Interaction of osteopontin with fibronectin and other extracellular matrix molecules.
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Animals↗
Biomedical subjects
S Beninati
Publications and source records attributed to S Beninati.
Identification of a substrate site for transglutaminases on the human protein synthesis initiation factor 5A.
Protein synthesis initiation factor 5A (eIF-5A) from human erythrocytes was found to be a substrate for both plasma transglutaminase (Factor XIIIa) and guinea pig liver transglutaminase (GPLTG). When purified eIF-5A was incubated with GPLTG or Factor XIIIa in the presence of succinylated beta-casein, a covalent complex was identified. By isolating and analysing the product of the transglutaminases (TGases) reaction, the site of modification on eIF-5A has been identified as the unique amino acid hypusine. The complex beta-casein.eIF-5A was enzymatically digested with proteinases and the predicted covalent cross-link of gamma-glutamyl-omega-hypusine was isolated from the digests by ion-exchange chromatography and purified by reversed-phase h.p.l.c. Acid hydrolysis of the purified dipeptide yielded equimolar amounts of hypusine and glutamic acid. Furthermore, fast atom bombardment m.s. analysis confirmed the isomer assignment to be gamma-glutamyl-omega-hypusine. These data indicate that hypusine-50 of the eIF-5A chain functions as acyl acceptor substrate for TGases, and reveal that eIF-5A may be cross-linked to intracellular proteins by TGases. Because the precise function of eIF-5A is still unknown, our results appear particularly stimulating in the light of the recent finding of a new biological role for this protein as a cellular factor binding specifically to the human immunodeficiency virus-1 Rev activation domain [Ruhl, Himmelspach, Bahr, Hammerschmid, Jaksche, Wolff, Auschauer, Farrington, Probst, Bevec and Hauber (1993) J. Cell Biol. 123, 1309-1320].
Animals↗
Transglutaminase-catalyzed polymerization of troponin in vitro.
In the presence of calcium ions, tissue transglutaminase catalyzes the polymerization of skeletal muscle troponin to high molecular weight insoluble aggregate. The specific action of transglutaminase is proved by the isolation of glutamyl-spermidine isopeptide derivatives. The process involves mainly the troponin T subunit (TnT), with formation of dimers and trimers of TnT, which were reactive with specific antibodies by immunoblotting. Furthermore when incubation is carried out in the presence of radioactive polyamines, the label is incorporated selectively into TnT subunits.
Animals↗
Polyamines in chloroplasts: identification of their glutamyl and acetyl derivatives.
Incubation of chloroplasts of Helianthus tuberosus with labelled putrescine and/or spermidine and proteolytic digestion of their trichloroacetate-soluble and -insoluble proteins revealed the presence of N-(gamma-glutamyl)-putrescine, N1,N4-bis-(gamma-glutamyl)-putrescine and N1,N8-bis-(gamma-glutamyl)spermidine. This finding may be regarded as unequivocal proof of the presence of transglutaminase activity in chloroplasts. In addition, the recovery of spermidine or putrescine and acetylspermidine from chloroplasts incubated with [3H]putrescine or [3H]spermidine respectively indicates the existence of biosynthetic and oxidative pathways. These results suggest that polyamines may have an important function in chloroplasts both in their free form and by covalently binding to proteins.
Acetylation↗
Plant transglutaminases.
The identification procedures, the characteristics and the potential function of the recently detected plant transglutaminases, are discussed in the light of the knowledge of animal transglutaminases. The enzyme has been studied occasionally in lower organisms (bacteria, fungi and green algae) and more extensively in Angiosperms.
Amino Acid Sequence↗
A protein produced by a monocytic human cell line can induce apoptosis on tumor cells.
A serum-free medium conditioned by U937, a human cell line of monocyte/macrophage origin, was found capable of inducing apoptosis on exponentially growing U937 cells themselves (autocrine suicide). The apoptosis-inducing agent is a macromolecule and possibly a protein (SKT factor), with a relative molecular mass in the range of 18-25 kDa. All human tumor cell lines examined have been induced to apoptosis with high efficiency, whereas non transformed human lymphocytes and monocytes are insensitive to the apoptosis-inducing activity; moreover, partially differentiated U937 are not killed but induced to full maturation. These observations suggest that the SKT factor could possibly be a cytokine with a specific cytotoxic tropism, that resembles in many respects the cytokine tumor necrosis factor (TNF), even though no TNF is detectable in the conditioned medium.
Apoptosis↗
Osteopontin: its transglutaminase-catalyzed posttranslational modifications and cross-linking to fibronectin.
Osteopontin (OP) is a component of extracellular, bone, and urinary stone matrices, but the mechanism by which it is stably incorporated into such matrices remains unknown. By SDS-PAGE analysis of [125I]OP, treated with a catalytic amount of TG, we first demonstrate both intra- and intermolecular covalent cross-linking of OP. Most importantly, the analysis of the products generated from reactions containing OP, Fn, and TG by SDS-PAGE, autoradiography, and Western blotting using either OP or Fn antibody, and quantitation of TG-catalyzed epsilon-(gamma-glutamyl)lysine isopeptide formation between OP and Fn demonstrate, for the first time, covalent cross-linking between these two proteins. Similar reactions in the presence of polyamine substrates of TG show OP-Fn intermolecular cross-linking via N,N-bis-(gamma-glutamyl)polyamine formation. Finally, immunoprecipitation of 125I-labeled NRK cell surface proteins with anti-OP and anti-Fn antibodies, SDS-PAGE analysis, and autoradiography provides critical evidence for nonreducible OP-Fn cross-linking in vivo. These results clearly suggest that TG-mediated cross-linking between OP and Fn represents one of the most likely mechanisms by which OP becomes covalently linked to bone matrix, urinary stone matrix, and to ECM.
Amino Acid Sequence↗
Cystamine potently suppresses in vitro HIV replication in acutely and chronically infected human cells.
We have investigated the effects of cystamine on the replication of human immunodeficiency virus (HIV) in human lymphocytes and macrophages, the natural targets of HIV in vivo. Treatment of chronically infected macrophages with cystamine, at a concentration (500 microM) that did not show any cytotoxic or cytostatic effects, strongly decreased (> 80%) HIV-p24 antigen production and completely abolished the production of infectious viral particles. Cystamine does not affect viral transcription, translation or protein processing; indeed, all HIV proteins are present in a pattern similar to that of nontreated cells. Instead, cystamine interferes with the orderly assembly of HIV virions, as shown by electron microscopy analysis, that reveals only defective viral particles in treated cells. Moreover, suppression of HIV replication, due to the inhibition of proviral DNA formation was observed in acutely infected lymphocytes and macrophages pretreated with cystamine. These results show that cystamine potently suppresses HIV replication in human cells by contemporaneously blocking at least two independent steps of the viral life cycle, without affecting cell viability, suggesting that this compound may represent a new possibility towards the treatment of HIV-1 infection.
Antiviral Agents↗
Differences in the post-translational modification of proteins by polyamines between weakly and highly metastatic B16 melanoma cells.
The identification of (gamma-glutamyl)polyamines in proteolytic digest of proteins from the cytosolic and particulate fractions of B16-F10 and B16-F10Lr6 cell lines, originating from a spontaneous tumor in C57BL/6 mice, indicates that polyamines are incorporated into melanoma cell proteins by transglutaminases (TGases-EC 2.3.2.13). The levels of spermidine-derived protein cross-links were found to be inversely related with the metastatic potential of the 2 melanoma lines. Characterization of TGase activity in the 2 tumor cell lines showed 3 types of enzyme. The soluble cellular TGase activity (TGase C) was higher, and increased more, during the growth of the least metastasizing clone B16-F10Lr6 than in the B16-F10 line, which is the most metastasizing. Consistently, N1,N8-bis(gamma-glutamyl) spermidine, which is responsible for protein cross-link formation, was present in greater amount in B16-F10Lr6 cells. The enhancement by theophylline of soluble-TGase activity and spermidine-dependent protein cross-links of B16-F10 cells reduced, with linear dose dependence, the ability of these cells to penetrate through human fibronectin-coated membrane in an in vitro assay of invasiveness. Our data confirm and extend earlier observations indicating that the propensity of a tumor to metastasize can be indirectly related to intracellular levels of TGase activity, and provide the basis for some speculation concerning the role of polyamines as modifiers of murine melanoma cell proteins in metastasis.
Animals↗
Transglutaminase-catalyzed incorporation of polyamines into phospholipase A2.
We have previously demonstrated that when phospholipase A2 is treated with either tissue transglutaminase or human plasma Factor XIIIa, a striking increase of its catalytic activity is observed, due to the formation of an intramolecular epsilon-(gamma-glutamyl)-lysine crosslink [Cordella-Miele et al. (1990) J. Biol. Chem. 265, 17180-17188]. Here, we report the effect of transglutaminase substrates such as mono-, di-, and polyamines on this transglutaminase-catalyzed post-translational modification of phospholipase A2. Incorporation of radioactively labeled polyamines into phospholipase A2 was demonstrated by using porcine pancreatic phospholipase A2 as a substrate in a conventional transglutaminase assay. These results were further confirmed by SDS-polyacrylamide gel electrophoresis followed by fluorography. Additionally, gamma-glutamyl-polyamine was detected and unequivocally identified in proteolytic digests of polyaminated phospholipase A2. When phospholipase A2 was incubated with transglutaminase in the presence of putrescine, spermine, spermidine, dansylcadaverine, or methylamine, a 2-3-fold increase in phospholipase A2 activity was observed. The increase of phospholipase A2 activity was found to be dependent upon the concentration of phospholipase A2, preincubation time, and the duration of the reaction. Increase in phospholipase A2 activity after transglutaminase treatment in the presence of polyamines was demonstrated using two different assay systems. Kinetic studies on phospholipase A2 pretreated with spermidine and transglutaminase demonstrated a significant increase of the apparent Vmax but no significant change in the apparent Km. Unlike phospholipase A2 pretreated with transglutaminase alone, polyaminated phospholipase A2 does not undergo non-covalent dimerization in solution. Polyaminated phospholipase A2 was further purified by chromatofocusing and was found to contain N-mono(gamma-glutamyl)-spermidine in a molar ratio of about 1:1 to phospholipase A2. Freshly purified, polyaminated phospholipase A2 had a specific activity approximately 3-fold higher than that of control phospholipase A2 treated in an identical way except for the absence of transglutaminase. To our knowledge, this is the first demonstration that transglutaminase catalyzes the incorporation of amines into a phospholipase, and that this post-translational modification increases phospholipase A2 activity.
Animals↗
A novel transglutaminase-catalyzed posttranslational modification of HIV-1 aspartyl protease.
We demonstrate that HIV-1 aspartyl protease (AP), the enzyme essential for the maturation of the AIDS virus, covalently incorporates spermidine catalyzed by guinea pig liver transglutaminase (TGase) and human coagulation factor XIIIa. Preliminary evidence indicates that there are at least three reactive glutamyl and lysyl residues in AP which act as acyl donor and acceptor respectively in a TGase reaction. SDS-PAGE and chromatographic analyses indicate that the two TGases tested catalyze the incorporation of radioactive spermidine into pure HIV-1 AP. The chemical identification and quantitation of (gamma-glutamyl) spermidine isopeptide provide conclusive evidence that the formation of this derivative is catalyzed by TGase. These results imply that TGase-catalyzed post-translational modification of HIV-1 AP may take place in a manner similar to the ones demonstrated in porcine pancreatic phospholipase A2.
Animals↗
N1N8-bis(gamma-glutamyl)spermidine cross-linking in epidermal-cell envelopes. Comparison of cross-link levels in normal and psoriatic cell envelopes.
N1N8-Bis(gamma-glutamyl)spermidine was found in exhaustive proteolytic digests of isolated cell envelopes from human epidermis at levels comparable with those of epsilon-(gamma-glutamyl)lysine. Significantly higher than normal amounts of these compounds, particularly the bis(gamma-glutamyl)polyamine, were observed in envelopes from afflicted areas (scales) of psoriatic patients. These findings support the notions that N1N8-bis(gamma-glutamyl)spermidine, like epsilon-(gamma-glutamyl)lysine, functions in cell envelopes as an enzyme-generated protein cross-link and stabilizing force and that individuals with the chronic, recurrent skin disease, psoriasis, exhibit in involved epidermis abnormal cell-envelope-protein cross-linking.
Amino Acids↗
Post-translational modification of apolipoprotein B by transglutaminases.
The major form of cross-link found in apolipoprotein B was identified as N1N12-bis-(gamma-glutamyl)spermine, a product known to be formed through the catalytic action of transglutaminases (EC 2.3.2.13). N1-(gamma-Glutamyl)spermine was present in a trace amount but epsilon-(gamma-glutamyl)lysine cross-links, which are formed during fibrin formation in plasma, were not detected. In the presence of catalytic amounts of plasma Factor XIIIa (a thrombin-dependent extracellular transglutaminase) or cellular transglutaminase (a cytosolic enzyme), apolipoprotein B and other plasma apolipoproteins (A-I, A-II and C) underwent covalently bridged polymerization and served as amine acceptor substrates. These results suggests that transglutaminases may participate in the covalent modification of apolipoproteins, either in the physiological state or during pathogenesis.
Animals↗
Stimulation of phospholipases A2 by transglutaminases.
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Animals↗
High-performance liquid chromatographic method for determination of hypusine and deoxyhypusine.
A selective and sensitive reversed-phase high-performance liquid chromatographic method is described for the determination of the amino acid hypusine which occurs ubiquitously in mammalian cells and for the simultaneous measurement of its immediate precursor deoxyhypusine. These amino acids, after their ion-exchange separation from the bulk of other amino acids in protein hydrolysates, are derivatized with o-phthalaldehyde and the fluorescent derivatives are separated by reverse-phase liquid chromatography. The sensitivity of this method allows detection of less than 5 pmol of each of these unusual amino acids. The method is applied to the determination of hypusine and deoxyhypusine in acid hydrolysates of cultured cells and tissues.
Animals↗
Age-related changes of glycosylation pattern in isolated rat hepatocytes.
The glycosylation pattern in isolated rat hepatocytes during pre- and post-natal development and senescence has been studied by following: the [14C]glucosamine and [3H]galactose incorporation into cellular glycoproteins and glycolipids and the activity of two microsomal enzymes, N-acetyl-glucosaminyl-1-P transferase and galactosyl transferase. The data show a lowered precursor incorporation into lipids and proteins in the fetus, newborn and old rats versus the adult. Only the galactosyl transferase activity is enhanced on the 19th and 22nd day of fetal life. The glucosamine and N-acetyl-glucosamine content in both soluble and protein bound fractions was increased, while the galactose content in lipids and proteins decreased in the fetal stage. The different sugar composition of the proteins, and the decreased glucosamine and galactose incorporation into the proteins, observed in the fetus, newborn and old rat, suggest a post-translational modification which may cause alterations in functions usually mediated by glycoproteins.
Aging↗
Inhibition of deoxyhypusine hydroxylase by polyamines and by a deoxyhypusine peptide.
The inhibition of deoxyhypusine hydroxylase was studied in vitro. Of the polyamines tested, spermine and its homologue thermine exhibited the strongest inhibition against the enzyme from rat testis. Kinetic analysis revealed that the inhibition by spermine was competitive (Ki, 0.25 +/- 0.02 mM) with respect to the deoxyhypusine protein substrate. Spermidine and its homologue caldine were also inhibitors, but less potent ones than spermine. The spermidine analogues with one or both primary amino groups replaced by the cyano group did not inhibit. A number of diamines, including putrescine, were found to display little or no inhibition. The observed effects of naturally occurring polyamines on deoxyhypusine hydroxylase activity is consistent with a suggestion of regulation of this enzymic activity by cellular levels of polyamines. A synthetic peptide Lys-Thr-Gly-deoxyhypusine-His-Gly-His-Ala-Lys, the amino acid sequence of which corresponds to that surrounding hypusine in eukaryotic initiation factor 4D, was found to display competitive-type inhibition (Ki, 0.44 +/- 0.02 mM) against deoxyhypusine hydroxylase from Chinese hamster ovary cells. Free hypusine and deoxyhypusine, on the other hand, possessed no inhibitory properties. A peptide analogous to the deoxyhypusine nonapeptide with lysine in place of deoxyhypusine had little effect on enzyme activity. The preparation of a derivative of deoxyhypusine, suitably protected for use in the solid-phase synthesis of deoxyhypusine peptides, is described.
Amino Acid Sequence↗
Transglutaminase expression in rat parotid gland after isoproterenol stimulation.
Transglutaminases (E.C. 2.3.2.13) are calcium-dependent enzymes that catalyze the covalent cross-linking of proteins, and occur in multiple molecular forms in a variety of tissues. Distribution of each form of transglutaminase varies with different tissues. Studies were undertaken to characterize the form of transglutaminase expressed in rat parotid gland, and to examine a possible physiological role for the enzyme. It was found that chronic treatment of rats with the beta-adrenergic agonist isoproterenol (IPR) resulted in the induction of parotid transglutaminase activity. The properties of this transglutaminase appeared to be distinct from those of the well-characterized guinea pig liver cytosol transglutaminase (TGase C). The findings that protein polymerization (observed on SDS-PAGE) and incorporation of radioactive putrescine, a polyamine, into protein occur in the presence of exogenous transglutaminase and calcium indicated that certain rat parotid salivary proteins are or could be substrates for this enzyme. Analysis of proteolytic digests of rat parotid salivary proteins on an amino acid analyzer and by high-performance liquid chromatography also indicated that these salivary proteins contain gamma-glutamyl derivatives of primary amines (e.g., polyamines or lysine), post-translational products of transglutaminase catalysis. The possible physiological function of this enzyme in the oral cavity might be stabilization of proteinaceous structures during normal oral homeostasis and/or woundhealing.
Animals↗