Department of Physiological Chemistry, University of Veterinary Medicine Hannover, Hannover, Germany.
Prostate. 2013 May;73(6):642-50. doi: 10.1002/pros.22605. Epub 2013 Jan 28.
Prostate-specific membrane antigen (PSMA) is a promising biomarker in the diagnosis of prostate cancer and a potential target for antibody-based therapeutic strategies. We isolated the canine PSMA cDNA and investigated the cellular and biochemical characteristics of the recombinant protein as a potential target for animal preclinical studies of antibody based-therapies.
Canine PSMA cDNA was isolated by PCR, cloned into expression vectors and transfected into COS-1 and MDCK cells. The biosynthesis and glycosylation of the recombinant protein were investigated in pulse-chase experiments, the cellular localization by confocal laser microscopy, the mode of association of PSMA with the membrane with solubilization in different detergents and its quaternary structure in sucrose-density gradients.
Canine PSMA shows 91% amino acid homology to human PSMA, whereby the major difference is a longer cytoplasmic tail of canine PSMA compared to its human counterpart. Canine PSMA is trafficked efficiently along the secretory pathway, undergoes homodimerization when it acquires complex glycosylated mature form. It associates with detergent-resistant membranes, which act as platforms along its intracellular trafficking. Confocal analysis revealed canine PSMA at the cell surface, Golgi, and the endoplasmic reticulum. A similar distribution is revealed for human PSMA, yet with reduced cell surface levels.
The cloning, expression, biosynthesis, processing and localization of canine PSMA in mammalian cells is described. We demonstrate that canine PSMA reveals similar characteristics to human PSMA rendering this protein useful as a translational model for investigations of prostate cancer as well as a suitable antigen for targeted therapy studies in dogs.
前列腺特异性膜抗原（PSMA）是前列腺癌诊断中很有前途的生物标志物，也是基于抗体的治疗策略的潜在靶点。我们分离了犬 PSMA cDNA，并研究了重组蛋白的细胞和生化特性，作为基于抗体治疗的动物临床前研究的潜在靶点。
通过 PCR 分离犬 PSMA cDNA，克隆到表达载体中，并转染 COS-1 和 MDCK 细胞。通过脉冲追踪实验研究重组蛋白的生物合成和糖基化，通过共聚焦激光显微镜研究细胞内定位，通过不同去污剂的溶解研究 PSMA 与膜的结合方式及其在蔗糖密度梯度中的四级结构。
犬 PSMA 与人类 PSMA 的氨基酸同源性为 91%，主要区别在于犬 PSMA 的细胞质尾巴比人类 PSMA 长。犬 PSMA 沿着分泌途径有效地运输，当获得复杂的糖基化成熟形式时，它会发生同源二聚化。它与去污剂抗性膜结合，作为其细胞内运输的平台。共聚焦分析显示犬 PSMA 位于细胞表面、高尔基体和内质网。人类 PSMA 也有类似的分布，但细胞表面水平降低。
本文描述了犬 PSMA 在哺乳动物细胞中的克隆、表达、生物合成、加工和定位。我们证明犬 PSMA 表现出与人类 PSMA 相似的特征，这使得该蛋白可作为前列腺癌研究的转化模型，也可作为犬靶向治疗研究的合适抗原。
