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L Lorand

Publications and source records attributed to L Lorand.

At least 37 records · Page 2Linked to original sources
PubMed1998

Cross-linking sites of the human tau protein, probed by reactions with human transglutaminase.

A portion of the neurofibrillary tangles of Alzheimer's disease has the characteristics of cross-linked protein. Because the principal component of these lesions is the microtubule-associated protein tau, and because a major source of cross-linking activity within neurons is supplied by tissue transglutaminase (TGase), it has been postulated that isopeptide bond formation is a major posttranslational modification leading to the formation of insoluble neurofibrillary tangles. Here we have mapped the sites on two isoforms of human tau protein (tau23 and tau40) capable of participating in human TGase-mediated isopeptide bond formation. Using dansyl-labeled fluorescent probes, it was shown that eight Gln residues can function as amine acceptor residues, with two major sites being Gln351 and Gln424. In addition, 10 Lys residues were identified as amine donors, most of which are clustered adjacent to the microtubule-binding repeats of tau in regions known to be solvent accessible in filamentous tau. The distribution of amine donors correlated closely with that of Arg residues, suggesting a link between neighboring positive charge and the TGase selectivity for donor sites in the protein substrate. Apart from revealing the sites that can be cross-linked during the TGase-catalyzed assembly of tau filaments, the results suggest a topography for the tau monomers so assembled.

Amino Acid Sequence↗
PubMed1997

Hydrolysis of gamma:epsilon isopeptides by cytosolic transglutaminases and by coagulation factor XIIIa.

Nepsilon-(gamma-glutamyl)lysine cross-links, connecting various peptide chain segments, are frequently the major products in transglutaminase-catalyzed reactions. We have now investigated the effectiveness of these enzymes for hydrolyzing the gamma:epsilon linkage. Branched compounds were synthesized, in which the backbone on the gamma-side of the cross-bridge was labeled with a fluorophor (5-(dimethylamino)-1-naphthalenesulfonyl or 2-aminobenzoyl) attached through an epsilon-aminocaproyl linker in the N-terminal position, and the other branch of the bridge was constructed with Lys methylamide or diaminopentane blocked by 2,4-dinitrophenyl at the Nalpha position. Hydrolysis of the cross-link could be followed in these internally quenched substrates by an increase in fluorescence. In addition to the thrombin and Ca2+-activated human coagulation Factor XIIIa, cytosolic transglutaminases from human red cells and from guinea pig liver were tested. All three enzymes were found to display good isopeptidase activities, with Km values of 10(-4) to 10(-5) M. Inhibitors of transamidation were effective in blocking the hydrolysis by the enzymes, indicating that expression of isopeptidase activity did not require unusual protein conformations. We suggest that transglutaminases may play a dynamic role in biology not only by promoting the formation but also the breaking of Nepsilon-(gamma-glutamyl)lysine isopeptides.

Animals↗
PubMed1997

Hierarchy of lens proteins requiring protection against heat-induced precipitation by the alpha crystallin chaperone.

Gel filtration of the water-soluble extract from bovine lens yields a group of proteins, emerging between the peaks of beta H and beta L crystallins, which show a considerably greater sensitivity to heat-induced aggregation/precipitation than the far more abundant beta and gamma crystallins. However, the small heat shock protein: alpha crystallin was effective in protecting these trace constituents of the lens from precipitating out of solution at 55 degrees C (measured under the standard conditions in a pH 7.5 buffer containing 50 mM sodium phosphate, 100 mM NaCl, 1 mM EDTA and 0.05% NaN3). Prominent components of the precipitate, formed in the absence of a recombinant alpha B crystallin chaperone could be resolved by one- and two-dimensional electrophoresis. Identification by amino acid sequencing revealed that the heat-sensitive group of lens proteins comprised glyceraldehyde-3-phosphate dehydrogenase (M(r) approximately 39 kDa), enolase (approximately 48 kDa), leucine aminopeptidase (approximately 52 kDa) and aldehyde dehydrogenase (approximately 53 kDa). These findings indicate for the first time that the aggregation of such minor lens constituents could possibly contribute to initiating the process of opacification in the development of cataracts.

Amino Acid Sequence↗
PubMed1997

A transglutaminase-related antigen associates with keratin filaments in some mouse epidermal cells.

A mouse monoclonal IgG, G82, directed against guinea pig liver transglutaminase recognizes a transglutaminase-related antigen that is associated with the keratin intermediate filament network in some primary mouse keratinocytes. The association can be seen at the resolution of individual keratin tonofibrils following fixation and staining for double-label indirect immunofluorescence. Western blots indicate that G82 reacts with two proteins of 95 kDa and 280 kDa, respectively, in extracts of these cells. The 95-kDa band is also recognized by a polyclonal antibody against purified guinea pig liver transglutaminase, and the 280-kDa protein seems to correspond to a similar protein that was shown to be recognized by G92.1.2 in the intermediate filament fraction of primary mouse fibroblasts. The transglutaminase-related antigen was shown by confocal microscopy to co-localize only with nonbasal cell specific keratin intermediate filaments.

Animals↗
PubMed1996

Cloning and regulation of cornifin beta, a new member of the cornifin/spr family. Suppression by retinoic acid receptor-selective retinoids.

In this study, we describe the isolation and characterization of a cDNA clone C12 that encodes a new member of the cornifin/small proline-rich protein (spr) family, which we have named cornifin beta. C12 encodes a 1.1-kilobase pair mRNA and a 24.3-kDa cytosolic protein with a high proline content (19%). Its total amino acid sequence exhibits a 37-66% identity while the first 30 amino acids at the amino terminus are 87% identical to that of members of the cornifin family. At its carboxyl terminus, cornifin beta contains 21 tandem repeats of an octapeptide. Cornifin beta expression is restricted to several squamous epithelia. It is highly expressed in esophagus, tongue, and oral mucosa but, in contrast to cornifin alpha, is not detectable in the epidermis. Both retinoic acid and a retinoid selective for the nuclear retinoic acid receptors were very potent suppressors of cornifin beta expression while an analog selective for the nuclear retinoid X receptors was much less effective, suggesting that a specific retinoid signaling pathway is involved in this suppression. Cornifin beta can function through some of its Gln residues as an amine acceptor in transglutaminase-catalyzed cross-linking reactions. These results indicate that cornifin beta functions as a cross-linked envelope precursor.

Amino Acid Sequence↗
PubMed1995

Contact with the N termini in the central E domain enhances the reactivities of the distal D domains of fibrin to factor XIIIa.

The reaction of Factor XIIIa with fibrin is the last enzyme-catalyzed step on the coagulation cascade, leading to the formation of a normal blood clot. The finding that fibrin is preferred by the cross-linking enzyme about 10-fold over the circulating fibrinogen suggests the operation of a unique substrate-level control for the orderly functioning of the physiological process in the forward direction. An important task is to elucidate the molecular mechanism for the transmission of the signal generated by the thrombin-catalyzed cleavage in the central E domain of fibrin to the distant Factor XIIIa-reactive glutamine residues. By focusing on the substrate sites present in gamma chain remnants of D type domains of fibrinogen and by employing the approach of fragment complementation with the regulatory E domain, which represents the thrombin-modified portion of fibrin, we have now succeeded in reconstructing in solution the phenomenon of kinetic enhancement for the reaction with Factor XIIIa. Two D type preparations (truncated fibrinogen, approximately 250 kDa and D', approximately 105 kDa) were obtained by digestion of human fibrinogen with endo Lys-C. Neither product could be cross-linked by Factor XIIIa, but as shown by the incorporation of dansylcadaverine, both were acceptor substrates for the enzyme. The plasmin-derived D (approximately 105-kDa) product, however, could be cross-linked into DD dimers. In all cases, the admixture of E fragments exerted a remarkable boosting effect on the reactions with Factor XIIIa. Even with native fibrinogen as substrate, cross-linking of gamma chains was enhanced in the presence of E. Nondenaturing electrophoresis was used to demonstrate the complex forming potential of E fragments with fibrinogen, truncated fibrinogen, D', or D. The GPRP tetrapeptide mimic of the GPRV N-terminal sequence of the alpha chains in the E fragments, abolished both complex formation and the kinetic boosting effect of E on the reactions of substrates with Factor XIIIa. Thus, the N-terminal alpha chain sequences seem to act as organizing templates for spatially orienting the D domains, probably during the protofibrillar assembly of the fibrin units, for favorable reaction with Factor XIIIa.

Binding Sites↗
PubMed1995

Identification of factor-XIIIa-reactive glutaminyl residues in the propolypeptide of bovine von Willebrand factor.

von Willebrand factor is a large multimeric plasma protein which plays important roles in platelet aggregation, blood coagulation and probably also in the adhesion of endothelial cells. A 100-kDa propeptide, called the propolypeptide of von Willebrand factor (pp-vWF), is generated during biosynthesis. We found that pp-vWF served as a substrate for transglutaminases including human factor XIIIa and guinea pig liver transglutaminase [Usui, T., Takagi, J. & Saito, Y. (1993) J. Biol. Chem. 268, 12311-12316]. As such, it could form cross-linked copolymers with the extracellular matrix protein, laminin, making it all the more likely that pp-vWF plays a role in cell adhesion phenomena [Takagi, J., Sudo, Y., Saito, T. & Saito, Y. (1994) Eur. J. Biochem. 222, 861-867]. In this work, we identified the Gln residues in pp-vWF specifically reacting with blood coagulation factor XIIIa as amine acceptors. The fluorescent amine, dansylcadaverine, was employed for labeling the enzyme-reactive sites of the protein. Following partial proteolysis, fragments containing the labeled Gln residues were isolated by passage through an anti-dansyl affinity chromatographic column. Amino acid sequence analyses of the fragments revealed that, out of about 40 Gln residues in pp-vWF, only four could be modified in the factor-XIIIa-catalyzed reaction.

Amino Acid Sequence↗
PubMed1995

Association of a transglutaminase-related antigen with intermediate filaments.

A mouse monoclonal antibody, G92.1.2, raised against guinea pig liver transglutaminase (TGase) recognizes an antigen present in primary mouse dermal fibroblasts. A filamentous pattern, bearing remarkable similarity to the vimentin intermediate filament (IF) network, is seen when these cells are fixed and processed for indirect immunofluorescence with the antibody. Double-label immunofluorescence reveals that the antigen reacting with the antibody colocalizes precisely with vimentin IF and that this colocalization is retained after the treatment of fibroblasts with colchicine, which induces a redistribution of the majority of IFs into perinuclear aggregates. These morphological observations are further supported by the finding that the protein reacting with G92.1.2 is retained in IF-enriched cytoskeletal preparations made by using nonionic detergent-containing high ionic strength solutions. Western blots of the IF fraction show that G92.1.2 recognizes a major band of approximately 280 kDa and does not cross react with vimentin. Furthermore, when the antibody is microinjected into live dermal fibroblasts, it causes a collapse of the vimentin IF network in the majority of injected cells. The results suggest that a form of TGase, or a TGase-related antigen, is closely associated with the vimentin IF network of primary cultures of mouse dermal fibroblasts.

Animals↗
PubMed1995

Prolactin-immunoglobulin G complexes from human serum act as costimulatory ligands causing proliferation of malignant B lymphocytes.

Several lines of evidence indicate that immunoglobulin-bound prolactin found in human serum is not a conventional complex between an anti-prolactin antibody and prolactin but a different type of association of prolactin with the Fab portion of IgG heavy chains. The complex of prolactin with IgG was purified from serum by anti-human prolactin affinity chromatography and was shown to contain close to 1 mole of N epsilon-(gamma-glutamyl)lysine crosslinks per mole of complex, a characteristic feature in structures crosslinked by transglutaminase. Interestingly, the complex caused a proliferation of cells from a subset of patients with chronic lymphocytic leukemia, while it was inactive in a cell proliferation prolactin bioassay. By contrast, human prolactin stimulated the proliferation of cells in the bioassay but had no effect on the complex-responsive cells from the patients. Competition studies with prolactin and free Fc fragment of IgG demonstrated a necessity for engaging both the prolactin and the immunoglobulin receptors for proliferation. More importantly, competition for the growth response by free prolactin and IgG suggests both possible reasons for the slow growth of this neoplasm as well as avenues for control of the disease.

B-Lymphocytes↗
PubMed1995

The fibronectin-binding domain of transglutaminase.

Guinea pig liver transglutaminase (EC 2.3.2.13) displays a Ca(2+)-independent binding (Ka = 10(7) M-1) to the same gelatin-binding domain of human plasma fibronectin that is known to form a very tight complex with the human red cell enzyme. The fibronectin-combining site of the liver transglutaminase was investigated by testing fragments obtained from the parent protein by controlled digestion with endoproteinase Lys-C. Overlay assays, probed with anti-fibronectin antibody, revealed that the fibronectin binding ability of the transglutaminase was encoded in a linear sequence in its 28-kDa N-terminal domain. Removal of the first 7 residues by further digestion of the purified 28-kDa material with endoproteinase Glu-C generated a 27-kDa fragment that, however, showed no binding activity. Thus, residues 1-7 in the liver enzyme seem to be of particular importance for influencing its ability to bind to fibronectin.

Amino Acid Sequence↗
PubMed1995

The pancornulins: a group of small proline rich-related cornified envelope precursors with bifunctional capabilities in isopeptide bond formation.

In this report, the pancornulins are identified as members of the spr (small, proline-rich) multigene family by amino acid sequence and mass spectrometry analyses. One of the pancornulins (14.9 kDa) is identical to the protein predicted by spr-1 clone 128. The other pancornulins (16.9 kDa and 22 kDa) are novel members of the spr family. Immunoelectron microscopy of purified cornified envelopes with a pancornulin-specific antibody established these proteins more definitively as cornified envelope precursors. In addition, two-dimensional electrophoretic analyses of keratinocyte extracts labeled enzymatically with dansylcadaverine (to identify amine acceptors) or dansylPGGQQIV (to identify amine donors) showed that both glutamine and lysine residues within the pancornulins participate in the isopeptide linkage characteristic of cornified envelope formation. These results contrasted with those obtained using involucrin, a prominent cornified envelope protein shown capable of acting only as an amine acceptor in this system. Novel partial cDNAs obtained after reverse transcription and polymerase chain reaction amplification of total messenger RNA with pancornulin-specific primers suggest that the spr multigene family may be even larger than previously described. The bifunctional reactivity of the pancornulins in cross-linking and the large number of family members identified to date suggest that the pancornulins and other spr-1-related proteins may be more important in cornified envelope formation than previously considered, perhaps functioning as "bridge" molecules during the early phases of cornified envelope assembly.

Amino Acid Sequence↗
PubMed1994

Effects of inhibiting transglutaminase during egg fertilization and development.

Transglutaminase inhibitor (1,3,4,5-tetramethyl-2-[(2-oxopropyl)thio]imidazolium chloride or L-682777) affected the appearance of the fertilization envelope (FE) and subsequent development in Sphaerechinus granularis and Arbacia punctulata in a concentration-dependent manner. Abnormalities were first visible in forming FE's at the lowest concentration (0.001 mM) of the inhibitor used. The wrinkled appearance of FE's suggested that the treatment prevented the I-T transition, whereas the finding of numerous denuded cells and empty FE's indicated that the inhibitor acted by rendering the FE's very fragile. Cell division by the 2-cell stage was also affected by this active-site-directed transglutaminase inhibitor. With 0.05 mM of the inhibitor, about 50% of all cells were polynucleated. In view of earlier findings that other transglutaminase inhibitors can produce abnormalities with regard to FE wrinkling and deranged cell division, it can be suggested that different forms of transglutaminases might be involved at various stages in embryonic development.

Animals↗
PubMed1994

Residue Gln-30 of human erythrocyte anion transporter is a prime site for reaction with intrinsic transglutaminase.

Elevating the intracellular concentration of Ca2+ ions in human erythrocytes leads to the formation of membrane-associated polymers, composed of skeletal and cytoplasmic proteins. The anion transporter band 3 serves as a membrane anchor for this N epsilon-(gamma-glutamyl)lysine cross-linked polymer. The reaction is catalyzed by an intrinsic transglutaminase, and it can be observed in broken cells merely by the addition of Ca2+. Certain primary amines, such as dansylcadaverine, inhibit the process by virtue of competition against the epsilon-lysyl or donor functionalities of the protein substrates. Dansylcadaverine itself becomes incorporated into the enzyme-specific gamma-glutaminyl or acceptor residues, blocking these from participating in the formation of protein-to-protein cross-links. This labeling procedure, coupled with an anti-dansyl antibody affinity procedure to isolate dansyl-labeled compounds, was employed to identify Gln-30 as the preferred transglutaminase-reactive acceptor site in the band 3 protein.

Amino Acids↗
PubMed1993

Type-2 plasminogen-activator inhibitor is a substrate for trophoblast transglutaminase and factor XIIIa. Transglutaminase-catalyzed cross-linking to cellular and extracellular structures.

Plasminogen-activator inhibitor type-2 (PAI-2), a serine-proteinase inhibitor, suppresses fibrinolysis by blocking both urokinase and tissue-type plasminogen activators. The 43-kDa PAI-2 molecule is an abundant cytosolic protein in certain cell types, but can upon appropriate stimulation be secreted as an approximately 60-70-kDa glycoprotein. However, in trophoblast membranes PAI-2 activity is associated with large covalent complexes (Jensen, P. H., Nykjaer, P., Andreasen P. A., Lund, L., Astedt, B. Lecander, I & Gliemann, J. (1989) Biochim. Biophys. Acta 986, 135-140). This study shows that PAI-2 can act as a substrate for both tissue transglutaminase and activated plasma factor XIII. In the presence of Ca2+, either of these will catalyze the incorporation of primary amines, such as putrescine, into PAI-2. Moreover, in reactions with tissue transglutaminase, PAI-2 homopolymers and, in conjunction with other biological substrates, heteropolymers were observed. As judged by the test of incorporating 125I-urokinase into SDS-resistant 125I-urokinase/PAI-2 complexes, polymerized PAI-2 retained its inhibitory activity. Furthermore, syncytiotrophoblast microvillous membranes and trophoblast detergent extracts incorporated 125I-PAI-2 into large structures in a reaction inhibited by putrescine and a synthetic inhibitor of transglutaminase. Trophoblast transglutaminase was identified as a tissue transglutaminase by non-denaturing gel electrophoresis and dansylcadaverine activity staining, fibronectin binding and Western blotting with a specific antibody. The transglutaminase-catalyzed and Ca(2+)-dependent anchoring of PAI-2 to extracellular membrane structures might have the purpose of focally regulating fibrinolysis.

Animals↗
PubMed1993

Affinity of human erythrocyte transglutaminase for a 42-kDa gelatin-binding fragment of human plasma fibronectin.

Complex formation between the human erythrocyte transglutaminase (protein-glutamine:amine gamma-glutamyltransferase, EC 2.3.2.13) and fibronectin or its fragments was examined by immunoanalytical procedures and by fluorescence polarization. A 42-kDa gelatin-binding structure, obtained from human plasma fibronectin by thermolytic digestion, showed as high an affinity for the cytosolic enzyme as the parent fibronectin chains themselves. A 21-kDa fragment comprising type I modules 8 and 9, the last two modules in the 42-kDa fragment, bound with an affinity 100-fold less than the 42-kDa fragment. Binding was remarkably specific and could be exploited for the affinity purification of transglutaminase directly from the hemoglobin-depleted erythrocyte lysate. In spite of the high affinity, it was possible to elute active enzyme from the 42-kDa fragment column with 0.25% monochloroacetic acid. This solvent might have general applicability in other systems involving separation of tightly bound ligands.

Antibodies, Monoclonal↗
PubMed1993

Association of the A subunits of recombinant placental factor XIII with the native carrier B subunits from human plasma.

Interactions of a recombinant human placental protein (rA2) expressed in yeast and considered to be identical to the catalytic A2 subunits of factor XIII, the fibrin stabilizing factor zymogen, were examined with the native carrier subunits (B2) of the factor isolated from human plasma. Nondenaturing electrophoresis and HPLC gel-filtration experiments showed a tight binding of rA2 to B2 for forming an ensemble similar to that of plasma factor XIII (A2B2). In the presence of excess B2, however, some higher ordered oligomers (rA2Bn, where n > 2) were also seen in electrophoresis. The same technique revealed a microheterogeneity in the rA2 preparation; nevertheless, all isoforms could bind to B2. By employing an ELISA procedure for measuring free B2 in mixtures with rA2, an apparent binding constant of 4 x 10(7) M-1 was derived for the association of rA2 with B2. Fluorescence depolarization was used to monitor the heterologous association of rA2 with fluorescein-labeled B2F as well as the dissociation of the rA2B2F structure. The former was characterized by an increase, and the latter by a decrease, in the fluorescence anisotropy of the system. Binding of rA2 to B2F (pH 7.5, mu = 0.315, 37 degrees C) was not influenced by low concentrations of Ca2+ (< or = 30 mM), and rA2B2F proved to be quite stable under these conditions. Much higher concentrations of Ca2+, as well as higher ionic strengths, were required to dissociate this assembly. By contrast, release of B2F from the thrombin-modified rA2'B2F occurred rapidly in the presence of low concentrations of Ca2+ at low ionic strength.(ABSTRACT TRUNCATED AT 250 WORDS)

Calcium↗