性依赖于N-糖基化，并且它可以被几种已知的GCPII抑制剂有效地抑制。与GCPII相比，GCPIII具有较低的N-乙酰-L-天冬氨酸水解酶活性，不同的pH和盐浓度依赖性，以及明显的底物特异性，表明这些同系物可能发挥不同的生物学作用。基于分子模型，我们解释了这两种酶的不同底物专一性，并通过定点突变检查了导致差异的氨基酸残基。这些结果可能有助于设计有效的和选择性的这两种酶的抑制剂。
Human glutamate carboxypeptidase II (GCPII) is a transmembrane metallopeptidase found mainly in the brain, small intestine, and prostate. In the brain, it cleaves N-acetyl-L-aspartyl-glutamate, liberating free glutamate. Inhibition of GCPII has been shown to be neuroprotective in models of stroke and other neurodegenerations. In prostate, it is known as prostate-specific membrane antigen, a cancer marker. Recently, human glutamate carboxypeptidase III (GCPIII), a GCPII homolog with 67% amino acid identity, was cloned. While GCPII is recognized as an important pharmaceutical target, no biochemical study of human GCPIII is available at present. Here, we report the cloning, expression, and characterization of recombinant human GCPIII. We show that GCPIII lacks dipeptidylpeptidase IV-like activity, its activity is dependent on N-glycosylation, and it is effectively inhibited by several known inhibitors of GCPII. In comparison to GCPII, GCPIII has lower N-acetyl-L-aspartyl-glutamate-hydrolyzing activity, different pH and salt concentration dependence, and distinct substrate specificity, indicating that these homologs might play different biological roles. Based on a molecular model, we provide interpretation of the distinct substrate specificity of both enzymes, and examine the amino acid residues responsible for the differences by site-directed mutagenesis. These results may help to design potent and selective inhibitors of both enzymes.