Role of mu-calpain in proteolytic cleavage of brain L-glutamic acid decarboxylase.
Glutamic acid decarboxylase (GAD) is the rate-limiting enzyme for gamma-aminobutyric acid (GABA) biosynthesis. Previously, we reported the presence of truncated forms of GAD in vivo and in vitro. In addition, an unidentified endogenous protease responsible for proteolytic cleavage of full-length GAD (fGAD) to its truncated form (tGAD) was also observed. In this communication, we report that mu-calpain is a good candidate for conversion of fGAD(67) to tGAD(67). This conclusion is based on the following observations: 1. purified recombinant GAD(67) is cleaved by mu-calpain at specific sites; 2. in brain synaptosomal preparation, GAD(67) is cleaved to its truncated form by an endogenous protease which is inhibited by specific calpain inhibitors; 3. in mu-calpain knockout mice, the level of tGAD in the brain is greatly reduced compared with the wild type; 4. when mu-calpain gene is silenced by siRNA, the level of tGAD is also markedly reduced compared to the control group; and 5. mu-calpain is activated by neuronal stimulation and Ca(2+)-influx. The physiological significance of calpain in regulation of GABA synthesis and GABAergic neurotransmission is also discussed.
Sha
Di
D
Department of Biomedical Sciences, Florida Atlantic University, Boca Raton, FL 33431, USA.
Jin
Ying
Y
Wu
Heng
H
Wei
Jianning
J
Lin
Chun-Hua
CH
Lee
Yi-Hsuan
YH
Buddhala
Chandana
C
Kuchay
Shafi
S
Chishti
Athar H
AH
Wu
Jang-Yen
JY
D000818:Animals; D001921:Brain / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D002118:Calcium / Q000378:metabolism; D002154:Calpain / Q000494:pharmacology / Q000502:physiology*; D002478:Cells, Cultured; D017868:Cyclic AMP-Dependent Protein Kinases / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D017353:Gene Deletion; D005968:Glutamate Decarboxylase / Q000187:drug effects / Q000378:metabolism*; D051379:Mice; D018345:Mice, Knockout / Q000235:genetics; D009474:Neurons / Q000187:drug effects / Q000502:physiology; D011499:Protein Processing, Post-Translational; D034741:RNA, Small Interfering / Q000235:genetics / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D013347:Subcellular Fractions; D013574:Synaptosomes / Q000187:drug effects / Q000378:metabolism; D014162:Transfection / Q000379:methods
D034741:RNA, Small Interfering; D017868:Cyclic AMP-Dependent Protein Kinases; D002154:Calpain; C449002:mu-calpain; D005968:Glutamate Decarboxylase; C402565:glutamate decarboxylase 1; D002118:Calcium