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

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 fGAD 67 to tGAD 67 . This conclusion is based on the following observations: 1. Purified recombinant GAD 67 is cleaved by μ-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 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; 5. μ-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. KeywordsCalpain; GABA synthesis; GAD; Proteolytic CleavageIn the central nervous system (CNS), γ-aminobutyric acid (GABA) is synthesized by a single enzymatic reaction catalyzed by L-glutamic decarboxylase (EC 4.1.1.15; GAD) (1,2). Mammalian species express two isoforms of GAD, namely, GAD 65 and GAD 67 , referring to GAD with a molecular weight of 65 kDa and 67 kDa, respectively. Previously, we reported **Address correspondence and reprint requests to Dr. Jang-Yen Wu, Florida Atlantic University, Department of Biomedical Science, 777 Glades Road, Boca Raton, FL 33431-0991, U.S. ; Email: jwu@fau.edu. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public Access NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript the presence of the truncated GAD (tGAD) derived from proteolytic cleavage of the full-length (fGAD) in vivo as well as in vitro (3,4). The presence of smaller forms of GAD was also observed from other laboratories (5-8). However, no information was reported regarding the identity of the endogenous proteases responsible for GAD cleavage and their physiological significance. Recently, we found that recombinant human brain GAD 65 is specifically cleaved at arginine 69 from the N-terminal of fGAD 65 to produce tGAD 65 , which is ∼2-3 fold more active than fGAD 65 (3). In addition, GAD 67 was found to be cleaved at two specific sites, one at arginine 70 and another at arginine 90, to produ...

Calpains and neuronal damage in the ischemic brain: The swiss knife in synaptic injury

“…Together, these results suggest that calpain-mediated cleavage of GAD65 may downregulate GABA-mediated phasic inhibition. Studies similar to those described above for GAD65 also showed the calpain-mediated cleavage of GAD67 (Sha et al, 2008), which may decrease the synthesis of GABA, as observed in in vitro experiments using purified human GAD67 and recombinant µ-calpain. Silencing of the m-calpain gene had no effect on the level of truncated GAD in cultured neurons isolated from the whole brain, whereas knock-down of μ-calpain greatly decreased the cleavage of both GAD isoforms.…”

“…GAD65 and GAD67 were shown to be cleaved after transient MCAO in rats, both in the core and penumbra regions (Buddhala et al, 2012), but the mechanisms involved were not elucidated. A role for calpain in GAD cleavage was first suggested based on studies showing i) a Ca 2+ -dependent cleavage of both GAD isoforms in rat synaptosomes incubated with the Ca 2+ ionophore ionomycin, and ii) a truncation of GAD65 following depolarization of the synaptosomes with KCl (Sha et al, 2008;Wei et al, 2006). Western blot experiments with an antibody against the C-terminus of GAD65 showed the formation of a calpain-mediated cleavage product with a molecular weight of 58 kDa.…”

“…Moreover, it was found that phosphorylation status can differentially regulate the calpain-mediated cleavage of GAD65 and GAD67: PKA-mediated phosphorylation of the latter isoform inhibits its cleavage by μ-calpain, while PKC-mediated GAD65 phosphorylation facilitates the process (Sha et al, 2008). In contrast with the full length protein, the human GAD67 lacking the first 70 or 90 amino acids (Δ1-70 or Δ1-90, respectively) is not cleaved by calpain, suggesting that the cleavage site resides in the N-terminus of the protein.…”

Augmented generation of protein fragments during wakefulness as the molecular cause of sleep: a hypothesis

“…Lys-GAD65-2 is the Ct fragment of the glutamic acid decarboxylase-65-2 (GAD65-2), which is bound to membranes of synaptic vesicles and mediates the synthesis of the inhibitory neurotransmitter c-aminobutyric acid (GABA) from the excitatory neurotransmitter glutamate. Calpain-generated Lys-GAD65-2 retains the enzymatic activity of uncleaved GAD65-2 but is no longer associated with synaptic vesicles [170][171][172] [see also Fig. 6(G)].…”

Calpain 2 Activated through N-Methyl-d-Aspartic Acid Receptor Signaling Cleaves CPEB3 and Abrogates CPEB3-Repressed Translation in Neurons

“…The resulting supernatant was collected and centrifuged at 12,000 ϫ g for 30 min. The synaptosome-enriched pellet was gently resuspended in Krebs-Ringer phosphate buffer (123 mM NaCl, 3 mM KCl, 0.4 mM MgCl 2 , 0.5 mM NaH 2 PO 4 , 0.25 mM Na 2 HPO 4 , and 1 mg/ml glucose [pH 7.4]) (48). The synaptosome solution was aliquoted and either stimulated by the addition of 50 M NMDA and 2.2 mM CaCl 2 at 37°C for the designated time or not stimulated and then lysed in Laemmli sample buffer for Western blot analysis.…”