Sácha P, Zámecník J, Barinka C, Hlouchová K, Vícha A, Mlcochová P, Hilgert I, Eckschlager T, Konvalinka J
Department of Biochemistry, Institute of Organic Chemistry and Biochemistry, Academy of Science of the Czech Republic, Flemingovo n.2, Prague 6, 16610 Czech Republic.
Neuroscience. 2007 Feb 23;144(4):1361-72. doi: 10.1016/j.neuroscience.2006.10.022. Epub 2006 Dec 5.
Glutamate carboxypeptidase II (GCPII) is a transmembrane glycoprotein expressed in various tissues. When expressed in the brain it cleaves the neurotransmitter N-acetylaspartylglutamate (NAAG), yielding free glutamate. In jejunum it hydrolyzes folylpoly-gamma-glutamate, thus facilitating folate absorption. The prostate form of GCPII, known as prostate specific membrane antigen (PSMA), is an established cancer marker. The NAAG-hydrolyzing activity of GCPII has been implicated in a number of pathological conditions in which glutamate is neurotoxic (e.g. amyotrophic lateral sclerosis, Huntington's disease, Alzheimer's disease, epilepsy, schizophrenia, and stroke). Inhibition of GCPII was shown to be neuroprotective in tissue culture and in animal models. GCPII is therefore an interesting putative therapeutic target. However, only very limited and controversial data on the expression and localization of GCPII in human brain are available. Therefore, we set out to analyze the activity and expression of GCPII in various compartments of the human brain using a radiolabeled substrate of the enzyme and the novel monoclonal antibody GCP-04, which recognizes an epitope on the extracellular portion of the enzyme and is more sensitive to GCPII than to the homologous GCPIII. We show that this antibody is more sensitive in immunoblots than the widely used antibody 7E11. By Western blot, we show that there are approximately 50-300 ng of GCPII/mg of total protein in human brain, depending on the specific area. Immunohistochemical analysis revealed that astrocytes specifically express GCPII in all parts of the brain. GCPII is enzymatically active and the level of activity follows the expression pattern. Using pure recombinant GCPII and homologous GCPIII, we conclude that GCPII is responsible for the majority of overall NAAG-hydrolyzing activity in the human brain.
谷氨酸羧肽酶II（GCPII）是一种在多种组织中表达的跨膜糖蛋白。当在大脑中表达时，它会裂解神经递质N - 乙酰天冬氨酰谷氨酸（NAAG），产生游离谷氨酸。在空肠中，它水解叶酰聚γ - 谷氨酸，从而促进叶酸吸收。GCPII的前列腺形式，即前列腺特异性膜抗原（PSMA），是一种公认的癌症标志物。GCPII的NAAG水解活性与许多谷氨酸具有神经毒性的病理状况有关（例如肌萎缩侧索硬化症、亨廷顿舞蹈病、阿尔茨海默病、癫痫、精神分裂症和中风）。在组织培养和动物模型中，GCPII的抑制被证明具有神经保护作用。因此，GCPII是一个有趣的潜在治疗靶点。然而，关于GCPII在人脑中的表达和定位，仅有非常有限且存在争议的数据。因此，我们着手使用该酶的放射性标记底物和新型单克隆抗体GCP - 04来分析GCPII在人脑各个区域的活性和表达，该抗体识别该酶细胞外部分的一个表位，并且对GCPII的敏感性高于同源的GCPIII。我们表明，这种抗体在免疫印迹中比广泛使用的抗体7E11更敏感。通过蛋白质免疫印迹法，我们表明，根据特定区域不同，人脑每毫克总蛋白中约有50 - 300纳克的GCPII。免疫组织化学分析显示，星形胶质细胞在大脑的所有部位都特异性表达GCPII。GCPII具有酶活性，且活性水平遵循表达模式。使用纯重组GCPII和同源GCPIII，我们得出结论，GCPII是人脑中大部分总体NAAG水解活性的原因。
