TAT-Mediated Delivery of Human Alanine:Glyoxylate Aminotransferase in a Cellular Model of Primary Hyperoxaluria Type I

Defects in liver peroxisomal alanine:glyoxylate aminotransferase (AGT), as a consequence of inherited mutations on the AGXT gene, lead to primary hyperoxaluria type I (PH1), a rare metabolic disorder characterized by the formation of calcium oxalate stones at first in the urinary tract and then in the whole body. The curative treatments currently available for PH1 are pyridoxine therapy, effective in only 10–30 % of the patients, and liver transplantation, an invasive procedure with potentially serious complications. A valid therapeutic option for PH1 patients would be the development of an enzyme administration therapy. However, the exogenous administration of the missing AGT would require the crossing of the plasma membrane to deliver the protein to liver peroxisomes. In this study, we constructed, purified and characterized the fusion protein of AGT with the membrane-penetrating Tat peptide (Tat-AGT). Although Tat-AGT shows subtle active site conformational changes as compared with untagged AGT, it retains a significant transaminase activity. Western-blot analyses, enzymatic assays and immunofluorescence studies show that active Tat-AGT can be successfully delivered to a mammalian cellular model of PH1 consisting of chinese hamster ovary cells expressing glycolate oxidase (CHO-GO), whereas untagged AGT cannot. Moreover, the intracellular transduced Tat-AGT makes CHO-GO cells able to detoxify endogenously produced glyoxylate to an extent similar to that of CHO-GO cells stably expressing AGT. Altogether, these results show that the Tat peptide is capable of delivering a functional AGT to mammalian cells, thus paving the way for the possibility to use Tat-AGT as an enzyme replacement therapy to counteract PH1.     

Structure of GroEL in Complex with an Early Folding Intermediate of Alanine Glyoxylate Aminotransferase

Primary hyperoxaluria type 1 is a rare autosomal recessive disease caused by mutations in the alanine glyoxylate aminotransferase gene (AGXT). We have previously shown that P11L and I340M polymorphisms together with I244T mutation (AGXT-LTM) represent a conformational disease that could be amenable to pharmacological intervention. Thus, the study of the folding mechanism of AGXT is crucial to understand the molecular basis of the disease. Here, we provide biochemical and structural data showing that AGXT-LTM is able to form non-native folding intermediates. The three-dimensional structure of a complex between the bacterial chaperonin GroEL and a folding intermediate of AGXT-LTM mutant has been solved by cryoelectron microscopy. The electron density map shows the protein substrate in a non-native extended conformation that crosses the GroEL central cavity. Addition of ATP to the complex induces conformational changes on the chaperonin and the internalization of the protein substrate into the folding cavity. The structure provides a three-dimensional picture of an in vivo early ATP-dependent step of the folding reaction cycle of the chaperonin and supports a GroEL functional model in which the chaperonin promotes folding of the AGXT-LTM mutant protein through forced unfolding mechanism.     

Bright Liver and Alanine Aminotransferase Are Associated with Metabolic Syndrome in Adults

Objectives: The metabolic syndrome has become a significant health problem worldwide. In this study, we investigated the relationship between the metabolic syndrome, bright liver (BL) by ultrasonography (US), and plasma alanine aminotransferase (ALT) levels among apparently healthy adults. Research Methods and Procedures: A total of 15, 430 nonalcoholic healthy adults without hepatitis B or C were recruited from four nationwide MJ Health Screening Centers in Taiwan in 2000. Metabolic syndrome was defined using the National Cholesterol Education Panel (NCEP) metabolic syndrome criteria or modified NCEP criteria. Based on liver US, subjects were classified into either having BL or not. The relationship between the metabolic syndrome, BL, and ALT levels was examined using multivariate logistic regression analysis. Results: The crude OR of the metabolic syndrome was 13.92 (12.19 using modified NCEP criteria), and the age‐BMI‐sex—adjusted OR was 3.77 (3.71 using modified NCEP criteria) in subjects with BL vs. subjects without BL, respectively. The ORs of the metabolic syndrome were significantly higher in subjects with elevated ALT levels than in those with normal ALT levels. After adjustment for age, sex, and BMI, BL and elevated ALT level were independently associated with increased risk of the metabolic syndrome. Discussion: Presence of BL and elevated plasma ALT level was independently associated with increased risk of the metabolic syndrome in adults. These factors contribute to a list of well‐known risk factors, including obesity, aging, and male sex, and thus can be applied as an additional evaluation for the metabolic syndrome in a clinical setting.     

Alanine Aminotransferase Variants Conferring Diverse NUE Phenotypes in Arabidopsis thaliana

Alanine aminotransferase (AlaAT, E.C. 2.6.1.2), is a pyridoxal-5’-phosphate-dependent (PLP) enzyme that catalyzes the reversible transfer of an amino group from alanine to 2-oxoglutarate to produce glutamate and pyruvate, or vice versa. It has been well documented in both greenhouse and field studies that tissue-specific over-expression of AlaAT from barley (Hordeum vulgare, HvAlaAT) results in a significant increase in plant NUE in both canola and rice. While the physical phenotypes associated with over-expression of HvAlaAT have been well characterized, the role this enzyme plays in vivo to create a more N efficient plant remains unknown. Furthermore, the importance of HvAlaAT, in contrast to other AlaAT enzyme homologues in creating this phenotype has not yet been explored. To address the role of AlaAT in NUE, AlaAT variants from diverse sources and different subcellular locations, were expressed in the wild-type Arabidopsis thaliana Col-0 background and alaat1;2 (alaat1-1;alaat2-1) knockout background in various N environments. The analysis and comparison of both the physical and physiological properties of AlaAT over-expressing transgenic plants demonstrated significant differences between plants expressing the different AlaAT enzymes under different external conditions. This analysis indicates that the over-expression of AlaAT variants other than HvAlaAT in crop plants could further increase the NUE phenotype(s) previously observed.     

Purification and Characterization of Methionine:Glyoxylate Aminotransferase from Brassica carinata and Brassica napus

The first step in the biosynthesis of allylglucosinolate from methionine in Brassica is thought to be the transamination of methionine to 2-keto-4-methylthiobutyrate. By using Q-Sepharose and Red Agarose, followed by high resolution anion exchange chromatography and chromatofocussing, a methionine:glyoxylate aminotransferase (MGAT) was purified to homogeneity from leaves of Brassica carinata var R-4218, and approximately 5000-fold from leaves of Brassica napus var Topas. The final purification was accomplished using nondenaturing polyacrylamide gel electrophoresis. The enzyme has a pl of 4.3, a native molecular mass of 230 to 290 kilodaltons, and a subunit molecular mass of approximately 50 kilodaltons. Four isozymes of the enzyme were identified in the six species of Brassica commonly cultivated. Nonglucosinolate producing species had only low levels of MGAT or an MGAT isozyme which was distinctly different from that in Brassica.     

Elevated Alanine Aminotransferase Is Strongly Associated with Incident Metabolic Syndrome: A Meta-Analysis of Prospective Studies

Background

The incidence of metabolic syndrome (MetS) is rapidly increasing worldwide and associated with alanine aminotransferase (ALT) activity. However, the impact of ALT activity on MetS incidence is inconsistent in published literature. We therefore estimated the association between elevated ALT activity and incident MetS through a meta-analysis of prospective cohort studies.

Methods/Principal Findings

All published prospective cohort studies on the association between elevated ALT activity and incident MetS were retrieved from Pubmed, Embase, and the Institute for Scientific Information (ISI). In all, seven prospective cohort studies, with 31545 participants and 2873 cases of incident MetS were recruited. If there was insignificant heterogeneity (P-value>0.05 and I²<50%), the fixed-effect model was used to calculate the pooled relative risks (RRs) of incident MetS induced by raised ALT. Otherwise, the random-effect model was used. The calculated RR was 1.81 (95% confidence interval [CI]: 1.49–2.14) when the incidence of MetS was compared between the highest versus the lowest classification of ALT activities. The pooled RR was 1.13 (95% CI: 1.11–1.16) in dose-response analysis with 5 units per liter (U/l) of ALT increment. Subgroup analysis suggested that gender disparity might be the main origin of heterogeneity in overall analysis (P = 0.007 between RRs of gender-specific subgroups evaluated with 5 U/l increments of ALT). Women had a higher dose-response risk of MetS incidence (1.38, 95% CI: 1.20–1.55) than men. Furthermore, sensitivity analysis confirmed the stability of results. No publication bias was found in our meta-analysis.

Conclusions/Significance

Current evidence from prospective studies supports the association between ALT elevation and increasing MetS incidence. This association is closer and more consistent in female population. Further studies are needed to confirm this association and to investigate the potential mechanism of ALT activity on MetS occurrence.     

Cerebral Alanine Transport and Alanine Aminotransferase Reaction: Alanine as a Source of Neuronal Glutamate

Abstract: Alanine transport and the role of alanine amino-transferase in the synthesis and consumption of glutamate were investigated in the preparation of rat brain synaptosomes. Alanine was accumulated rapidly via both the high-and low-affinity uptake systems. The high-affinity transport was dependent on the sodium concentration gradient and membrane electrical potential, which suggests a cotransport with Na⁺. Rapid accumulation of the Na⁺-alanine complex by synaptosomes stimulated activity of the Na⁺/K⁺ pump and increased energy utilization; this, in turn, activated the ATP-producing pathways, glycolysis and oxidative phosphorylation. Accumulation of Na⁺ also caused a small depolarization of the plasma membrane, a rise in [Ca²⁺]₁, and a release of glutamate. Intra-synaptosomal metabolism of alanine via alanine aminotransferase, as estimated from measurements of N fluxes from labeled precursors, was much slower than the rate of alanine uptake, even in the presence of added oxoacids. The velocity of [¹⁵N]alanine formation from [¹⁵N]glutamine was seven to eight times higher than the rate of [¹⁵N]glutamate generation from [¹⁵N]alanine. It is concluded that (a) overloading of nerve endings with alanine could be deleterious to neuronal function because it increases release of glutamate; (b) the activity of synaptosomal alanine aminotransferase is much slower than that of glutaminase and hence unlikely to play a major role in maintaining [glutamate] during neuronal activity; and (c) alanine aminotransferase might serve as a source of glutamate during recovery from ischemia/hypoxia when the alanine concentration rises and that of glutamate falls.     

Four of the Most Common Mutations in Primary Hyperoxaluria Type 1 Unmask the Cryptic Mitochondrial Targeting Sequence of Alanine:glyoxylate Aminotransferase Encoded by the Polymorphic Minor Allele

The gene encoding the liver-specific peroxisomal enzyme alanine:glyoxylate aminotransferase (AGT, EC. 2.6.1.44) exists as two common polymorphic variants termed the “major” and “minor” alleles. The P11L amino acid replacement encoded by the minor allele creates a hidden N-terminal mitochondrial targeting sequence, the unmasking of which occurs in the hereditary calcium oxalate kidney stone disease primary hyperoxaluria type 1 (PH1). This unmasking is due to the additional presence of a common disease-specific G170R mutation, which is encoded by about one third of PH1 alleles. The P11L and G170R replacements interact synergistically to reroute AGT to the mitochondria where it cannot fulfill its metabolic role (i.e. glyoxylate detoxification) effectively. In the present study, we have reinvestigated the consequences of the interaction between P11L and G170R in stably transformed CHO cells and have studied for the first time whether a similar synergism exists between P11L and three other mutations that segregate with the minor allele (i.e. I244T, F152I, and G41R). Our investigations show that the latter three mutants are all able to unmask the cryptic P11L-generated mitochondrial targeting sequence and, as a result, all are mistargeted to the mitochondria. However, whereas the G170R, I244T, and F152I mutants are able to form dimers and are catalytically active, the G41R mutant aggregates and is inactive. These studies open up the possibility that all PH1 mutations, which segregate with the minor allele, might also lead to the peroxisome-to-mitochondrion mistargeting of AGT, a suggestion that has important implications for the development of treatment strategies for PH1.     

Alanine Aminotransferase Is Associated with an Adverse Nocturnal Blood Glucose Profile in Individuals with Normal Glucose Regulation

Objective

Although the association between alanine aminotransferase (ALT) levels and risk of type 2 diabetes is well-studied, the effects of slightly increased ALT levels within the normal range on the temporal normal glucose profile remains poorly understood.

Methods

A total of 322 Chinese subjects without impaired glucose tolerance or previous diagnoses of diabetes were recruited for study from 10 hospitals in urban areas across China. All subjects wore a continuous glucose monitoring (CGM) system for three consecutive days. The diurnal (06∶00–20∶00) and nocturnal (20∶00–06∶00) mean blood glucose (MBG) levels were calculated. Subjects were stratified by ALT quartile level and correlation analyses were performed.

Results

The median ALT level was 17 IU/L, and subjects with ALT ≥17 IU/L had higher nocturnal MBG level than those with ALT <17 IU/L (P<0.05). Nocturnal MBG was positively correlated with ALT levels (Pearson correlation analysis: r = 0.187, P = 0.001), and the correlation remained significant after correction for the homeostatic model assessment of insulin resistance index (HOMA-IR) (r = 0.105, P = 0.041). No correlations were found between diurnal MBG and ALT, and nocturnal or diurnal MBG and aspartate aminotransferase or gamma-glutamyltransferase (all, P>0.05). Multivariate stepwise regression analysis of elevated nocturnal MBG identified increased HOMA-IR, elevated ALT levels, and decreased homeostatic model assessment of ß-cell function as independent factors (all, P<0.05).

Conclusions

Mildly elevated ALT levels, within the normal range, are associated with unfavorable nocturnal glucose profiles in Chinese subjects with normal glucose regulation.     

Alanine Aminotransferase in Bovine Brain: Purification and Properties

Abstract: Mitochondrial and cytosolic alanine aminotransferases (EC 2.6.1.2) were partially purified (140- and 180-fold, respectively) from bovine brain cortex by means of (NH₄)₂SO₄ precipitation, gel filtration on Sephadex G-150, and ion-exchange chromatography on DEAE A-50 and characterized. The enzymes exhibited identical molecular weights (110,000 ± 10,000) and pH optima (7.8), but were eluted from CM Sephadex C-50 at different ionic strengths. Isoelectric focusing of the enzymes indicated a pi value of 5.2 for the cytosolic enzyme and 7.2 for the mitochondrial enzyme. The K _m values of the mitochondrial enzyme were 5.1 m M , 6.6 m M , 0.7 m M , and 0.4 m M and of the cytosolic isozyme were 30.3 m M , 4.3 m M , 0.7 m M , and 0.5 m M for alanine, glutamate, 2-oxoglutarate, and pyruvate, respectively. The results indicated that two forms of alanine aminotransferase exist in nerve tissue, which suggests that they may play different roles in the cellular metabolism of nerve tissue.     

Influence of Convulsants on Rat Brain Activities of Alanine Aminotransferase and Aspartate Aminotransferase

There exist differences between 12-day-old and adult rats in the onset of seizures induced by some inhibitors of glutamate decarboxylase (GAD). The aim of study was to investigate if there are differences between both groups in activities of rat brain alanine aminotransferase (ALT) and aspartate aminotransferase (AST), the enzymes involved in glutamate metabolism, after the administration of 3-mercaptopropionic acid as specific GAD inhibitor or isoniazid as less specific general inhibitor of pyridoxal enzymes. Activities of both aminotransferases in a supernatant 20,000 g of the whole brain (containing predominantly cytosolic isoforms of enzymes) were increased at the beginning of 3-mercaptopropionic acid-induced generalized tonic-clonic seizures. At isoniazid-induced generalized tonic-clonic seizures, a significant increase in both enzyme activities was observed in adult rat brain. In the 12-day-old rat brain, ALT and AST activities reached about 40% and about 50–60% of adult control levels, respectively. In in vitro experiments, no influence of 3-mercaptopropionic acid on transaminase activities was found and an inhibitory effect of isoniazid on the enzymes was confirmed. Increased aminotransferase activities might participate in the enhanced synthesis of excitatory amino acid neurotransmitters in the nervous system, which may take a part in the initiation of epileptic seizures. Alternatively, the increased AST activity may be connected with an increased transport of NADH from the cytosol to mitochondria, while the increased ALT activity would represent the transformation of pyruvate to alanine as a consequence of increased glycolysis.     

丙氨酸氨基转移酶同工酶的研究

<正> 丙氨酸氨基转移酶(ALT)(EC.2.6.1.2)是催化L-丙氨酸与α-酮戊二酸反应生成丙酮酸和谷氨酸的可逆反应的酶,在临床上是最常用酶之一。到目前为止,对于ALT同工酶的研究国内外文献报导甚少,国外文献仅限于动物。本文旨在探索人类ALT同工酶的存在及其在细胞内的分布。     

Stabilization of Rat Liver Mitochondrial Alanine Aminotransferase with Ethanol and Trehalose

Rat liver mitochondrial alanine aminotransferase (mALT) is known to be a very unstable enzyme, a property that has hindered efforts to purify it. In this report we examine the possibility of stabilizing mALT with ethanol, trehalose, and protease inhibitors. The presence of ethanol was shown to slow down the inactivation of mALT, increasing its half-life from 1 to 4 h. Trehalose was found to greatly enhance the stability of mALT in a concentration-dependent manner. In the presence of 36.5% trehalose, the half-life of mALT was 85 h. Of the protease inhibitors tested only antipain and chymostatin slowed down the inactivation of mALT but only within the first 24 h following preparation of the crude enzyme. It is concluded that the inclusion of ethanol and trehalose in purification protocols could aid the purification of the enzyme. It is also concluded that the inclusion of protease inhibitors in purification protocols of mALT may not be necessary as its inactivation does not seem to be due to protease activity.     

Purification and Characterization of L-Alanine: 4,5-Dioxovalerate (Glyoxylate) Aminotransferase from Radish (Raphanus sativus L.) Seedlings

L-Alanine:4,5-dioxovalerate (DOVA) aminotransferase was purified131-fold and characterized from greening seedlings of radish(Raphanus sativus L.). The enzyme was shown to be identicalwith alanine:glyoxylate aminotransferase. The rate of activityof DOVA aminotransferase was 15 times less than that of glyoxylateaminotransferase. Its molecular weight was estimated to be approximately123,000 with two identical subunits, and exhibited a single,broad pH optimum at 8.0. DOVA aminotransferase activity wascompetitively inhibited by glyoxylate. A kinetic study of theenzyme at different alanine concentrations suggested a pingpong reaction mechanism. The K_m values for DOVA and L-alaninewere 0.71 and 1.7 mM, respectively. The activity ratio of transamination under various conditions,the cellular localization of the enzyme and the lack of correlationbetween the activity of this enzyme and chlorophyll synthesis,indicate that DOVA aminotransferase in radish is not involvedin 5-aminolevulinate synthesis. (Received July 7, 1984; Accepted September 11, 1984)     

Influence of Proline on Rat Brain Activities of Alanine Aminotransferase,Aspartate Aminotransferase and Acid Phosphatase

Hyperprolinemia type II (HPII) is an autosomal recessive disorder caused by the severe deficiency of enzyme ¹-pyrroline-5-carboxylic acid dehydrogenase leading to tissue accumulation of proline. Chronic administration of Pro led to significant reduction of cytosolic ALT activity of olfactory lobes (50.57%), cerebrum (40%) and medulla oblongata (13.71%) only. Whereas mitochondrial ALT activity was reduced significantly in, all brain regions such as olfactory lobes (73.23%), cerebrum (70.26%), cerebellum (65.39%) and medulla oblongata (65.18%). The effect of chronic Pro administration on cytosolic AST activity was also determined. The cytosolic AST activity from olfactory lobes, cerebrum and medulla oblongata reduced by 75.71, 67.53 and 76.13%, respectively while cytosolic AST activity from cerebellum increased by 28.05%. The mitochondrial AST activity lowered in olfactory lobes (by 72.45%), cerebrum (by 78%), cerebellum (by 49.56%) and medulla oblongata (by 69.30%). In vitro studies also showed increase in brain tissue proline and decrease in glutamate levels. In vitro studies indicated that proline has direct inhibitory effect on these enzymes and glutamate levels in brain tissue showed positive correlation with AST and ALT activities. Acid phosphatase (ACP) activity reduced significantly in olfactory lobes (40.33%) and cerebrum (20.82%) whereas it elevated in cerebellum (97.32%) and medulla oblongata (76.33%). The histological studies showed degenerative changes in brain. Following proline treatment, the animals became sluggish and showed low responses to tail pricks and lifting by tails and showed impaired balancing. These observations indicate influence of proline on AST, ALT and ACP activities of different brain regions leading to lesser synthesis of glutamate thereby causing neurological dysfunctions.     

Alanine Aminotransferase and Risk of the Metabolic Syndrome: A Linear Dose-Response Relationship

Background

Elevated baseline circulating alanine aminotransferase (ALT) level has been demonstrated to be associated with an increased risk of the metabolic syndrome (MetS), but the nature of the dose-response relationship is uncertain.

Methods

We performed a systematic review and meta-analysis of published prospective cohort studies to characterize in detail the nature of the dose-response relationship between baseline ALT level and risk of incident MetS in the general population. Relevant studies were identified in a literature search of MEDLINE, EMBASE, and Web of Science up to December 2013. Prospective studies in which investigators reported relative risks (RRs) of MetS for 3 or more categories of ALT levels were eligible. A potential nonlinear relationship between ALT levels and MetS was examined using restricted cubic splines.

Results

Of the 489 studies reviewed, relevant data were available on 29,815 non-overlapping participants comprising 2,125 incident MetS events from five prospective cohort studies. There was evidence of a linear association (P for nonlinearity = 0.38) between ALT level and risk of MetS, characterised by a graded increase in MetS risk at ALT levels 6–40 U/L. The risk of MetS increased by 14% for every 5 U/L increment in circulating ALT level (95% CI: 12–17%). Evidence was lacking of heterogeneity and publication bias among the contributing studies.

Conclusions

Baseline ALT level is associated with risk of the MetS in a linear dose-response manner. Studies are needed to determine whether the association represents a causal relationship.     

Variants in the ASB10 Gene Are Associated with Primary Open Angle Glaucoma

Background

Recently nonsynonymous coding variants in the ankyrin repeats and suppressor of cytokine signaling box-containing protein 10 (ASB10) gene were found to be associated with primary open angle glaucoma (POAG) in cohorts from Oregon and Germany, but this finding was not confirmed in an independent cohort from Iowa. The aim of the current study was to assess the role of ASB10 gene variants in Pakistani glaucoma patients.

Methods

Sanger sequencing of the coding exons and splice junctions of the ASB10 gene was performed in 30 probands of multiplex POAG families, 208 sporadic POAG patients and 151 healthy controls from Pakistan. Genotypic associations of individual variants with POAG were analyzed with the Fisher’s exact or Chi-square test.

Results

In total 24 variants were identified in POAG probands and sporadic patients, including 11 novel variants and 13 known variants. 13 of the variants were nonsynonymous, 6 were synonymous, and 5 were intronic. Three nonsynonymous variants (p.Arg49Cys, p.Arg237Gly, p.Arg453Cys) identified in the probands were not segregating in the respective families. This is not surprising since glaucoma is a multifactorial disease, and multiple factors are likely to be involved in the disease manifestation in these families. However a nonsynonymous variant, p.Arg453Cys (rs3800791), was found in 6 sporadic POAG patients but not in controls, suggesting that it infers increased risk for the disease. In addition, one synonymous variant was found to be associated with sporadic POAG: p.Ala290Ala and the association of the variant with POAG remained significant after correction for multiple testing (uncorrected p-value 0.002, corrected p-value 0.047). The cumulative burden of rare, nonsynonymous variants was significantly higher in sporadic POAG patients compared to control individuals (p-value 0.000006).

Conclusions

Variants in ASB10 were found to be significantly associated with sporadic POAG in the Pakistani population. This supports previous findings that sequence variants in the ASB10 gene may act as a risk factor for glaucoma.