Prostate-specific membrane antigen: a novel folate hydrolase in human prostatic carcinoma cells.
Prostate-specific membrane antigen: a novel folate hydrolase in human prostatic carcinoma cells.
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发表时间：
1996-09
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通讯作者：
J. Pinto;Brian S. Suffoletto;T. Berzin;C. Qiao;Silan Lin;W. Tong;Florian May;Bhaskar Mukherjee;W. Heston
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作者：
J. Pinto;Brian S. Suffoletto;T. Berzin;C. Qiao;Silan Lin;W. Tong;Florian May;Bhaskar Mukherjee;W. Heston
一种新的单克隆抗体已经开发出来，它与人前列腺癌，特别是高级别肿瘤有强烈的反应。这种抗体（7 E11 C-5）目前正作为转移性疾病的显像剂进行III期试验。我们克隆了编码被7 E11 C-5单克隆抗体识别的抗原的基因，并将这种独特的蛋白质命名为前列腺特异性膜（PSM）抗原。PSM抗原是一种推定的II类跨膜糖蛋白，分子大小为Mr 94，000。在功能上，II类膜蛋白充当转运或结合蛋白或具有水解活性。初步研究表明，蝶酰单谷氨酸（叶酸）与膜组分的结合也与PSM单克隆抗体发生交叉反应。我们观察到与PSM抗原相关的叶酸水解酶具有显着的羧肽酶活性。我们的研究的目的是证明，人前列腺癌细胞表达PSM抗原表现出叶酸水解酶活性使用甲氨蝶呤三谷氨酸（MTXGlu 3）和蝶酰五谷氨酸（PteGlu 5）作为底物。使用毛细管电泳检测来自四种人前列腺癌细胞系（LNCaP、PC-3、TSU-Prl和杜克-145）的分离的膜级分的叶酸水解酶活性。在各种pH范围内以及在存在和不存在硫醇试剂的情况下定时孵育后，用硼酸钠和SDS的电解质实现蝶酰基（谷氨酸）n衍生物的分离，同时在300 nm处监测吸光度。结果清楚地表明，高度表达PSM的LNCaP细胞水解MTXGlu 3的γ-谷氨酰键。膜结合酶是一种外肽酶，因为随着MTX和PteGlu 1的积累，它分别从MTX Glu 3和PteGlu 5中逐渐释放谷氨酸。半纯化的酶在pH 2.5至9.5范围内具有广泛的活性，并在pH 5和8时表现出最大活性。酶活性在还原型谷胱甘肽、高半胱氨酸和对羟基苯甲酸汞（0.05-0.5 mm）存在下得以维持，但受到DTT（>/=0.2 mm）的微弱抑制。与LNCaP细胞膜相反，从其他人前列腺腺癌细胞（PC-3、杜克-145和TSU-Pr 1）分离的膜不表现出可比较的水解酶活性，它们也不与7 E11-C5单克隆抗体反应。在用亚克隆到pREP 7真核表达载体中的全长2.65-kb PSM cDNA转染PC-3细胞后，非PSM抗原表达PC-3细胞产生了对7 E11-C5单克隆抗体的免疫反应性，并表现出与LNCaP细胞相同的叶酸水解酶活性和最适pH活性曲线。来自LNCaP转染细胞和PC-3转染细胞的膜结合酶也具有从N-乙酰基-α-乙酰基谷氨酸水解α-连接的谷氨酰基部分的能力。我们已经确定PSM抗原是蝶酰聚-γ-谷氨酰羧肽酶（叶酸水解酶），并在人前列腺癌中强烈表达。表达这种酶的癌细胞对甲氨蝶呤治疗有抗性。那些开发利用叶酸拮抗剂治疗前列腺癌的未来治疗策略的人需要考虑这种耐药机制。
A novel monoclonal antibody has been developed that reacts strongly with human prostatic cancer, especially tumors of high grade. This antibody (7E11C-5) is currently in Phase 3 trials as an imaging agent for metastatic disease. We have cloned the gene that encodes the antigen that is recognized by the 7E11C-5 monoclonal antibody and have designated this unique protein prostate-specific membrane (PSM) antigen. PSM antigen is a putative class II transmembranous glycoprotein exhibiting a molecular size of Mr 94,000. Functionally, class II membrane proteins serve as transport or binding proteins or have hydrolytic activity. Preliminary studies have demonstrated binding of pteroylmonoglutamate (folate) to membrane fractions that also cross-reacted with the PSM monoclonal antibody. We observed substantial carboxypeptidase activity as folate hydrolase associated with PSM antigen. The purpose of our study was to demonstrate that human prostatic carcinoma cells expressing PSM antigen exhibit folate hydrolase activity using methotrexate triglutamate (MTXGlu3) and pteroylpentaglutamate (PteGlu5) as substrates. Isolated membrane fractions from four human prostate cancer cell lines (LNCaP, PC-3, TSU-Prl, and Duke-145) were examined for folate hydrolase activity using capillary electrophoresis. After timed incubations at various pH ranges and in the presence and absence of thiol reagents, separation of pteroyl(glutamate)n derivatives was achieved with an electrolyte of sodium borate and SDS, while absorbance was monitored at 300 nm. The results demonstrate clearly that LNCaP cells, which highly express PSM, hydrolyze gamma-glutamyl linkages of MTXGlu3. The membrane-bound enzyme is an exopeptidase, because it progressively liberates glutamates from MTXGlu3 and PteGlu5 with accumulation of MTX and PteGlu1, respectively. The semipurified enzyme has a broad activity from pH 2.5 to 9.5 and exhibits activity maxima at pH 5 and 8. Enzymatic activity is maintained in the presence of reduced glutathione, homocysteine, and p-hydroxymercuribenzoate (0.05-0.5 mm) but was inhibited weakly by DTT (>/=0.2 mm). By contrast to LNCaP cell membranes, membranes isolated from other human prostate adenocarcinoma cells (PC-3, Duke-145, and TSU-Pr1) did not exhibit comparable hydrolase activity, nor did they react with 7E11-C5 monoclonal antibody. After transfection of PC-3 cells with a full-length 2.65-kb PSM cDNA subcloned into a pREP7 eukaryotic expression vector, non-PSM antigen-expressing PC-3 cells developed immunoreactivity to 7E11-C5 monoclonal antibody and demonstrated folate hydrolase activities and optimum pH activity profiles identical to those of LNCaP cells. The membrane-bound enzymes from both LNCaP- and PC-3-transfected cells also have a capacity to hydrolyze an alpha-linked glutamyl moiety from N-acetyl-alpha-aspartylglutamate. We have identified that PSM antigen is a pteroyl poly-gamma-glutamyl carboxypeptidase (folate hydrolase) and is expressed strongly in human prostate cancer. Cancer cells that express this enzyme are resistant to methotrexate therapy. Those developing future therapeutic strategies in the treatment of prostate cancer that utilize folate antagonists need to consider this mechanism of resistance.