Abstrakt
Glutamate carboxypeptidase II (GCPII), also known as prostate specific membrane antigen (PSMA), is type II transmembrane glycoprotein with short intracellular and transmembrane domain and large extracellular part possessing carboxypeptidase activity. GCPII is expressed predominantly in the brain, kidney, small intestine and prostate.
In the brain, it cleaves the most abundant peptide neurotrasmitter, N-acetyl-L-aspartyl-Lglutamate (NAAG), to yield N-acetyl-L-aspartate and an important neurotransmitter L-glutamate. In small intestine, it cleaves poly-gamma-glutamylated folates and thus enables folate absorption.
Nevertheless, the biological role of GCPII in other tissues remains to be elucidated. To date, four different attempts to generate GCPII KO mice have been reported.
The results are rather controversial. While deletion of exons 1 and 2 or exons 9 and 10 was reported to be embryonic lethal, other groups reported that adding 3 stop codons between exon 1 and 2 or deletion of exons 3 to 5 resulted in generation of viable GCPII KO mice with no obvious phenotype.
Further characterization of these viable KO mice revealed their decreased susceptibility to peripheral neuropathies and stroke as well as severe impairment of angiogenesis. Interestingly, residual NAAG peptidase activity has been observed suggesting compensatory expression of a homolog of GCPII also capable of NAAG cleavage - GCPIII - in the brain.
To shed the light on this controversy as well as to decipher other potential roles of GCPII, we attempted to generate GCPII KO mice using TALEN technology by targeting exon 11 of GCPII encoding gene (FolhI) and established reliable genotyping method based on nested PCR. The GCPII KO mice are viable, breed normally and do not show any obvious phenotype.
Here, we present basic characterization of these mice using qPCR, Western blot and enzymatic activity analysis.