The
peptide neurotransmitter N-acetylaspartylglutamate is inactivated by extracellular
peptidase activity following synaptic release. It is speculated that the
enzyme,
glutamate carboxypeptidase II (GCPII, EC 3.14.17.21), participates in this inactivation. However, CGCPII knockout mice appear normal in standard neurological tests. We report here the cloning and characterization of a mouse
enzyme (tentatively identified as
glutamate carboxypeptidase III or GCPIII) that is homologous to an
enzyme identified in a human lung
carcinoma. The mouse
peptidase was cloned from two non-overlapping EST clones and mouse brain
cDNA using PCR. The sequence (GenBank, AY243507) is 85% identical to the human
carcinoma enzyme and 70% homologous to mouse GCPII. GCPIII sequence analysis suggests that it too is a
zinc metallopeptidase. Northern blots revealed message in mouse ovary, testes and lung, but not brain. Mouse cortical and cerebellar neurons in culture expressed GCPIII message in contrast to the glial specific expression of GCPII. Message levels of GCPIII were similar in brains obtained from wild-type mice and mice that are null mutants for GCPII. Chinese hamster ovary (CHO) cells transfected with rat GCPII or mouse GCPIII expressed membrane bound
peptidase activity with similar V(max) and K(m) values (1.4 micro m and 54 pmol/min/mg; 3.5 micro m and 71 pmol/min/mg, respectively). Both
enzymes are activated by a similar profile of
metal ions and their activities are blocked by
EDTA. GCPIII message was detected in brain and spinal cord by RT-PCR with highest levels in the cerebellum and hippocampus. These data are consistent with the hypothesis that nervous system cells express at least two differentially distributed homologous
enzymes with similar pharmacological properties and affinity for NAAG.