Rapid detection of glyoxalase I (GLO) on cellulose acetate gel and the distribution of GLO variants in a Dutch population

Summary A rapid electrophoretic procedure is described for detecting the human red cell glyoxalase I variants (GLO 1, GLO 2-1, and GLO 2) on cellulose acetate gel (cellogel) on which the sites of enzymed activity are visualized as purple bands against white background. The frequency of GLO ¹ gene in a Dutch population living in and around Leiden was found to be 0.4544.     

On the population genetics of the red cell glyoxalase I (GLO)

Summary The GLO phenotype distribution was studied in the population from the Rostock area (n=233). The gene frequencies of GLO alleles were estimated to be GLO¹=0.4249, GLO²=0.5751. The electrophoretic separation was achieved on horizontal starch gel.     

Gene diversity in Indian populations

Summary The phenotypes of glyoxalase I (GLO) were determined in a random population from Hessen (Germany) by high-voltage agarose gel electrophoresis. The gene frequencies in 1150 unrelated individuals were 0.4391 for GLO¹ and 0.5609 for GLO². Rare phenotypes were not observed. The segregation of phenotypes in 50 families and 32 mother-child combinations supports the assumed autosomal codominant inheritance. The possibility of a simultaneous typing for GLO, esterase D (EsD), and carbonic anhydrase₂ (CA₂) on one gel is discussed.     

miR-137 inhibits melanoma cell proliferation through downregulation of GLO1

Late-stage melanoma is refractory to current therapies. MicroRNAs (miRNAs) can modulate many physiological and pathological processes of melanoma. Studies have demonstrated that miR-137 acts as a tumor suppressor by inhibiting the proliferation of melanoma cells through targeting multiple mRNAs. The glyoxalase system member glyoxalase 1 (GLO1) is the principal scavenging enzyme of methylglyoxal (MG), a toxic byproduct of glycolysis. Using ³⁵S in vivo/vitro labelling analysis for dynamic proteomics (SiLAD), we found that miR-137 downregulated the expression of GLO1 in melanoma cells. Bioinformatics analysis predicted that GLO1 is a direct target of miR-137. This was validated by dual luciferase reporter assay. Quantitative RT-PCR (qRT-PCR) and western blot analysis indicated that miR-137 could decrease endogenous GLO1 expression. Furthermore, siRNA targeting of GLO1 mimicked inhibition of melanoma cell proliferation caused by miR-137 overexpression. Re-expression of GLO1 was able to restore miR-137-mediated suppression of melanoma cell proliferation. Therefore, these results suggest that miR-137 inhibits the proliferation of melanoma cells by targeting GLO1.     

Distribution of glyoxalase I (GLO) variants in Western Europe and the Indian subcontinent

Summary English, Italian (including Sardinian), and Spanish populations from Europe and Muslim, Hindu, Sikh, Punjabi, and other populations from the Indian subcontinent currently living either in Birmingham or in India were screened for electrophoretically detectable genetic variants of red cell glyoxalase I (GLO), and their frequencies were reported. All the western European populations investigated, including those reported, exhibited an incidence of close to 44% for the GLO ¹ gene. The frequency distribution of the GLO ¹ gene in various populations from the Indian subcontinent, in contrast, was found to range between 0.15 and 0.33. These observations suggest that the European populations in general are genetically more homogeneous than are the populations of the Indian subcontinent.     

Identification of glyoxalase 1 polymorphisms associated with enzyme activity

The glyoxalase system and its main enzyme, glyoxalase 1 (GLO1), protect cells from advanced glycation end products (AGEs), such as methylglyoxal (MG) and other reactive dicarbonyls, the formation of which is increased in diabetes patients as a result of excessive glycolysis. MG is partly responsible for harmful protein alterations in living cells, notably in neurons, leading to their dysfunction, and recent studies have shown a negative correlation between GLO1 expression and tissue damage. Neuronal dysfunction is a common diabetes complication due to elevated blood sugar levels, leading to high levels of AGEs. The aim of our study was to determine whether single nucleotide polymorphisms (SNPs) in the GLO1 gene influence activity of the enzyme. In total, 125 healthy controls, 101 type 1 diabetes, and 100 type 2 diabetes patients were genotyped for three common SNPs, rs2736654 (A111E), rs1130534 (G124G), and rs1049346 (5′-UTR), in GLO1. GLO1 activity was determined in whole blood lysates for all participants of the study.     

The dagestan gene pool: Analysis of the frequencies of classical genetic markers in Avars

This study is part of long-term research in the gene pool of Dagestan ethnic groups. The phenotype (in percent), gene, and haplotype frequencies in three Avar populations are reported. A total of 37 alleles of 13 loci of immune and biochemical genetic marker systems (ABO, Rhesus, P, Lewis, HP, GC, C′3, TF, 6PGD, GLO1, ESD, ACP, and PGM1) have been studied. Rare haplotypes of the Rhesus system (CDE, Cde and cdE) have been found in the populations studied. In two out of three local populations (Khunzakh and Kharakhi), a typically “Caucasoid” rare gene ACP1 ^c of the AcP1 locus has proved to be relatively frequent (0.030 and 0.023, respectively). The frequencies of the allele variantsP ² , le, and Hp ¹ of loci of the P, Lewis, and HP systems, respectively, have been found to be lower than in other Caucasian ethnic groups and the total northern Eurasian population. The mean allele frequencies for the GC, C′3, TF, 6PGD, GLO1, and ESD systems in the populations studied are comparable wit those for both Caucasian ethnic groups and the total population of the European historical ethnographic province. Statistical analysis of the results has shown 11 cases of significant deviations of the observed phenotype frequencies from the Hardy-Weinberg equilibrium.     

Glyoxalase 1 (GLO) in the chicken: Genetic variation and lack of linkage to the MHC

Electrophoretic variation of glyoxalase 1 (GLO) has been detected in chicken red-cell lysates. Three phenotypes are shown to be inherited through a diallelic system, just as in humans and mice. The chicken GLO phenotypes differ from their mammalian counterparts in that one of the homozygotes is devoid of GLO activity. The heterozygote produces two bands, while the other homozygote yields a single band of GLO activity with mobility equal to the faster of these two bands. In noninbred White Leghorn birds, the GLO ^*2 allele occurred significantly more often in birds homozygous for the B ^*1 allele at the chicken MHC than in those homozygous for B ^*19, suggesting that the products of these loci may have population associations in the chicken. Absence of close linkage between the GLO and B loci was, however, demonstrated by appropriate test crosses.     

Recent advances in the discovery and development of glyoxalase I inhibitors

Glyoxalase I (GLO1) is a homodimeric Zn²⁺-metalloenzyme that catalyses the transformation of methylglyoxal (MG) to d-lacate through the intermediate S-d-lactoylglutathione. Growing evidence indicates that GLO1 has been identified as a potential target for the treatment cancer and other diseases. Various inhibitors of GLO1 have been discovered or developed over the past several decades including natural or natural product-based inhibitors, GSH-based inhibitors, non-GSH-based inhibitors, etc. The aim of this review is to summarize recent achievements of concerning discovery, design strategies, as well as pharmacological aspects of GLO1 inhibitors with the target of promoting their development toward clinical application.     

Methylglyoxal,the foe and friend of glyoxalase and Trx/TrxR systems in HT22 nerve cells

Methylglyoxal (MGO) is a major glycating agent that reacts with basic residues of proteins and promotes the formation of advanced glycation end products (AGEs) which are believed to play key roles in a number of pathologies, such as diabetes, Alzheimer's disease, and inflammation. Here, we examined the effects of MGO on immortalized mouse hippocampal HT22 nerve cells. The endpoints analyzed were MGO and thiol status, the glyoxalase system, comprising glyoxalase 1 and 2 (GLO1/2), and the cytosolic and mitochondrial Trx/TrxR systems, as well as nuclear Nrf2 and its target genes. We found that nuclear Nrf2 is induced by MGO treatment in HT22 cells, as corroborated by induction of the Nrf2-controlled target genes and proteins glutamate cysteine ligase and heme oxygenase 1. Nrf2 knockdown prevented MGO-dependent induction of glutamate cysteine ligase and heme oxygenase 1. The cystine/glutamate antiporter, system x_c^–, which is also controlled by Nrf2, was also induced. The increased cystine import (system x_c^–) activity and GCL expression promoted GSH synthesis, leading to increased levels of GSH. The data indicate that MGO can act as both a foe and a friend of the glyoxalase and the Trx/TrxR systems. At low concentrations of MGO (0.3 mM), GLO2 is strongly induced, but at high MGO (0.75 mM) concentrations, GLO1 is inhibited and GLO2 is downregulated. The cytosolic Trx/TrxR system is impaired by MGO, where Trx is downregulated yet TrxR is induced, but strong MGO-dependent glycation may explain the loss in TrxR activity. We propose that Nrf2 can be the unifying element to explain the observed upregulation of GSH, GCL, HO1, TrxR1, Trx2, TrxR2, and system x_c^– system activity.     

Glyoxalase I reduces glycative and oxidative stress and prevents age‐related endothelial dysfunction through modulation of endothelial nitric oxide synthase phosphorylation

Endothelial dysfunction is a major contributor to cardiovascular disease (CVD), particularly in elderly people. Studies have demonstrated the role of glycation in endothelial dysfunction in nonphysiological models, but the physiological role of glycation in age‐related endothelial dysfunction has been poorly addressed. Here, to investigate how vascular glycation affects age‐related endothelial function, we employed rats systemically overexpressing glyoxalase I (GLO1), which detoxifies methylglyoxal (MG), a representative precursor of glycation. Four groups of rats were examined, namely young (13 weeks old), mid‐age (53 weeks old) wild‐type, and GLO1 transgenic (WT/GLO1 Tg) rats. Age‐related acceleration in glycation was attenuated in GLO1 Tg rats, together with lower aortic carboxymethyllysine (CML) and urinary 8‐hydroxydeoxyguanosine (8‐OHdG) levels. Age‐related impairment of endothelium‐dependent vasorelaxation was attenuated in GLO1 Tg rats, whereas endothelium‐independent vasorelaxation was not different between WT and GLO1 Tg rats. Nitric oxide (NO) production was decreased in mid‐age WT rats, but not in mid‐age GLO1 Tg rats. Age‐related inactivation of endothelial NO synthase (eNOS) due to phosphorylation of eNOS on Thr495 and dephosphorylation on Ser1177 was ameliorated in GLO1 Tg rats. In vitro, MG increased phosphorylation of eNOS (Thr495) in primary human aortic endothelial cells (HAECs), and overexpression of GLO1 decreased glycative stress and phosphorylation of eNOS (Thr495). Together, GLO1 reduced age‐related endothelial glycative and oxidative stress, altered phohphorylation of eNOS, and attenuated endothelial dysfunction. As a molecular mechanism, GLO1 lessened inhibitory phosphorylation of eNOS (Thr495) by reducing glycative stress. Our study demonstrates that blunting glycative stress prevents the long‐term impact of endothelial dysfunction on vascular aging.     

Polymorphism of red cell glyoxalase I (EI: 4.4.1.5); a new genetic marker in man. Investigation of 169 mother-child combinations.

The polymorphism of glyoxalase I was investigated in 169 mother-child combinations from southwestern Germany. Glyoxalase I (GLO) has 3 common phenotypes: GLO 1, GLO 2-1, and GLO 2. The results are in good agreement with the formal hypothesis: Two alleles GLO1 and GLO2 at an autosomal locus. The GLO1 gene frequency was estimated to be 0.39. From the electrophoretic pattern the GLO-molecule appears to consist of two subunits.     

Glycolate oxidase isozymes are coordinately controlled by GLO1 and GLO4 in rice

Glycolate oxidase (GLO) is a key enzyme in photorespiratory metabolism. Four putative GLO genes were identified in the rice genome, but how each gene member contributes to GLO activities, particularly to its isozyme profile, is not well understood. In this study, we analyzed how each gene plays a role in isozyme formation and enzymatic activities in both yeast cells and rice tissues. Five GLO isozymes were detected in rice leaves. GLO1 and GLO4 are predominately expressed in rice leaves, while GLO3 and GLO5 are mainly expressed in the root. Enzymatic assays showed that all yeast-expressed GLO members except GLO5 have enzymatic activities. Further analyses suggested that GLO1, GLO3 and GLO4 interacted with each other, but no interactions were observed for GLO5. GLO1/GLO4 co-expressed in yeast exhibited the same isozyme pattern as that from rice leaves. When either GLO1 or GLO4 was silenced, expressions of both genes were simultaneously suppressed and most of the GLO activities were lost, and consistent with this observation, little GLO isozyme protein was detected in the silenced plants. In contrast, no observable effect was detected when GLO3 was suppressed. Comparative analyses between the GLO isoforms expressed in yeast and the isozymes from rice leaves indicated that two of the five isozymes are homo-oligomers composed of either GLO1 or GLO4, and the other three are hetero-oligomers composed of both GLO1 and GLO4. Our current data suggest that GLO isozymes are coordinately controlled by GLO1 and GLO4 in rice, and the existence of GLO isozymes and GLO molecular and compositional complexities implicate potential novel roles for GLO in plants.     

Glyoxalase-I is a novel prognosis factor associated with gastric cancer progression

Glyoxalase I (GLO1), a methylglyoxal detoxification enzyme, is implicated in the progression of human malignancies. The role of GLO1 in gastric cancer development or progression is currently unclear. The expression of GLO1 was determined in primary gastric cancer specimens using quantitative polymerase chain reaction, immunohistochemistry (IHC), and western blotting analyses. GLO1 expression was higher in gastric cancer tissues, compared with that in adjacent noncancerous tissues. Elevated expression of GLO1 was significantly associated with gastric wall invasion, lymph node metastasis, and pathological stage, suggesting a novel role of GLO1 in gastric cancer development and progression. The 5-year survival rate of the lower GLO1 expression groups was significantly greater than that of the higher expression groups (log rank P = 0.0373) in IHC experiments. Over-expression of GLO1 in gastric cancer cell lines increases cell proliferation, migration and invasiveness. Conversely, down-regulation of GLO1 with shRNA led to a marked reduction in the migration and invasion abilities. Our data strongly suggest that high expression of GLO1 in gastric cancer enhances the metastasis ability of tumor cells in vitro and in vivo, and support its efficacy as a potential marker for the detection and prognosis of gastric cancer.     

我国汉族人群的乙二醛酶Ⅰ遗传多态性

用琼脂糖平板凝胶电泳技术分析了我国六个城市的汉族共1238人的红细胞乙二醛酶I的遗传多态性。每个汉族人群的受检人数和GLO1*1基因频率分别为:郑州200人(0.1023),哈尔滨195人(0.1103),呼和浩特210人(0.1259),贵阳204人(0.1373),兰州210人(0.1476),西安199人(0.1508)。中国不同地区汉族人群之间的GLOI基因频率分布存在显著性差异,但这种差异明显小于中国不同少数民族之间的差异。     

Population genetic studies in three Chinese minorities

Three minority ethnic groups from China (Mongolians, Koreans, Zhuang) were examined with respect to the genetic markers GLO, GPT, ACP, ESD, 6-PGD, PGM₁ subtypes, C3, and TF. Significant variations were noted for the gene frequencies of GLO, GPT, ESD, sub PGM₁ between Zhuang and Mongolians; for GPT, ACP, ESD, sub PGM₁ between Zhuang and Koreans; and for GLO between Mongolians and Koreans.     

Effects of R219K polymorphism of ATP-binding cassette transporter 1 gene on serum lipids ratios induced by a high-carbohydrate and low-fat diet in healthy youth

Background

Diets are the important players in regulating plasma lipid profiles. And the R219K polymorphism at the gene of ATP-binding cassette transporter 1(ABCA1) was reported to be associated with the profiles. However, no efforts have been made to investigate the changes of lipid profiles after a high-carbohydrate and low-fat diet in different subjects with different genotypes of this polymorphism. This study was to evaluate the effects of ABCA1 R219K polymorphism on serum lipid and apolipoprotein (apo) ratios induced by a high-carbohydrate/low-fat (high-CHO) diet. After a washout diet of 54.1% carbohydrate for 7 days, 56 healthy young subjects (22.89 ± 1.80 years old) were given a high-CHO diet of 70.1% carbohydrate for 6 days. Height, weight, waist circumference, hip circumference, glucose (Glu), triglyceride (TG), total cholesterol (TC), high density lipoprotein cholesterol (HDL-C), low density lipoprotein cholesterol (LDL-C), apoA-1 and apoB-100 were measured on the 1^st, 8^th and 14^th days of this study. Body mass index (BMI), waist-to-hip ratios (WHR), log(TG/HDL-C), TC/HDL-C, LDL-C/HDL-C and apoA-1/apoB-100 were calculated. ABCA1 R219K was analyzed by a PCR-RFLP method.

Results

The results indicate that the male subjects of all the genotypes had higher WHR than their female counterparts on the 1^st, 8^th and 14^th days of this study. The male K carriers had higher log(TG/HDL-C) and TC/HDL-C than the female carriers on the 1^st and 14^th days, and higher LDL-C/HDL-C on the 14^th day. When compared with that on the 8^th day, TC/HDL-C was decreased regardless of the genotypes and genders on the 14^th day. Log(TG/HDL-C) was increased in the males with the RR genotype and the female K carriers. Lowered BMI, Glu and LDL-C/HDL-C were found in the male K carriers, but only lowered BMI in the female K carriers and only lowered LDL-C/HDL-C in the females with the RR genotype.

Conclusions

These results suggest that ABCA1 R219K polymorphism is associated differently in males and females with elevated log(TG/HDL-C) and decreased LDL-C/HDL-C induced by the high-CHO diet.     

Cloning and characterization of gluconolactone oxidase of Penicillium cyaneo-fulvum ATCC 10431 and evaluation of its use for production of D-erythorbic acid in recombinant Pichia pastoris

A D-erythorbic acid-forming soluble flavoprotein, gluconolactone oxidase (GLO), was purified from Penicillium cyaneo-fulvum strain ATCC 10431 and partially sequenced. Peptide sequences were used to isolate a cDNA clone encoding the enzyme. The cloned gene exhibits high levels of similarity with the genes encoding other known eukaryotic lactone oxidases and also with the genes encoding some putative prokaryotic lactone oxidases. Analysis of the coding sequence of the GLO gene indicated the presence of a typical secretion signal sequence at the N terminus of GLO. No other targeting or anchoring signals were found, suggesting that GLO is the first known lactone oxidase that is secreted rather than targeted to the membranes of the endoplasmic reticulum or mitochondria. Experimental evidence, including the N-terminal sequence of mature GLO and data on glycosylation and localization of the enzyme in native and recombinant hosts, supports this analysis. The GLO gene was expressed in Pichia pastoris, and recombinant GLO was produced by using the strong methanol-induced AOX1 promoter. In order to evaluate the suitability of purified GLO for production of D-erythorbic acid, we immobilized it on N-hydroxysuccinimide-activated Sepharose and found that the immobilized GLO retained full activity during immobilization but was rather unstable under reaction conditions. Our results show that both soluble and immobilized forms of GLO can, in principle, be used for production of D-erythorbic acid from D-glucono-delta-lactone or (in combination with glucose oxidase and catalase) from glucose. We also demonstrated the feasibility of glucose-D-erythorbic acid fermentation with recombinant strains coexpressing GLO and glucose oxidase genes, and we analyzed problems associated with construction of efficient D-erythorbic acid-producing hosts.     

Polymorphism of BF,C2, and GLO in Japanese patients with insulin-dependent diabetes mellitus: Confirmation of an increase of BF *FT

Summary The distribution of phenotypes controlled by three HLA-linked loci BF, C2, and GLO has been studied in Japanese patients with insulin-dependent diabetes mellitus (IDDM). A slight but significant higher incidence of a rare varian BF ^*FT (=^* F075) in patients was confirmed in the combined data with our previous study (Tokunaga et al. 1981 b). No significant association of C2 and GLO alleles with IDDM was found.