In 2022 the World Health Organization declared a Public Health Emergency for an outbreak of mpox, the zoonotic Orthopoxvirus (OPV) affecting at least 104 nonendemic locations worldwide. Serologic ...detection of mpox infection is problematic, however, due to considerable antigenic and serologic cross-reactivity among OPVs and smallpox-vaccinated individuals. In this report, we developed a high-throughput multiplex microsphere immunoassay using a combination of mpox-specific peptides and cross-reactive OPV proteins that results in the specific serologic detection of mpox infection with 93% sensitivity and 98% specificity. The New York State Non-Vaccinia Orthopoxvirus Microsphere Immunoassay is an important tool to detect subclinical mpox infection and understand the extent of mpox spread in the community through retrospective analysis.
γ-Glutamyl hydrolase (GGH) plays a central role in folate metabolism and antifolate action. Increased GGH activity has been found in rat hepatoma cells resistant to the cancer drug methotrexate ...(MTX). The aim of this study was to identify polymorphisms in the
GGH gene that modulate GGH activity and that may affect methotrexate resistance. Exons of the human γ-glutamyl hydrolase (
hGGH) gene were amplified by polymerase chain reaction (PCR) from breast cancer tissue and leukemia cell lines. Single-stranded conformational polymorphism (SSCP) analysis was performed, and PCR products containing different patterns were cloned and sequenced. Six single nucleotide polymorphisms (SNPs) were identified, at bases −401C>T, −354G>T, −124T>G, +16T>C, +452C>T, and +1102A>G, relative to the A of the translation start codon being considered as +1. The SNP at +16, which changes codon −19 (relative to the start of the mature hGGH protein) in the endoplasmic reticulum targeting sequence of hGGH protein from cysteine to arginine, has previously been identified in this laboratory. The SNP at +452 changes the conserved hGGH protein codon 127 from threonine to isoleucine. The functions of SNPs in the promoter of the
hGGH gene were studied by site-directed mutagenesis of a 516-bp region of the
hGGH gene promoter in a luciferase reporter vector and transfection into HepG2 and MCF-7 cells. All of the promoter polymorphisms enhanced the production of luciferase compared to the wild-type
hGGH gene promoter in HepG2 cells, and −401C>T and −124T>G enhanced luciferase expression in MCF-7 cells, suggesting that polymorphisms in the
hGGH gene promoter may increase expression of hGGH protein.
Human γ-glutamyl hydrolase (hGH) is a key enzyme in the metabolism of folic acid and in the pharmacology of many antifolate drugs. hGH catalyzes removal of the poly-γ-glutamate chains of ...intracellular folic acid and antifolates. hGH crystallized as a homodimer with two putative active sites. However, the quaternary structure and the number of species of the enzyme in solution have not been determined. hGH has now been characterized using analytical ultracentrifugation and dynamic light scattering. HisTag fusion proteins of wild-type hGH, rat GH, and hGH expressed as a glycosylated protein were studied. Analyses of HisTag wild-type hGH were conducted over a range of protein concentrations (1.4–200
μM), ionic strengths (0–1
M NaCl), and pH (4.5–8.5). A single species with a molecular mass consistent with a homodimer was observed. Glycosylated hGH and HisTag rat γ-glutamyl hydrolase also formed very stable homodimers. The lack of dissociation of the dimer, the large monomer–monomer interface, and the presence of catalytically essential Tyr-36 in the homodimer interface sequences suggest that homodimer formation is required for the hGH monomer to fold into an active conformation. The conservation of hGH monomer–monomer interface sequences in other mammalian and plant γ-glutamyl hydrolase molecules suggests that they also exist as stable homodimers.
Urbanisation en Mélanésie Adams, Christophe; Alevêque, Guillaume; Bambridge, Tamatoa ...
Journal de la Société des océanistes,
12/2017
144-145
Journal Article
Human γ-glutamyl hydrolase (hGH) is a central enzyme in folyl and antifolylpoly-γ-glutamate metabolism, which functions by catalyzing the cleavage of the γ-glutamyl chain of substrates. We previously ...reported that Cys-110 is essential for activity. Using the sequence of hGH as a query, alignment searches of protein data bases were made using the SSearch and TPROBE programs. Significant similarity was found between hGH and the glutamine amidotransferase type I domain of Escherichia colicarbamoyl phosphate synthetase. The resulting hypothesis is that the catalytic fold of hGH is similar to the folding of this domain in carbamoyl phosphate synthetase. This model predicts that Cys-110 of hGH is the active site nucleophile and forms a catalytic triad with residues His-220 and Glu-222. The hGH mutants C110A, H220A, and E222A were prepared. Consistent with the model, mutants C110A and H220A were inactive. However, the Vmax of the E222A hGH mutant was reduced only 6-fold relative to the wild-type enzyme. The model also predicted that His-171 in hGH may be involved in substrate binding. The H171N hGH mutant was found to have a 250-fold reducedVmax. These studies to determine the catalytic mechanism begin to define the three dimensional interactions of hGH with poly-γ-glutamate substrates.
Gamma-glutamyl hydrolase (GH), which hydrolyses the gamma-glutamyl conjugates of folic acid, is a key enzyme in the maintenance of cellular folylpolyglutamate concentrations. The catalytic mechanism ...of GH is not known. Consistent with earlier reports that GH is sulphydryl-sensitive, we found that recombinant human GH is inhibited by iodoacetic acid, suggesting that at least one cysteine is important for activity Rhee, Lindau-Shepard, Chave, Galivan and Ryan (1998) Mol. Pharmacol. 53, 1040-1046. Using site-directed mutagenesis, the cDNA for human GH was altered to encode four different proteins each with one of four cysteine residues changed to alanine. Three of the mutant proteins had activities similar to wild-type GH and were inhibited by iodoacetic acid, whereas the C110A mutant had no activity. Cys-110 is conserved among the human, rat and mouse GH amino acid sequences. The wild-type protein and all four mutants had similar intrinsic fluorescence spectra, indicating no major structural changes had been introduced. These results indicate that Cys-110 is essential for enzyme activity and suggest that GH is a cysteine peptidase. These studies represent the first identification of the essential Cys residue in this enzyme and provide the beginning of a framework to determine the catalytic mechanism, important in defining GH as a therapeutic target.
γ-Glutamyl hydrolase (GH) plays an important role in the metabolism of folic acid and the pharmacology of antifolates such as methotrexate. We have previously cloned and characterized the human GH ...cDNA. In this report, the complete organization and structure of the human GH gene was determined. The human GH gene spans 24
kb in the human genome, with nine exons sized from 51 to 371
bp. All of exon–intron splice junctions follow the GT–AG rule. The sequence upstream of exon 1 consists of a promoter-like, GC-rich region and a number of putative
cis active elements including Sp1, AP1, and MZF1 sites. A TATA sequence in the 5′ region of human GH gene was not observed, similar to housekeeping genes known to be tissue-specific and differentially expressed. S1 nuclease protection analysis with human liver, prostate, brain, and mammary gland revealed a major transcription start point at nucleotide −125 relative to the ATG start codon and several minor transcription start points. Analysis of GH cDNA isolated from human liver indicated a nucleotide change, T→C, in the leader sequence of GH, which suggested a polymorphism. Studies of cDNA from different human tissue sources provided evidence that there is a single spliced cDNA species in human.
γ-Glutamyl hydrolase catalyzes the cleavage of the γ-glutamyl chain of folylpoly-γ-glutamyl substrates and is a central enzyme in folyl and antifolyl poly-γ-glutamate metabolism. The crystal ...structure of human γ-glutamyl hydrolase, determined at 1.6-Å resolution, reveals that the protein is a homodimer. The overall structure of human γ-glutamyl hydrolase contains 11 α-helices and 14 β-strands, with a fold in which a central eight-stranded β-sheet is sandwiched by three and five α-helices on each side. The topology is very similar to that of the class I glutamine amidotransferase domains, with the only major differences consisting of extensions in four loops and at the C terminus. These insertions are important for defining the substrate binding cleft and/or the dimer interface. Two sequence motifs are found in common between human γ-glutamyl hydrolase and the class I glutamine amidotransferase family and include the catalytically essential residues, Cys-110 and His-220. These residues are located in the center of a largel-shaped cleft that is closed at one end and open at the other. Several conserved residues, including Glu-114, His-171, Gln-218, and Lys-223, may be important for substrate binding. Modeling of a methotrexate thioester intermediate, based on the corresponding complex of the glutamate thioester intermediate of Escherichia coli carbamoyl-phosphate synthetase, indicates that the substrate binds in an orientation with the pteroyl group toward the open end of the cleft.
gamma-Glutamyl hydrolase catalyzes the cleavage of the gamma-glutamyl chain of folylpoly-gamma-glutamyl substrates and is a central enzyme in folyl and antifolyl poly-gamma-glutamate metabolism. The ...crystal structure of human gamma-glutamyl hydrolase, determined at 1.6-A resolution, reveals that the protein is a homodimer. The overall structure of human gamma-glutamyl hydrolase contains 11 alpha-helices and 14 beta-strands, with a fold in which a central eight-stranded beta-sheet is sandwiched by three and five alpha-helices on each side. The topology is very similar to that of the class I glutamine amidotransferase domains, with the only major differences consisting of extensions in four loops and at the C terminus. These insertions are important for defining the substrate binding cleft and/or the dimer interface. Two sequence motifs are found in common between human gamma-glutamyl hydrolase and the class I glutamine amidotransferase family and include the catalytically essential residues, Cys-110 and His-220. These residues are located in the center of a large l-shaped cleft that is closed at one end and open at the other. Several conserved residues, including Glu-114, His-171, Gln-218, and Lys-223, may be important for substrate binding. Modeling of a methotrexate thioester intermediate, based on the corresponding complex of the glutamate thioester intermediate of Escherichia coli carbamoyl-phosphate synthetase, indicates that the substrate binds in an orientation with the pteroyl group toward the open end of the cleft.
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