Mouse glutamate carboxypeptidase II (GCPII) has a similar enzyme activity and inhibition profile but a different tissue distribution to human GCPII

Abstract

Glutamate carboxypeptidase II ( GCPII ), also known as prostate‐specific membrane antigen ( PSMA ) or folate hydrolase, is a metallopeptidase expressed predominantly in the human brain and prostate. GCPII expression is considerably increased in prostate carcinoma, and the enzyme also participates in glutamate excitotoxicity in the brain. Therefore, GCPII represents an important diagnostic marker of prostate cancer progression and a putative target for the treatment of both prostate cancer and neuronal disorders associated with glutamate excitotoxicity. For the development of novel therapeutics, mouse models are widely used. However, although mouse GCPII activity has been characterized, a detailed comparison of the enzymatic activity and tissue distribution of the mouse and human GCPII orthologs remains lacking. In this study, we prepared extracellular mouse GCPII and compared it with human GCPII . We found that mouse GCPII possesses lower catalytic efficiency but similar substrate specificity compared with the human protein. Using a panel of GCPII inhibitors, we discovered that inhibition constants are generally similar for mouse and human GCPII . Furthermore, we observed highest expression of GCPII protein in the mouse kidney, brain, and salivary glands. Importantly, we did not detect GCPII in the mouse prostate. Our data suggest that the differences in enzymatic activity and inhibition profile are rather small; therefore, mouse GCPII can approximate human GCPII in drug development and testing. On the other hand, significant differences in GCPII tissue expression must be taken into account when developing novel GCPII ‐based anticancer and therapeutic methods, including targeted anticancer drug delivery systems, and when using mice as a model organism.

A novel PSMA/GCPII‐deficient mouse model shows enlarged seminal vesicles upon aging

“…On the other hand, the amount of PSMA/GCPII in the epididymis was as high as 180 pg per 1 μg total protein. In comparison, we previously showed that the amount of PSMA/GCPII in the male kidney and brain is 100‐250 pg and 2.5‐10 pg in 1 μg total protein, respectively . We thus visualized and localized PSMA/GCPII in the epididymis by immunohistochemistry using two different antibodies against mouse PSMA/GCPII—GCPII‐02 and GCPII‐04 (Figure ).…”

“…The extracellular portion of mouse GCPII (rm‐GCPII) was prepared as previously described . Briefly, large scale expression was performed using Drosophila S2 (BiP‐BirA‐KDEL) cells stably transfected with a plasmid encoding rm‐GCPII (pMT/BiP/AvimGCPII) . Biotinylated rm‐GCPII was purified from S2 cell media using Streptavidin Mutein Matrix (Roche, Basel, Switzerland) .…”

“…Site‐directed mutagenesis was carried out using pMT/BiP/AvimGCPII as a template according to the manufacturer's protocol (Phusion Site‐Directed Mutagenesis Kit, Thermo Fisher Scientific, Waltham, MA). Four consecutive mutageneses were performed.…”

“…The recombinant protein rm‐GCPIIdel17 was prepared similarly as previously described for rm‐GCPII . Briefly, Drosophila S2 (BiP‐BirA‐KDEL) cells were co‐transfected with the plasmids pMT/BiP/AvimGCPIIdel17 and pCoBlast (Invitrogen), followed by cell culture selection using cultivation of the transfectants in the presence of blasticidin (Invitrogen) and hygromycin B (Invitrogen).…”

“…The recombinant protein rm-GCPIIdel17 was prepared similarly as previously described for rm-GCPII. 31 Briefly, Drosophila S2 (BiP-BirA-KDEL) cells 32 were co-transfected with the plasmids pMT/BiP/ AvimGCPIIdel17 and pCoBlast (Invitrogen), followed by cell culture selection using cultivation of the transfectants in the presence of blasticidin (Invitrogen) and hygromycin B (Invitrogen). Prepared stably transfected cultures of S2 cells were tested for their ability to express rm-GCPIIdel17 using Western blot, and the cell culture expressing the highest amount of rm-GCPIIdel17 was used for large-scale expression.…”