Identification and functional analysis of truncated human glutamic acid decarboxylase 65

Human brain glutamate decarboxylase 65 (hGAD65) was found to exist as full-length and truncated forms when the glutathione S-transferase-tagged hGAD65 fusion protein was subjected to factor Xa cleavage. The truncated form is produced by cleavage at arginine 69 based on N-terminal amino acid sequence analysis, and has a molecular weight of 58 kD. It is resistant to further factor Xa cleavage or mild trypsin treatment and is more active and more stable than the full-length form. Both the full-length and truncated forms of GAD are also observed in brain preparations in the presence of protease inhibitors. Furthermore, full-length GAD could be converted to the truncated form by endogenous proteases, suggesting that the conversion of full-length to truncated GAD mediated by endogenous protease may represent an important mechanism in the regulation of GABA biosynthesis in the brain.     

Effect of apocalmodulin on recombinant human brain glutamic acid decarboxylase

In this work, we report that the recombinant glutathione S-transferase (GST)-human L-glutamic acid decarboxylase (HGAD) isoforms, 65-kDa L-glutamic acid decarboxylase (GAD) (GST-HGAD65) fusion protein or free truncated HGAD65, were activated by apocalmodulin (ApoCaM) to an extent of 60%. Both truncated forms of GAD67 (tGAD67), HGAD67(Delta1-70) and HGAD67(Delta1-90), were markedly activated by ApoCaM to an extent of 141 and 85%, respectively, while GST-HGAD67 was not significantly affected. The activation appears to be due to an increase of GAD affinity for its cofactor, pyridoxal phosphate (PLP). This conclusion is based on the following observations. Firstly, the V(max) of GAD was increased when ApoCaM was present whereas the affinity for the substrate, glutamate, was not affected. Secondly, the affinity of GAD for PLP was increased in the presence of ApoCaM. Thirdly, results from calmodulin-agarose affinity column chromatography studies indicated a direct interaction or binding between ApoCaM and GAD. Fourthly, ApoCaM was found to be copurified with GAD65/GAD67 by anti-GAD65/67 immunoaffinity column using rat brain extract. Hence, it is proposed that a conformational change is induced when ApoCaM interacts with GAD65 or tGAD67, resulting in an increase of GAD affinity for PLP and the activation of GAD. The physiological significance of the interaction between GAD and ApoCaM is discussed.     

谷氨酸脱羧酶研究进展

谷氨酸脱羧酶(glutamic acid decarboxylase,GAD,EC4.1.1.15)在生物体内广泛存在,其催化产物γ-氨基丁酸(γ-aminobutyric acid,GABA)是哺乳动物体内一种重要的抑制性神经递质。在对自身免疫性疾病以及糖尿病研究中,特别是1型糖尿病,GAD、GABA以及谷氨酸脱羧酶抗体(glutamic acid decarboxylase-antibody,GAD-Ab)等的水平作为病理分析、疾病诊断、免疫治疗的重要参数,历来备受研究者关注。本文就GAD及其催化产物GABA的研究进展进行了综述,为更好地研究自身免疫性疾病的发病机理,探索更加有效安全的治疗方法提供参考。     

Calpain cleavage of brain glutamic acid decarboxylase 65 is pathological and impairs GABA neurotransmission

Previously, we have shown that the GABA synthesizing enzyme, L-glutamic acid decarboxylase 65 (GAD65) is cleaved to form its truncated form (tGAD65) which is 2-3 times more active than the full length form (fGAD65). The enzyme responsible for cleavage was later identified as calpain. Calpain is known to cleave its substrates either under a transient physiological stimulus or upon a sustained pathological insult. However, the precise role of calpain cleavage of fGAD65 is poorly understood. In this communication, we examined the cleavage of fGAD65 under diverse pathological conditions including rats under ischemia/reperfusion insult as well as rat brain synaptosomes and primary neuronal cultures subjected to excessive stimulation with high concentration of KCl. We have shown that the formation of tGAD65 progressively increases with increasing stimulus concentration both in rat brain synaptosomes and primary rat embryo cultures. More importantly, direct cleavage of synaptic vesicle - associated fGAD65 by calpain was demonstrated and the resulting tGAD65 bearing the active site of the enzyme was detached from the synaptic vesicles. Vesicular GABA transport of the newly synthesized GABA was found to be reduced in calpain treated SVs. Furthermore, we also observed that the levels of tGAD65 in the focal cerebral ischemic rat brain tissue increased corresponding to the elevation of local glutamate as indicated by microdialysis. Moreover, the levels of tGAD65 was also proportional to the degree of cell death when the primary neuronal cultures were exposed to high KCl. Based on these observations, we conclude that calpain-mediated cleavage of fGAD65 is pathological, presumably due to decrease in the activity of synaptic vesicle - associated fGAD65 resulting in a decrease in the GABA synthesis - packaging coupling process leading to reduced GABA neurotransmission.     

Structural and functional analysis of cysteine residues in human glutamate decarboxylase 65 (GAD65) and GAD67

Previously, we have shown that brain glutamate decarboxylase (GAD) is greatly inhibited by sulfhydryl reactive reagent suggesting cysteine residue(s) may play an important role in GAD function. In this report, we determined the role of cysteine residues in the recombinant human 65-kDa GAD isoform (hGAD65) and 67-kDa GAD isoform (hGAD67), using a combination of matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry and site-directed mutagenesis. Here, we report that cysteine 446 (C446) in hGAD65 is important for its activity and is present as free sulfhydryl group. This conclusion is based on the following observations: (i) mutation of C446 in hGAD65 to alanine reduced hGAD65 activity by more than 90%, (ii) MALDI-TOF analysis of the non-reduced, trypsin-digested GAD65 revealed that C446 is present as a free sulfhydryl group as indicated by a peak at m/z (mass/charge) 647.3446 (peptide 443-448) and, when GAD65 was treated with sulfhydryl reagent, N-ethylmaleimide (NEM), the peak is shifted to m/z 772.3702,a mass increase of 125.1 daltons (Da) as a result of modification of cysteine by NEM. Parallel studies have also been conducted with hGAD67. Cysteine 455 was found to be important for GAD67 activity.     

A structural model for the inhibition of calpain by calpastatin: crystal structures of the native domain VI of calpain and its complexes with calpastatin peptide and a small molecule inhibitor

The Ca(2+)-dependent cysteine protease calpain along with its endogenous inhibitor calpastatin is widely distributed. The interactions between calpain and calpastatin have been studied to better understand the nature of calpain inhibition by calpastatin, which can aid the design of small molecule inhibitors to calpain. Here we present the crystal structure of a complex between a calpastatin peptide and the calcium-binding domain VI of calpain. DIC19 is a 19 residue peptide, which corresponds to one of the three interacting domains of calpastatin, which is known to interact with domain VI of calpain. We present two crystal structures of DIC19 bound to domain VI of calpain, determined by molecular replacement methods to 2.5A and 2.2A resolution. In the process of crystallizing the inhibitor complex, a new native crystal form was identified which had the homodimer 2-fold axis along a crystallographic axis as opposed to the previously observed dimer in the asymmetric unit. The crystal structures of the native domain VI and its inhibitor PD150606 (3-(4-iodophenyl)-2-mercapto-(Z)-2-propenoic acid) complex were determined with the help of molecular replacement methods to 2.0A and 2.3A resolution, respectively. In addition, we built a homology model for the complex between domain IV and DIA19 peptide of calpastatin. Finally, we present a model for the calpastatin-inhibited calpain.     

Is there a high molecular weight glutamic acid decarboxylase?

Gel-filtration chromatography from crude extracts of mouse brain indicates the presence of a high molecular weight (HMW) (more than 750 kd) and a low molecular weight (LMW) (87.5 Kd) glutamic acid decarboxylase (GAD) when they are concentrated prior chromatography either by precipitation with salts or ethanol. Kinetically both peaks of GAD-activity exhibit an almost identical Km for L-glutamate. Only LMW-GAD appears if the concentration step is carried out by ultrafiltration or if the extract is chromatographied without the concentrating step. HMW-GAD disappears from the chromatographic profiles if ethanolic extracts of GAD are treated with Triton X-100 before the chromatographic procedure. The sodium sulfate precipitation of a previously separated LMW-GAD gives rise to the reappearance of a HMW-GAD peak. Apparently HMW-GAD does not exist as a different molecular entity; indeed it may be an artefactual aggregation of LMW-GAD.     

The effects of adrenalectomy and thermal stress on glutamic acid decarboxylase activity in different regions of the rat brain

Glutamic acid decarboxylase (GAD) enzyme activity was measured in synaptosomes prepared from the hypothalamus, the hippocampus, the striatum and the cerebral cortex of control, adrenalectomized and rat exposed to a thermal stress. Adrenalectomy caused a statistically significant decrease in the enzyme activity in the striatum, while it had no effect in the other three brain areas. On the other hand, exposure to the thermal stress resulted in a dramatic increase of GAD specific activity in all brain areas examined. This thermal stress-induced increase in enzyme activity was observed in both non-operated and adrenalectomized animals, which implies that it is not mediated by glucocorticoids.Abbreviations used GAD glutamic acid decarboxylase - GABA -aminobutyric acid - AET 2-aminoethylisourethonium bromide - ADX adrenalectomized - rpm revolutions per minute     

Inverse polymerase chain reaction mediated chromosome walking within the human glutamic acid decarboxylase gene

Using inverse polymerase chain reaction (PCR), we have cloned partial intronic sequences from human glutamic acid decarboxylase (GAD) gene. A small 153 bp core region was selected from the GAD cDNA sequence to design outward primers corresponding to its 3′ and 5′ ends. EcoRI digested human DNA which had been circularized by self-ligation and then linearized withSacII was used as a substrate to can.y out PCR. This gave a 900 bp long product which was cloned into pUC19. The sequence analysis of this fragment revealed the presence of introns in the region flanking the selected core DNA. In this work we used this technique to walk into the upsteam region of the GAD gene using sequence information from its cloned cDNA.     

Cloning, expression, purification and characterization of the cholera toxin B subunit and triple glutamic acid decarboxylase epitopes fusion protein in Escherichia coli

Induction of specific immunological unresponsiveness by oral autoantigens such as glutamic acid decarboxylase 65 (GAD65) is termed oral tolerance and may be a potential therapy for autoimmune diabetes. However, the requirement for large amounts of protein will limit clinical testing of autoantigens, which are difficult to produce. Mucosal adjuvants such as cholera toxin B subunit (CTB) may lower the level of autoantigens required. Here we describe cloning, expression, purification and identification study of the CTB and triple GAD_531–545 epitopes fusion gene. The fusion gene was ligated via a flexible hinge tetrapeptide and expressed as a soluble protein in Escherichia coli BL21 (DE3) driven by the T7 promoter. We purified the recombination protein from the cell lysate and obtained approximately 2.5 mg of CTB–GAD_(531–545)3 per liter of culture with greater than 90% purity by a Ni–NTA resin column. The bacteria produced this protein as the pentameric form, which retained the GM1-ganglioside binding affinity and the native antigenicity of CTB and GAD65. Further studies revealed that oral administration of bacterial CTB–GAD_(531–545)3 fusion protein showed the prominent reduction in pancreatic islet inflammation in non-obese diabetic mice. The results presented here demonstrate that the bacteria bioreactor is an ideal production system for an oral protein vaccine designed to develop immunological tolerance against autoimmune diabetes.     

Influence of protein nutrition on calpain activity of mouse kidney: Modulation by calpastatin

The effect of protein depletion and refeeding with a normal diet on calpain activity was examined in mouse kidney soluble homogenate. In terms of units per gram of protein, it increased 2.9 times with depletion and decreased upon refeeding. After a DEAE-Sephacel chromatography, the homogenate yielded three enzymatic activities. Their sum, assessed as total calpain activity, was higher than the activity measured before fractionation and did not appreciably change during protein depletion and refeeding. Because the proportion of total activity displayed by the complete homogenate increased with depletion and decreased with refeeding, a low calpastatin content in depleted kidney was envisaged. This was confirmed by direct estimations: depleted kidney had 6 times less calpastatin compared to both normal and 16 h refed tissue. We concluded that a decrease in calpastatin content contributes to an increased calpain activity related to degradable protein in protein depleted kidney. In view of this, it seems not unlikely that the in vivo rate of protein breakdown depicted by kidney during protein depletion and refeeding is in part effected through modulation of the calpain proteolytic system. (Mol Cell Biochem 166: 95-99, 1997)     

Inhibition of calpain expression by E-64d in the rat retina subjected to ischemia/reperfusion injury

To investigate the effect of E-64d, a selective inhibitor of calpain, on the expression of calpain and calpastatin in rat retina was subjected to ischemia/reperfusion injury (IRI). An animal model of retinal IRI was set up by increasing the intraocular pressure (110 mm Hg) of a rat eye for 1 h. The retinal thickness and morphologic changes were detected by histology. The protein expression of m-calpain (a calpain isoform) in the retina was assessed by immunohistochemistry and Western blot assay. The mRNA of m-calpain, as well as calpastatin (an endogenous protein inhibitor of calpain), in the retina was assessed by RT-PCR, and the ratio of m-calpain/calpastatin was then calculated. To evaluate the effect of E-64d on the expression of calpain, the drug (5 μl of 100 μM) was injected intravitreously immediately after IRI. There were retinal edematous changes, particularly in the inner plexiform layer after IRI. The protein expression of m-calpain in the retina was increased 24 h after IRI, an effect that was inhibited by E-64d (P < 0.05). The mRNA expression of m-calpain and calpastatin was also increased 24 h and 3 h after IRI, respectively. Neither m-calpain nor calpastatin mRNA expression was influenced by E-64d (P > 0.05). The mRNA ratio of m-calpain to calpastatin was increased at the 6 h, 24 h and 72 h after IRI, and only at 24 h the increase of the ratio of m-calpain to calpastatin was inhibited by E-64d (P < 0.05). In the rat retina of IRI, E-64d inhibits the increase of m-calpain protein expression, as well as the mRNA ratio increase of m-calpain to calpastatin. E-64d also inhibited the retinal damage induced by IRI, suggesting a role for E-64d in the protection of the retinal apoptosis induced by IRI. Published in Russian in Molekulyarnaya Biologiya, 2008, Vol. 42, No. 2, pp. 258–264. The text was submitted by the authors in English.     

Comparative expression and purification of human glutamic acid decarboxylase from Saccharomyces cerevisiae and Pichia pastoris

The yeast cell factory is a potentially useful source of proteins in general. They include glutamic acid decarboxylase (GAD), which is one of the major autoantigens for Type 1 diabetes. We have created a hybrid form of GAD consisting of amino acids 1–101 of the human GAD67 protein fused to amino acids 96–585 of the human GAD65 protein, and have modified this to include a C-terminal hexa-Histidine (H6) tag sequence. This hybrid GAD67/65-H6 was expressed in two yeast hosts: constitutively under the control of the plasmid phosphoglycerate kinase promoter (PGK1) in Saccharomyces cerevisiae, and inducibly under the control of the chromosomal alcohol oxidase promoter (AOX1) in Pichia pastoris. Enzymatically active hybrid GAD was prepared from yeast lysates by purification either on an affinity column based on the GAD-1 monoclonal antibody, or by metal-affinity chromatography. The purified GAD67/65-H6 was radiolabelled with iodine-125 and tested with Type 1 diabetes sera in a radioimmunoprecipitation assay, and results were compared with those using untagged GAD67/65 and those using porcine brain GAD. The results of enzymatic and immunological assays show hybrid GAD67/65 is isolated at high specific activity and moderate yield, and the addition of the H6 tag sequences or the choice of yeast strain did not appreciably affect enzyme activity, percentage recovery of GAD, protein purification, or the utility in diagnosis of diabetes in terms of specificity and sensitivity to the various sera.     

Phosphorylation and activation of brain aromatic L-amino acid decarboxylase by cyclic AMP-dependent protein kinase

Aromatic L-amino acid decarboxylase (AAAD), an enzyme required for the synthesis of catecholamines, indoleamines, and trace amines, is rapidly activated by cyclic AMP-dependent pathways in striatum and midbrain in vivo, suggesting enzyme phosphorylation. We now report that the catalytic subunit of cyclic AMP-dependent protein kinase (PKA) directly phosphorylated AAAD immunoprecipitated from homogenates prepared from the mouse striatum and midbrain in vitro. Under the same phosphorylation conditions, the catalytic subunit of PKA also phosphorylated a recombinant AAAD protein expressed in Escherichia coli transfected with an AAAD cDNA isolated from the bovine adrenal gland. The PKA-induced AAAD phosphorylation of immunoprecipitates from striatum and midbrain was time and concentration dependent and blocked by a specific PKA peptide inhibitor. Incubation of the catalytic subunit of PKA with striatal homogenates increased enzyme activity by approximately 20% in a time- and concentration-dependent manner. Moreover, incubation of the catalytic subunit of PKA with recombinant AAAD increased activity by approximately 70%. A direct phosphorylation of AAAD protein by PKA might underlie the cyclic AMP-induced rapid and transient activation of AAAD in vivo.     

Developmental changes of calpain and calpastatin in rabbit brain

A major part of the Ca-activated proteolytic activity in the soluble fraction from rabbit brain could be due to the activity of the neutral thiol-proteases calpain I and II. The activity of calpains exceeded that of the endogenous inhibitor, calpastatin, at all developmental stages studied. The level of calpains increased rapidly from the prenatal stage to reach a peak 10–20 days postnatally. From this period the level of calpains decreased slowly to reach the adult levels. The level of calpastatin increased steadily from the prenatal stage to old age.     

The structural and functional heterogeneity of glutamic acid decarboxylase: A review

Studies of the GABA-synthetic enzyme glutamate decarboxylase (glutamic acid decarboxylase; GAD; E.C.4.1.1.15) began in 1951 with the work of Roberts and his colleagues. Since then, many investigators have demonstrated the structural and functional heterogeneity of brain GAD. At least part of this heterogeneity derives from the existence of two GAD genes.In honor of the 70th birthday of Dr. Eugene Roberts     

Genotypic effects of calpain 1 and calpastatin on the tenderness of cooked M. longissimus dorsi steaks from Jersey x Limousin, Angus and Hereford-cross cattle

Single nucleotide polymorphisms (SNPs) in the calpain 1 (CAPN1) and calpastatin (CAST) genes were studied to determine their effects on meat tenderness in Bos taurus cattle. Strip loins (M. longissimus dorsi) were removed from cattle in four resource populations after slaughter (n = 1042), aged under controlled conditions until fixed times after rigor mortis, cooked and measured using a tenderometer. Animals were genotyped for the CAPN1 SNP c.947C>G (p.Ala316Gly; AF252504) and for the CAST SNP c.2959A>G (AF159246). Frequencies of CAPN1 C alleles ranged from 23% to 68%, and CAST A alleles from 84% to 99.5%. From all data combined, the CAPN1 CC genotype (compared with the GG genotype) was associated with a 20.1 +/- 1.7% reduced average shear force at intermediate stages of ageing (P < 0.001) and with a 9.5 +/- 1.3% reduction near ultimate tenderness (P < 0.001). The heterozygote was intermediate. For CAST, corresponding values for AA compared with AG genotypes were reductions of 8.6 +/- 2.0% and 5.1 +/- 1.6% respectively (both P < 0.001), but there were too few GG genotypes for comparison. There were small interactions between the CAPN1 and CAST genotypes. For the CAPN1 and CAST genotypes combined, the maximal genotype effect in average shear force was 25.7 +/- 5.5% (P < 0.001) at intermediate stages and 15.2 +/- 4.8% near ultimate tenderness (P < 0.01).