Elsevier

Brain Research

Volume 1207, 1 May 2008, Pages 9-18
Brain Research

Research Report
Role of μ-calpain in proteolytic cleavage of brain l-glutamic acid decarboxylase

https://doi.org/10.1016/j.brainres.2008.02.033Get rights and content

Abstract

Glutamic acid decarboxylase (GAD) is the rate-limiting enzyme for γ-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 μ-calpain is a good candidate for conversion of fGAD67 to tGAD67. This conclusion is based on the following observations: 1. purified recombinant GAD67 is cleaved by μ-calpain at specific sites; 2. in brain synaptosomal preparation, GAD67 is cleaved to its truncated form by an endogenous protease which is inhibited by specific calpain inhibitors; 3. in μ-calpain knockout mice, the level of tGAD in the brain is greatly reduced compared with the wild type; 4. when μ-calpain gene is silenced by siRNA, the level of tGAD is also markedly reduced compared to the control group; and 5. μ-calpain is activated by neuronal stimulation and Ca2+-influx. The physiological significance of calpain in regulation of GABA synthesis and GABAergic neurotransmission is also discussed.

Section snippets

Cleavage of GAD67 under neuronal stimulation condition

When the rat brain synaptosomes were stimulated by 55 mM K+ and 2.2 mM Ca2+ for 5 min, the extent of the conversion of fGAD67 to the truncated form was increased in comparison with the non-stimulation condition (Fig. 1A). The amounts of the truncated GAD67 were significantly increased under stimulation for an additional 5 min (Fig. 1A). When 2 mM EDTA/EGTA was present during the period of stimulation, no significant cleavage was observed 6 (Fig. 1A), suggesting that the cleavage of GAD67 in rat

Discussion

An important mechanism involved in the regulation of enzyme activity is through proteolytic cleavage of enzyme proteins. For instance, protein tyrosine phosphatases (PTPs) is regulated by a proteolytic mechanism and the cleaved proteins show higher activity (Gu and Majerus, 1996). In striatum neurons, striatal tyrosine phosphatase (STEP) is proteolytically cleaved by glutamate-induced calcium influx and calpain activation. The cleaved STEP can reach its targets and perform its function. In this

Materials

Protein inhibitor cocktail, ionomycin, μ-calpain, calpain inhibitor I, polyclonal anti-μ-calpain, polyclonal anti-m-calpain, goat anti-rabbit IgG conjugated with horseradish peroxidase (HRP), ABTS [2,2′-azino-bis(3-ethylbenziazoline-6-sulfonic acid)], and Igepal CA 630 were purchased from Sigma (St. Louise, MO). Calpastatin peptide was purchased from Calbiochem (San Diego, CA). Lipofectamine, Opti-MEM medium, TRIZOL and the cDNA synthesis kit were from Invitrogen (Carlsbad, CA). pfu polymerase

Acknowledgments

This work was supported in part by National Institutes of Health Grant NS37851 (to J.-Y.W.), National Science Foundation grant IBN-9723079 (to J.-Y.W), the Schmidt Family Foundation and the Center of Excellence for Biomedical and Marine Biotechnology at Florida Atlantic University.

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