Modulation of the in situ activity of tissue transglutaminase by calcium and GTP.
AuthorsZhang J, Lesort M, Guttmann RP, Johnson GV
JournalJ Biol Chem
PubMed ID9442073
'Tissue transglutaminase (tTG) is a calcium-dependent enzyme that catalyzes the posttranslational modification of proteins by transamidation of specific polypeptide-bound glutamine residues. Previous in vitro studies have demonstrated that the transamidating activity of tTG requires calcium and is inhibited by GTP. To investigate the endogenous regulation of tTG, a quantitative in ... More
Tissue transglutaminase does not contribute to the formation of mutant huntingtin aggregates.
AuthorsChun W, Lesort M, Tucholski J, Ross CA, Johnson GV
JournalJ Cell Biol
PubMed ID11285271
'The cause of Huntington''s disease (HD) is a pathological expansion of the polyglutamine domain within the NH(2)-terminal region of huntingtin. Neuronal intranuclear inclusions and cytoplasmic aggregates composed of the mutant huntingtin within certain neuronal populations are a characteristic hallmark of HD. Because in vitro expanded polyglutamine repeats are glutaminyl-donor substrates ... More
Analysis of the catalytic activity of human factor XIIIa by site-directed mutagenesis.
AuthorsHettasch JM, Greenberg CS
JournalJ Biol Chem
PubMed ID7961769
'Factor XIIIa (FXIIIa) stabilizes fibrin clots by covalently cross-linking fibrin molecules. The purpose of this study was to determine the amino acid requirements at the active site of FXIIIa for catalysis. We selected amino acids 310-317 (Arg-Tyr-Gly-Gln-Cys-Trp-Val-Phe) in the human FXIII A-chain sequence for analysis based on the high degree ... More
Single-molecule imaging of RNA polymerase-DNA interactions in real time.
AuthorsHarada Y, Funatsu T, Murakami K, Nonoyama Y, Ishihama A, Yanagida T
JournalBiophys J
PubMed ID9929475
'Using total internal reflection fluorescence microscopy, we have directly observed individual interactions of single RNA polymerase molecules with a single molecule of lambda-phage DNA suspended in solution by optical traps. The interactions of RNA polymerase molecules were not homogeneous along DNA. They dissociated slowly from the positions of the promoters ... More
A simple assay and histochemical localization of transglutaminase activity using a derivative of green fluorescent protein as substrate.
AuthorsFurutani Y, Kato A, Notoya M, Ghoneim MA, Hirose S
JournalJ Histochem Cytochem
PubMed ID11156693
'Histidine-tagged green fluorescent protein (His(6)-Xpress-GFP), a widely used fluorescent probe, was found to be a good substrate for transglutaminase, an enzyme that catalyzes covalent crosslinking of proteins. GFP alone did not serve as a substrate but its derivative His(6)-Xpress-GFP was readily crosslinked through the Gln and Lys residues present in ... More
A series of biotinylated tracers distinguishes three types of gap junction in retina.
AuthorsMills SL, Massey SC
JournalJ Neurosci
PubMed ID11069972
'Gap junctions serve many important roles in various tissues, but their abundance and diversity in neurons is only beginning to be understood. The tracer Neurobiotin has revealed many different networks interconnected by gap junctions in retina. We compared the relative permeabilities of five different retinal gap junctions by measuring their ... More
Transglutaminase type 1 and its cross-linking activity are concentrated at adherens junctions in simple epithelial cells.
AuthorsHiiragi T, Sasaki H, Nagafuchi A, Sabe H, Shen SC, Matsuki M, Yamanishi K, Tsukita S
JournalJ Biol Chem
PubMed ID10567386
'Transglutaminase type 1 was identified as a tyrosine-phosphorylated protein from the isolated junctional fraction of the mouse liver. This enzyme was reported to be involved in the covalent cross-linking of proteins in keratinocytes, but its expression and activity in other cell types have not been examined. Northern blotting revealed that ... More
Transglutaminase activity in the eye: cross-linking in epithelia and connective tissue structures.
AuthorsRaghunath M, Cankay R, Kubitscheck U, Fauteck JD, Mayne R, Aeschlimann D, Schlötzer-Schrehardt U
JournalInvest Ophthalmol Vis Sci
PubMed ID10549636
'PURPOSE: To assess the distribution of transglutaminase (TGase) activity in ocular tissues and the target structures for cross-linking. METHODS: Cryosections from human and cynomolgus monkey eyes were incubated with the biotinylated amine donor substrate cadaverine (biotC), which was subsequently visualized with streptavidin-peroxidase. Confocal laser scanning was used to colocalize biotC ... More
Carboxyl-terminal truncation of recombinant factor XIII A-chains. Characterization of minimum structural requirement for transglutaminase activity.
AuthorsLai TS, Achyuthan KE, Santiago MA, Greenberg GS
JournalJ Biol Chem
PubMed ID7929131
'A series of truncation mutants lacking 218, 229, 250, and 269 amino acid residues from the carboxyl terminus of blood coagulation factor XIII A-chains (FXIII A), designated as delta K513, delta A502, delta Y481, and delta K462, respectively, were expressed in Escherichia coli to define the minimum structure required for ... More
Differential properties of two gap junctional pathways made by AII amacrine cells.
AuthorsMills SL, Massey SC
JournalNature
PubMed ID7477263
'The retina is sensitive to light stimuli varying over more than 12 log units in intensity. It accomplishes this, in part, by switching between rod-dominated circuits designed for maximum utilization of scarce photons and cone circuits designed for greater acuity. Rod signals are integrated into the cone pathways through AII ... More
Sphingosylphosphocholine reduces the calcium ion requirement for activating tissue transglutaminase.
AuthorsLai TS, Bielawska A, Peoples KA, Hannun YA, Greenberg CS
JournalJ Biol Chem
PubMed ID9195933
'Tissue transglutaminase (tTG) catalyzes a Ca2+-dependent transglutaminase reaction resulting in the formation of gamma-glutamyl-epsilon-lysine bonds and is activated during apoptosis to catalyze the formation of apoptotic body. We investigate whether lipids that are membrane components and involved in cell signaling could modify the Ca2+-dependent activation of tTG. We found that ... More
Redirecting lipoic acid ligase for cell surface protein labeling with small-molecule probes.
'Live cell imaging is a powerful method to study protein dynamics at the cell surface, but conventional imaging probes are bulky, or interfere with protein function, or dissociate from proteins after internalization. Here, we report technology for covalent, specific tagging of cellular proteins with chemical probes. Through rational design, we ... More
Identification of transglutaminase substrates in HT29 colon cancer cells: use of 5-(biotinamido)pentylamine as a transglutaminase-specific probe.
AuthorsLee KN, Maxwell MD, Patterson MK, Birckbichler PJ, Conway E
JournalBiochim Biophys Acta
PubMed ID1353685
'A biotinamine probe, 5-(biotinamido)pentylamine, was used for biotin-labeling of proteins in HT29 colon cancer cell extracts by endogenous transglutaminase activity. The biotin-labeled protein substrates were isolated and recovered by avidin-affinity chromatography. The proteins were separated using SDS-polyacrylamide gel electrophoresis, electroblotted onto a polyvinylidene difluoride membrane, visualized using Coomassie blue, cut ... More
A novel in situ method for the detection of deficient transglutaminase activity in the skin.
AuthorsRaghunath M, Hennies HC, Velten F, Wiebe V, Steinert PM, Reis A, Traupe H
JournalArch Dermatol Res
PubMed ID9860283
'Autosomal recessive congenital ichthyoses are disorders of epidermal cornification, but are clinically and etiologically heterogeneous. Some cases, known as lamellar ichthyosis, are caused by mutations in the TGM1 gene encoding transglutaminase 1, which result in markedly diminished or lost enzyme activity and/or protein. In some cases, this enzyme is present ... More
In situ analysis of carboxyl and sulfhydryl groups of extracellular polymeric secretions by confocal laser scanning microscopy.
AuthorsKawaguchi T, Decho AW
JournalAnal Biochem
PubMed ID12009705
Methods for determining the modification of protein thiols by reactive lipids.
AuthorsOh J, Johnson MS, Landar A,
JournalMethods Cell Biol
PubMed ID17445707
Biotin-based tagging techniques have been applied with success to monitor the posttranslational modification of reactive protein thiols in cell signaling proteins and enzymes by ROS/RNS and electrophilic compounds. One of the most versatile techniques is to attach biotin covalently to thiol-reactive molecules, and label thiol-containing proteins. After separation ... More
Biotin-protein bond: instability and structural modification to provide stability for in vivo applications.
AuthorsMock DM, Bogusiewicz A,
JournalMethods Mol Biol
PubMed ID18287661
Biotinylation of proteins is a powerful tool for investigating biological phenomenon, both in vitro and in vivo. Biotinylating reagents that form covalent bonds with several types of amino acid residues are commercially available. However, most, if not all, of these commercially available biotinylating agents produce biotin-protein bonds that are susceptible ... More
Surface plasmon resonance (SPR) as a tool for antibody conjugate analysis.
AuthorsAdamczyk M, Mattingly PG, Shreder K, Yu Z
JournalBioconjug Chem
PubMed ID10563772
Surface plasmon resonance (SPR) analysis was used to assess the immunoreactivity of anti-biotin (4) and anti-fluorescein (5) monoclonal antibody after conjugation with the N-hydroxysuccinimide ester of acridinium-9-carboxamide 1. Only minor changes in the apparent equilibrium dissociation constants of the antibody conjugates for their ligands resulted from the conjugation process. However, ... More
SOI optical microring resonator with poly(ethylene glycol) polymer brush for label-free biosensor applications.
AuthorsDe Vos K, Girones J, Popelka S, Schacht E, Baets R, Bienstman P,
JournalBiosens Bioelectron
PubMed ID19200711
Label-free monitoring of biomolecular interactions has become of key importance for the emerging proteomics field. Monitoring real time interaction kinetics and high throughput screening of complex samples is of major importance for a variety of applications. We previously reported the use of Silicon-on-Insulator photonics microring resonators for cheap disposable biosensors ... More
Reversibility of covalent electrophile-protein adducts and chemical toxicity.
AuthorsLin D, Saleh S, Liebler DC,
JournalChem Res Toxicol
PubMed ID19548357
The biotin-tagged electrophiles 1-biotinamido-4-(4'-[maleimidoethylcyclohexane]-carboxamido)butane (BMCC) and N-iodoacetyl-N-biotinylhexylenediamine (IAB) have been used as model electrophile probes in complex proteomes to identify protein targets associated with chemical toxicity. Whereas IAB activates stress signaling and apoptosis in HEK293 cells, BMCC does not. Cysteine Michael adducts formed from BMCC and nonbiotinylated analogues rapidly disappeared ... More
A new method for the chemical labelling of nucleic acid with biotin to produce non-radioactive probes has been developed. NN'-Bis-(3-aminopropyl)butane-1,4-diamine (spermine) and long-chain diamino compounds (diaminohexane, diaminodecane and diaminododecane) were linked covalently to biotin and the resultant conjugates were attached to nucleic acid by using a cross-linking reagent (glutaraldehyde or ... More
A microtiter plate transglutaminase assay utilizing 5-(biotinamido)pentylamine as substrate.
AuthorsSlaughter TF, Achyuthan KE, Lai TS, Greenberg CS
JournalAnal Biochem
PubMed ID1359806
Transglutaminases belong to an important family of enzymes involved in hemostasis, skin formation, and wound healing. We describe a technique for the measurement of transglutaminase activity using polystyrene microtiter plates coated with N,N'-dimethylcasein. The substrate 5-(biotinamido)pentylamine is covalently incorporated into N,N'-dimethylcasein by transglutaminase in a calcium-dependent reaction. The biotinylated product ... More
The mitogen-activated protein kinase kinase kinase dual leucine zipper-bearing kinase (DLK) acts as a key regulator of keratinocyte terminal differentiation.
AuthorsRobitaille H, Proulx R, Robitaille K, Blouin R, Germain L
JournalJ Biol Chem
PubMed ID15695824
In the skin, epithelial cells undergo a terminal differentiation program leading to the formation of the stratum corneum. Although it is expected that the last phases of this process must be tightly regulated since it results in cell death, the signaling pathways involved in this induction remain ill defined. We ... More
Novel protein-disulfide isomerases from the early-diverging protist Giardia lamblia.
Protein-disulfide isomerase is essential for formation and reshuffling of disulfide bonds during nascent protein folding in the endoplasmic reticulum. The two thioredoxin-like active sites catalyze a variety of thiol-disulfide exchange reactions. We have characterized three novel protein-disulfide isomerases from the primitive eukaryote Giardia lamblia. Unlike other protein-disulfide isomerases, the giardial ... More
Site-directed mutagenesis of the calcium-binding site of blood coagulation factor XIIIa.
AuthorsLai TS, Slaughter TF, Peoples KA, Greenberg CS
JournalJ Biol Chem
PubMed ID10455172
Blood coagulation factor XIIIa is a calcium-dependent enzyme that covalently ligates fibrin molecules during blood coagulation. X-ray crystallography studies identified a major calcium-binding site involving Asp(438), Ala(457), Glu(485), and Glu(490). We mutated two glutamic acid residues (Glu(485) and Glu(490)) and three aspartic acid residues (Asp(472), Asp(476), and Asp(479)) that are ... More
15-Deoxy-Delta 12,14-prostaglandin J2 inhibition of NF-kappaB-DNA binding through covalent modification of the p50 subunit.
AuthorsCernuda-Morollón E, Pineda-Molina E, Cañada FJ, Pérez-Sala D
JournalJ Biol Chem
PubMed ID11466314
Cyclopentenone prostaglandins display anti-inflammatory activities and interfere with the signaling pathway that leads to activation of transcription factor NF-kappaB. Here we explore the possibility that the NF-kappaB subunit p50 may be a target for the cyclopentenone 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)). This prostaglandin inhibited the DNA binding ability of recombinant p50 in ... More
Analysis of factor XIII substrate specificity using recombinant human factor XIII and tissue transglutaminase chimeras.
AuthorsHettasch JM, Peoples KA, Greenberg CS
JournalJ Biol Chem
PubMed ID9312126
Human factor XIII (FXIII) and tissue transglutaminase (tTG) are homologous proteins. FXIII requires thrombin for activation and cross-links the gamma chains of fibrin(ogen) more efficiently than the Aalpha chains. On the other hand, tTG is thrombin-independent and forms predominantly Aalpha and Aalpha-gamma chain complexes. Previous work from this laboratory demonstrated ... More
Distinct nuclear localization and activity of tissue transglutaminase.
AuthorsLesort M, Attanavanich K, Zhang J, Johnson GV
JournalJ Biol Chem
PubMed ID9575137
Tissue transglutaminase is a calcium-dependent transamidating enzyme that has been postulated to play a role in the pathology of expanded CAG repeat disorders with polyglutamine expansions expressed within the affected proteins. Because intranuclear inclusions have recently been shown to be a common feature of many of these codon reiteration diseases, ... More
The Caenorhabditis elegans ERp60 homolog protein disulfide isomerase-3 has disulfide isomerase and transglutaminase-like cross-linking activity and is involved in the maintenance of body morphology.
AuthorsEschenlauer SC, Page AP
JournalJ Biol Chem
PubMed ID12424233
A novel protein disulfide isomerase gene, pdi-3, was isolated from the nematode Caenorhabditis elegans. This gene encodes an enzyme related to the ERp60 class of thioredoxin proteins and was found to exhibit unusual enzymatic properties. Recombinant protein displayed both disulfide bond isomerase activity and calcium-dependent transglutaminase-like cross-linking activity. The pdi-3 ... More
Functionalization of covalent DNA-streptavidin conjugates by means of biotinylated modulator components.
AuthorsNiemeyer CM, Ceyhan B, Blohm D
JournalBioconjug Chem
PubMed ID10502335
Covalent DNA-streptavidin conjugates are versatile biomolecular coupling reagents, since they have binding capacity for both a complementary nucleic acid and four molecules of biotin. The DNA-streptavidin hybrid molecules have been investigated for their capabilities to bind two different types of biotinylated components. Thus, (i) a functional biomolecule, e.g., a single-stranded ... More
Gliadin T cell epitope selection by tissue transglutaminase in celiac disease. Role of enzyme specificity and pH influence on the transamidation versus deamidation process.
AuthorsFleckenstein B, Molberg Ø, Qiao SW, Schmid DG, von der Mülbe F, Elgstøen K, Jung G, Sollid LM
JournalJ Biol Chem
PubMed ID12093810
Tissue transglutaminase (TG2) can modify proteins by transamidation or deamidation of specific glutamine residues. TG2 has a major role in the pathogenesis of celiac disease as it is both the target of disease-specific autoantibodies and generates deamidated gliadin peptides that are recognized by CD4(+), DQ2-restricted T cells from the celiac ... More