The cloning and characterization of a second brain enzyme with NAAG peptidase activity.
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.
Bzdega
Tomasz
T
Department of Biology, Georgetown University, Washington, D.C., USA.
Crowe
Samantha L
SL
Ramadan
Epolia R
ER
Sciarretta
Kathryn H
KH
Olszewski
Rafal T
RT
Ojeifo
Olumide A
OA
Rafalski
Victoria A
VA
Wroblewska
Barbara
B
Neale
Joseph H
JH
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D001921:Brain / Q000201:enzymology*; D002462:Cell Membrane / Q000201:enzymology; D002478:Cells, Cultured; D003001:Cloning, Molecular; D006224:Cricetinae; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D043425:Glutamate Carboxypeptidase II / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008670:Metals / Q000494:pharmacology; D051379:Mice; D008969:Molecular Sequence Data; D009928:Organ Specificity; D012333:RNA, Messenger / Q000096:biosynthesis; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017385:Sequence Homology; D013116:Spinal Cord / Q000201:enzymology; D014162:Transfection
D004791:Enzyme Inhibitors; D008670:Metals; D012333:RNA, Messenger; D043425:Glutamate Carboxypeptidase II