Identification of glycinin in vivo as a polyamine-conjugated protein via a gamma-glutamyl linkage.
Ontology highlight
ABSTRACT:
SUBMITTER: Kang H
PROVIDER: S-EPMC1219502 | biostudies-other | 1998 Jun
REPOSITORIES: biostudies-other
Similar Datasets
Project description:BackgroundCarboxylation is a modification of glutamate (Glu) residues which occurs post-translation that is catalyzed by γ-glutamyl carboxylase in the lumen of the endoplasmic reticulum. Vitamin K is a critical co-factor in the post-translational conversion of Glu residues to γ-carboxyglutamate (Gla) residues. It has been shown that the process of carboxylation is involved in the blood clotting cascade, bone growth, and extraosseous calcification. However, studies in this field have been limited by the difficulty of experimentally studying substrate site specificity in γ-glutamyl carboxylation. In silico investigations have the potential for characterizing carboxylated sites before experiments are carried out.ResultsBecause of the importance of γ-glutamyl carboxylation in biological mechanisms, this study investigates the substrate site specificity in carboxylation sites. It considers not only the composition of amino acids that surround carboxylation sites, but also the structural characteristics of these sites, including secondary structure and solvent-accessible surface area (ASA). The explored features are used to establish a predictive model for differentiating between carboxylation sites and non-carboxylation sites. A support vector machine (SVM) is employed to establish a predictive model with various features. A five-fold cross-validation evaluation reveals that the SVM model, trained with the combined features of positional weighted matrix (PWM), amino acid composition (AAC), and ASA, yields the highest accuracy (0.892). Furthermore, an independent testing set is constructed to evaluate whether the predictive model is over-fitted to the training set.ConclusionsIndependent testing data that did not undergo the cross-validation process shows that the proposed model can differentiate between carboxylation sites and non-carboxylation sites. This investigation is the first to study carboxylation sites and to develop a system for identifying them. The proposed method is a practical means of preliminary analysis and greatly diminishes the total number of potential carboxylation sites requiring further experimental confirmation.
| S-EPMC3278826 | biostudies-literature
Project description:Glutathione (GSH) is often upregulated in cancer, where it serves to mitigate oxidative stress. γ-glutamyl-transferase (GGT) is a key enzyme in GSH homeostasis, and compared to normal brain its expression is elevated in tumors, including in primary glioblastoma. GGT is therefore an attractive imaging target for detection of glioblastoma. The goal of our study was to assess the value of hyperpolarized (HP) γ-glutamyl-[1-13C]glycine for non-invasive imaging of glioblastoma. Nude rats bearing orthotopic U87 glioblastoma and healthy controls were investigated. Imaging was performed by injecting HP γ-glutamyl-[1-13C]glycine and acquiring dynamic 13C data on a preclinical 3T MR scanner. The signal-to-noise (SNR) ratios of γ-glutamyl-[1-13C]glycine and its product [1-13C]glycine were evaluated. Comparison of control and tumor-bearing rats showed no difference in γ-glutamyl-[1-13C]glycine SNR, pointing to similar delivery to tumor and normal brain. In contrast, [1-13C]glycine SNR was significantly higher in tumor-bearing rats compared to controls, and in tumor regions compared to normal-appearing brain. Importantly, higher [1-13C]glycine was associated with higher GGT expression and higher GSH levels in tumor tissue compared to normal brain. Collectively, this study demonstrates, to our knowledge for the first time, the feasibility of using HP γ-glutamyl-[1-13C]glycine to monitor GGT expression in the brain and thus to detect glioblastoma.
| S-EPMC7148357 | biostudies-literature
Project description:We have cloned a 2.4-kilobase cDNA from a human placental cDNA library by using a gamma-glutamyl transpeptidase [GGT; gamma-glutamyltransferase, (5-glutamyl)-peptide:amino acid 5-glutamyltransferase, EC 2.3.2.2] probe. The deduced amino acid sequence of this cDNA, GGT-rel, exhibited an overall similarity of 39.5% with human GGT. Sequences that could represent a heavy and a light chain, analogous to GGT, as well as a putative transmembrane region were identified in GGT-rel. Transfectants overexpressing GGT-rel were tested for their ability to catalyze cleavage of the gamma-glutamyl moiety from natural and synthetic substrates for GGT. Experiments with glutathione added to the medium suggested that GGT-rel could hydrolyze the gamma-glutamyl moiety. More definitive evidence was obtained in experiments in which this protein converted leukotriene C4 to leukotriene D4. However, GGT-rel did not convert synthetic substrates that are commonly used to assay GGT. Our results indicate that GGT can no longer be considered the only enzyme capable of cleaving the gamma-glutamyl linkage of leukotriene C4 and, most likely, of other natural compounds.
| S-EPMC52071 | biostudies-other
Project description:The role of glutathione (GSH) in eukaryotic cells is well known. The biosynthesis of this γ-glutamine tripeptide is well studied. However, other γ-glutamyl peptides were found in various sources, and the pathways of their formation were not always clear. The aim of the present study was to determine whether Saccharomyces cerevisiae can produce γ-glutamyl tripeptides other than GSH and to identify the pathways associated with the formation of these peptides. The tripeptide γ-Glu-Val-Gly (γ-EVG) was used as a model. Wild-type yeast cells were shown to produce this peptide during cultivation in minimal synthetic medium. Two different biosynthetic pathways for this peptide were identified. The first pathway consisted of two steps. In the first step, γ-Glu-Val (γ-EV) was produced from glutamate and valine by the glutamate-cysteine ligase (GCL) Gsh1p or by the transfer of the γ-glutamyl group from GSH to valine by the γ-glutamyltransferase (GGT) Ecm38p or by the (Dug2p-Dug3p)2 complex. In the next step, γ-EV was combined with glycine by the glutathione synthetase (GS) Gsh2p. The second pathway consisted of transfer of the γ-glutamyl residue from GSH to the dipeptide Val-Gly (VG). This reaction was carried out mainly by the (Dug2p-Dug3p)2 complex, whereas the GGT Ecm38p did not participate in this reaction. The contribution of each of these two pathways to the intracellular pool of γ-EVG was dependent on cultivation conditions. In this work, we also found that Dug1p, previously identified as a Cys-Gly dipeptidase, played an essential role in the hydrolysis of the dipeptide VG in yeast cells. It was also demonstrated that γ-EV and γ-EVG could be effectively imported from the medium and that γ-EVG was imported by Opt1p, known to be a GSH importer. Our results demonstrated that γ-glutamyl peptides, particularly γ-EVG, are produced in yeast as products of several physiologically important reactions and are therefore natural components of yeast cells.
| S-EPMC6508711 | biostudies-literature
Project description:Gamma-glutamylamine cyclotransferase (GGACT) is an enzyme that converts gamma-glutamylamines to free amines and 5-oxoproline. GGACT shows high activity toward gamma-glutamyl-epsilon-lysine, derived from the breakdown of fibrin and other proteins cross-linked by transglutaminases. The enzyme adopts the newly identified cyclotransferase fold, observed in gamma-glutamylcyclotransferase (GGCT), an enzyme with activity toward gamma-glutamyl-alpha-amino acids (Oakley, A. J., Yamada, T., Liu, D., Coggan, M., Clark, A. G., and Board, P. G. (2008) J. Biol. Chem. 283, 22031-22042). Despite the absence of significant sequence identity, several residues are conserved in the active sites of GGCT and GGACT, including a putative catalytic acid/base residue (GGACT Glu(82)). The structure of GGACT in complex with the reaction product 5-oxoproline provides evidence for a common catalytic mechanism in both enzymes. The proposed mechanism, combined with the three-dimensional structures, also explains the different substrate specificities of these enzymes. Despite significant sequence divergence, there are at least three subfamilies in prokaryotes and eukaryotes that have conserved the GGCT fold and GGCT enzymatic activity.
| S-EPMC2843214 | biostudies-literature
Project description:We isolated from a tomato cDNA library the tomPRO1 locus, which encodes gamma-glutamyl kinase (GK) and gamma-glutamyl phosphate reductase (GPR). This locus is unusual among eukaryotic genetic elements because it contains two open reading frames, and thus resembles prokaryotic polycistronic operons. The first open reading frame, specifying GK, is terminated by a TAA codon, which is followed by five nucleotides, an ATG translation initiation codon, and the second open reading frame, encoding GPR. DNA sequence analysis of fragments obtained by PCR amplification confirmed that the internal TAA and neighboring sequences are present in the endogenous tomPRO1 sequence in tomato. We demonstrated with RNase protection assays that the tomPRO1 locus is transcribed in tomato tissue culture cells, into a product that contains the internal stop codon. In Escherichia coli, tomPRO1 directed the synthesis of two proteins, a 33-kDa GK and a 44-kDa GPR. Antibodies against the 44-kDa GPR purified from E. coli recognized a 70-kDa product in tomato tissue culture cells and a 60-kDa product in leaves and roots. These results suggest that in tomato tissues, GPR is made as part of a longer polypeptide by some translational mechanism that enables bypass of the internal stop codon, such as frameshifting or ribosome hopping. The tomPRO1 locus may be the first example of a nuclear genetic element in plants that encodes two functional enzymes in two distinct open reading frames.
| S-EPMC21589 | biostudies-literature
Project description:We report on the discovery of interactions of porcine endometrial 17 beta-oestradiol dehydrogenase with actin. The 17 beta-oestradiol dehydrogenase of porcine uteri is an essentially unidirectional enzyme compounded in specialized organelles. The enzyme activity in Brij 35 extracts of the particulate fraction of epithelial cells sedimenting between 1800 and 11,000 g(av). was collected by immunoadsorption and eluted at low pH. The eluate contained three proteins of 32, 45 and 80 kDa as shown by SDS/PAGE and silver staining. They were identified by amino acid sequencing and immunotyping as oestradiol dehydrogenase (32 kDa), actin (45 kDa) and a covalent dehydrogenase-actin complex (80 kDa). Disulphides, aldimines, periodate-degradable bonds and hydrophobic interactions were excluded as linkages in the 80 kDa protein. The epsilon-(gamma-glutamyl)-lysine nature of the covalent cross-link was recognized by narrow-bore h.p.l.c. analysis of enzymic digests of electro-eluted 80 kDa material. An involvement of the actin anchor in positioning of the oestradiol dehydrogenase-containing organelles according to metabolic requirements is discussed.
| S-EPMC1137765 | biostudies-other
Project description:Gamma-glutamyl transferase (GGT5) was discovered due to its ability to convert leukotriene C4 (LTC4, a glutathione S-conjugate) to LTD4 and may have an important role in the immune system. However, it was not known which cells express the enzyme in humans. We have developed a sensitive and specific antibody that can be used to detect human GGT5 on Western blots and in fixed tissue sections. We localized GGT5 expression in normal human tissues. We observed GGT5 expressed by macrophages present in many tissues, including tissue-fixed macrophages such as Kupffer cells in the liver and dust cells in the lung. GGT5 was expressed in some of the same tissues that have been shown to express gamma-glutamyl transferase (GGT1), the only other enzymatically active protein in this family. But, the two enzymes were often expressed by different cell types within the tissue. For example, GGT5 was expressed by the interstitial cells of the kidney, whereas GGT1 is expressed on the apical surface of the renal proximal tubules. Other tissues with GGT5-positive cells included: adrenal gland, salivary gland, pituitary, thymus, spleen, liver, bone marrow, small intestine, stomach, testis, prostate and placenta. GGT5 and GGT1 are cell surface enzymes. The different pattern of expression results in their access to different extracellular fluids and therefore different substrates. GGT5 has access to substrates in blood and intercellular fluids, while GGT1 has access primarily to fluids in ducts and glands throughout the body. These data provide new insights into the different functions of these two related enzymes.
| S-EPMC4393757 | biostudies-literature
Project description:Pseudoxanthoma elasticum (PXE), a heritable multi-system disorder manifesting with ectopic mineralization of soft connective tissues, is caused by mutations in the ABCC6/MRP6 gene/protein system, but the mechanisms how the ABCC6 mutations lead to aberrant mineralization are currently unknown. In this study, we utilized a transgenic mouse model, Abcc6-/-, to examine the mineralization processes. We focused on matrix gla protein (MGP) which has been shown to be critical, when activated by gamma-carboxylation of glutamyl residues, for prevention of unwanted mineralization. The concentration of MGP in the serum of Abcc6-/- mice was significantly reduced when compared to wild-type controls (p<0.004). More importantly, MGP isolated from the liver of Abcc6-/- mice was largely under-carboxylated and therefore possesses no activity. Finally, examination of the Abcc6-/- mice revealed association of total and under-carboxylated forms of MGP with ectopic mineralization while the gamma-carboxylated form was essentially absent. These results suggest that MGP in Abcc6-/- mice is largely in inactive form and is unable to prevent the unwanted mineralization of connective tissues in PXE.
| S-EPMC2700335 | biostudies-literature
Project description:Radical addition of H3PO2 to N-/C-protected vinyl glycine led to the corresponding H-phosphinic acid in excellent yield. The non-nucleophilic H-phosphinic acid was converted to a nucleophilic P(III) species, RP(OTMS)2, which was used in two approaches to the target phosphinic acid containing pseudopeptide. New methodology was developed that led to excellent yields in the reaction of RP(OTMS)2 with unactivated electrophiles, including an acyclic homoallylic bromide. However, en route to the target pseudopeptide, Arbuzov reaction of RP(OTMS)2 with a cyclic homoallylic bromide, (R)-3-(bromomethyl)-cyclopent-1-ene, led to a rearranged allylic phosphinic acid rather than the desired homoallylic derivative, a putative glutarate surrogate. Conjugate addition of RP(OTMS)2 to alpha-methylene glutarate containing a chiral auxiliary resulted in only modest diastereoselectivity. Purification by flash chromatography provided protected derivatives of both diastereomers of the pseudopeptide. Following global deprotection, coupling of (S)-H-Glu-gamma-[Psi(P(O)(OH)(CH2))]-(S)-Glu-OH and (S)-H-Glu-gamma-[Psi(P(O)(OH)(CH2))]-(R)-Glu-OH to (4-amino-4-deoxy-10-methyl)pteroyl azide led to the target compounds for biochemical study as inhibitors of the ATP-dependent ligase, folylpoly-gamma-glutamate synthetase.
| S-EPMC1989106 | biostudies-literature