βB1 crystallin is an amine-donor substrate for tissue transglutaminase

The effects of tissue transglutaminase on the water-soluble proteins in bovine lens homogenates are described. Addition of liver transglutaminase and Ca²⁺ to calf lens homogenates resulted not only in the appearance of 50- and 57-kDa dimers, but also in a decrease in the amount of βB1 crystallin and the almost complete disappearance of βB3 and βA3. This is not the result of Ca²⁺-induced proteolysis, since histamine completely inhibits this phenomenon. It may be concluded that these polypeptides are involved in β-crystallin crosslinking by transglutaminase. This notion was confirmed by using βB1- and βBp-specific antisera. Both sera reacted with the 57-kDa dimer; the βBp-specific antiserum also reacted with the 50-kDa dimer. No reaction in the region 50–57 kDa was detectable when EDTA was used instead of Ca²⁺. Using reconstituted mixtures of βB1- and βBp-crystallin chains, and N-terminally truncated derivatives thereof, it was shown that in the βB1/βBp dimer, glutamine residue -9 of βBp crosslinks to one of the lysine residues in the N-terminal extension of βB1.     

The carboxy-terminal lysine of alpha B-crystallin is an amine-donor substrate for tissue transglutaminase.

A hexapeptide, corresponding to the sequence around the glutamine in beta A3-crystallin that functions as amine-acceptor for transglutaminase, was synthesized. This peptide was biotinylated and used as a probe to identify amine-donor substrates for transglutaminase among lens proteins. It was found that Ca(2+)-activated transglutaminase linked this peptide not only to several beta-crystallins but, unexpectedly, also to alpha B-crystallin. The C-terminal lysine residue of alpha B-crystalline could be identified as the site of linkage. This strengthens the notion that, at least in crystallins, all transglutaminase substrate residues are located in terminal extensions of the polypeptides. It was shown that in lens homogenate, alpha B-crystallin can be covalently crosslinked to beta-crystallins by transglutaminase. The transglutaminase-mediated crosslinking of alpha B-crystallin may have implications for its involvement in normal and pathological processes in lens and other tissues.     

The p53 oncoprotein is a substrate for tissue transglutaminase kinase activity

Increased expression and activity of the ubiquitous enzyme, tissue transglutaminase (TG2), is consistently seen in a variety of models of apoptosis. The p53 oncoprotein is also involved in apoptosis. Here we investigated the interaction of TG2 with p53 and show that the p53 is a substrate for the recently identified serine/threonine kinase activity of TG2. Phosphospecific antibodies indicated that TG2 phosphorylated p53 at Ser(15) and Ser(20), residues that are critically important in the interaction of p53 with Mdm2. The TG2-induced phosphorylation was abrogated by high Ca(2+) concentrations and inhibited by cystamine, a known inhibitor of TG2 cross-linking activity. Furthermore, we demonstrate that TG2-induced phosphorylation of p53 reduces the ability of p53 to interact with Mdm2. Although TG2 cross-linking activity has been clearly implicated in apoptosis, our observations reported here suggest TG2 modification of p53 could be an additional mechanism whereby TG2 could facilitate apoptosis.     

Lipoprotein (a) is a substrate for factor XIIIa and tissue transglutaminase.

The mechanisms which mediate deposition of lipoprotein (a) (Lp(a)), an atherogenic lipoprotein particle, onto the vessel wall and cell surfaces are unknown. An irreversible deposition of Lp(a) may require the presence of enzymes that catalyze its binding to surface-oriented structures. Transglutaminases catalyze cross-linking of proteins as well as incorporation of primary amines into protein substrates. We studied whether tissue transglutaminase and/or activated Factor XIII (plasma derived or recombinant FXIIIa) incorporate primary amines into Lp(a). In the presence of Ca2+, Factor XIIIa and tissue transglutaminase catalyze incorporation of monodansylcadaverine or [14C]putrescine into purified Lp(a) in a specific and time-dependent manner. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated that monodansylcadaverine became incorporated into the apo(a) portion of Lp(a). Lp(a) purified from five different donors showing different apo(a) phenotypes were substrates for tissue transglutaminases (TG). Western blot analysis confirmed that apo(a) was the major monodansylcadaverine carrying protein moiety of Lp(a). Tissue TG also extensively cross-linked the apo(a) portion of the Lp(a) particle. Characterization of the specificity of tissue TG showed that fibronectin, alpha 2-plasmin inhibitor, and apo(a) could be readily labeled with monodansylcadaverine by tissue TG, but other proteins including low density lipoprotein, IgG, alpha 1-proteinase inhibitor, and albumin showed poor or no reactivity. Direct comparison of Lp(a) with low density lipoprotein showed that apoB 100 was a poor substrate for transglutaminases. Recombinant apolipoprotein (a) proved to be an excellent substrate for TGs in that 1 mol of recombinant apolipoprotein (a) incorporated as much as 15 mol of [14C]putrescine, which corresponded to five times the amount of amine incorporated into Lp(a). The susceptibility of Lp(a) to transglutaminases suggests a mechanism whereby the interaction of Lp(a) with surface receptors and other surface oriented structures could be enzymatically altered.     

Detyrosinated Glu-tubulin is a substrate for cellular Factor XIIIA transglutaminase in differentiating osteoblasts

Microtubule components α- and β-tubulin undergo a number of posttranslational modifications that modulate their dynamics and cellular functions. These modifications include polyamination and covalent crosslinking by transglutaminase enzymes. We have demonstrated previously that the less dynamic and more stable tubulin form—detyrosinated Glu-tubulin—is found in high molecular weight, oligomeric complexes in bone-forming osteoblasts during differentiation and along with deposition of collagenous extracellular matrix. In this study, we report that oligomeric Glu-tubulin has high nocodazole tolerance, indicating further increased stability. We show that α-tubulin, which gives rise to Glu-tubulin, is a transglutaminase substrate in in vitro assays and that it is crosslinked into oligomers (dimers, trimers and tetramers) by transglutaminase 2 and Factor XIIIA; β-tubulin was not crosslinked by transglutaminase activity. The oligomeric Glu-tubulin was specifically localized to the plasma membrane of osteoblasts as analyzed by subcellular fractionation, cell surface biotinylation experiments and total internal reflection fluorescence (TIRF) microscopy. Glu- and α-tubulin co-localized with cellular Factor XIIIA as analyzed by conventional and TIRF microscopy. The Factor XIIIA-specific substrate peptide bF11 co-localized with α-tubulin and acted as a competitive inhibitor to oligomerization of Glu-tubulin, attenuating its formation in cells. This was associated with significantly decreased type I collagen deposition and decreased secretory activity as measured by synaptotagmin VII levels on the osteoblast plasma membrane. Our results suggest that Glu-tubulin may exist as covalently stabilized form which may be linked to the secretion and elaboration of collagenous extracellular matrix.     

C1 inhibitor cross-linking by tissue transglutaminase

C1 inhibitor, a plasma proteinase inhibitor of the serpin superfamily involved in the regulation of complement classical pathway and intrinsic blood coagulation, has been shown to bind to several components of the extracellular matrix. These reactions may be responsible for C1 inhibitor localization in the perivascular space. In the study reported here, we have examined whether C1 inhibitor could function as a substrate for plasma (factor XIIIa) or tissue transglutaminase. We made the following observations: 1) SDS-polyacrylamide gel electrophoresis and autoradiography showed that C1 inhibitor exposed to tissue transglutaminase (but not to factor XIIIa) incorporated the radioactive amine donor substrate [(3)H]putrescine in a calcium-dependent manner; 2) the maximum stoichiometry for the uptake of [(3)H]putrescine by C1 inhibitor was 1:1; 3) proteolytic cleavage and peptide sequencing of reduced and carboxymethylated [(3)H]putrescine-C1 inhibitor identified Gln(453) (P'9) as the single amine acceptor residue; 4) studies with (125)I-labeled C1 inhibitor showed that tissue transglutaminase was also able to cross-link C1 inhibitor to immobilized fibrin; and 5) C1 inhibitor cross-linked by tissue transglutaminase to immobilized fibrin had inhibitory activity against its target enzymes. Thus, tissue transglutaminase-mediated cross-linking of C1 inhibitor to fibrin or other extracellular matrix components may serve as a mechanism for covalent serpin binding and influence local regulation of the proteolytic pathways inhibited by C1 inhibitor.     

Tissue transglutaminase is an integrin-binding adhesion coreceptor for fibronectin

The protein cross-linking enzyme tissue transglutaminase binds in vitro with high affinity to fibronectin via its 42-kD gelatin-binding domain. Here we report that cell surface transglutaminase mediates adhesion and spreading of cells on the 42-kD fibronectin fragment, which lacks integrin-binding motifs. Overexpression of tissue transglutaminase increases its amount on the cell surface, enhances adhesion and spreading on fibronectin and its 42-kD fragment, enlarges focal adhesions, and amplifies adhesion-dependent phosphorylation of focal adhesion kinase. These effects are specific for tissue transglutaminase and are not shared by its functional homologue, a catalytic subunit of factor XIII. Adhesive function of tissue transglutaminase does not require its cross-linking activity but depends on its stable noncovalent association with integrins. Transglutaminase interacts directly with multiple integrins of beta1 and beta3 subfamilies, but not with beta2 integrins. Complexes of transglutaminase with integrins are formed inside the cell during biosynthesis and accumulate on the surface and in focal adhesions. Together our results demonstrate that tissue transglutaminase mediates the interaction of integrins with fibronectin, thereby acting as an integrin-associated coreceptor to promote cell adhesion and spreading.     

An involucrin-like protein in hepatocytes serves as a substrate for tissue transglutaminase during apoptosis.

Cornified envelopes and apoptotic bodies are transglutaminase-cross-linked end-products of physiological cell death pathways. The two structures have similar amino acid composition. Involucrin has been considered as a cornified envelope precursor protein expressed specifically in terminally differentiating keratinocytes and squamous epithelia. We report the presence in hepatocytes of an involucrin-like protein which could be purified from dog liver with procedures characteristic to involucrins. When compared to purified dog esophagus involucrin, the liver protein also reacts with anti-involucrin antibodies, has the same relative molecular mass, possesses similar amino acid composition, and shows almost identical peptide mapping pattern. The involucrin-like protein is detectable by immunohistochemistry in normal and apoptotic hepatocytes, is a substrate of tissue transglutaminase, and is incorporated into cross-linked apoptotic bodies. These results suggest that there are overlapping molecular components in the two characteristic forms (cornification and apoptosis) of naturally occurring cell death.     

Fibronectin is a component of the sodium dodecyl sulfate-insoluble transglutaminase substrate

Liver plasma membranes contain a morphologically distinct protein complex which serves as a substrate for the plasma membrane-associated transglutaminase. The complex, which appears as a two-dimensional sheet, is insoluble in sodium dodecyl sulfate and reducing agents and has been named SITS for sodium dodecyl sulfate-insoluble transglutaminase substrate (Tyrrell, D. J., Sale, W. S., and Slife, C. W. (1988) J. Biol. Chem. 263, 1946-1951). Polyclonal antibodies raised against SITS were used to probe for soluble constituents of the matrix. Immunoblots showed that proteins of 230, 35, and 32 kDa reacted with the anti-SITS antiserum when the soluble fraction from a liver homogenate was examined. The 230-kDa protein was identified as fibronectin after observing cross-reactivity of anti-SITS antiserum with authentic fibronectin and cross-reactivity of anti-fibronectin antiserum with the 230-kDa cytosolic protein and purified SITS. Preincubating anti-SITS antiserum with purified fibronectin decreased immunostaining of the 230-kDa cytosolic protein and authentic fibronectin. Immunoblots of the plasma membrane fraction using anti-SITS and anti-fibronectin antisera showed that both antisera reacted with proteins at the top of the stacking gel (SITS) and of 230 kDa. In addition, the anti-SITS antiserum reacted with proteins of 85, 35, and 32 kDa. Immunofluorescence microscopy revealed that the anti-SITS and anti-fibronectin antisera both react with isolated SITS and with the same filamentous structures associated with intact plasma membranes. These studies show that fibronectin is a component of the plasma membrane matrix, SITS. This finding is consistent with the proposed role of this matrix which is to mediate cell-cell adhesion between hepatocytes in the tissue.     

Human tissue transglutaminase is inhibited by pharmacologic and chemical acetylation

Human tissue transglutaminase (TGM2) is implicated in the pathogenesis of several neurodegenerative disorders including Alzheimer's, Parkinson's and expanded polyglutamine (polyQ) diseases. TGM2 promotes formation of soluble and insoluble high molecular weight aggregates by catalyzing a covalent linkage between peptide‐bound Q residues in polyQ proteins and a peptide‐bound Lys residue. Therapeutic approaches to modulate the activity of TGM2 are needed to proceed with studies to test the efficacy of TGM2 inhibition in disease processes. We investigated whether acetylation of Lys‐residues by sulfosuccinimidyl acetate (SNA) or aspirin (ASA) would alter the crosslinking activity of TGM2. Acetylation by either SNA and/or ASA resulted in a loss of >90% of crosslinking activity. The Lys residues that were critical for inhibition were identified by mass spectrometry as Lys⁴⁴⁴, Lys⁴⁶⁸, and Lys⁶⁶³. Hence, acetylation of Lys‐residues may modulate the enzymatic function of TGM2 in vivo and offer a novel approach to treatment of TGM2 mediated disorders.     

Cell-surface-associated tissue transglutaminase is a target of MMP-2 proteolysis

MT1-MMP, a prototypic member of a membrane-type metalloproteinase subfamily, is an invasion promoting protease and an activator of MMP-2. In addition, MT1-MMP proteolysis regulates the functionality of cell-surface adhesion/signaling receptors including tissue transglutaminase (tTG). tTG is known to serve as an adhesion coreceptor for beta1/beta3 integrins and as an enzyme that catalyzes the cross-linking of proteins and the conjugation of polyamines to proteins. Here, we report that MMP-2, functioning in concert with MT1-MMP, hydrolyzes cell-surface-associated tTG, thereby further promoting the effect initiated by the activator of MMP-2. tTG, in return, preferentially associates with the activation intermediate of MMP-2. This event decreases the rate of MMP-2 maturation and protects tTG against proteolysis by MMP-2. Our cell culture, in vitro experiments, and in silico modeling indicate that the catalytic domain of MMP-2 directly associates with the core enzymatic domain II of tTG (the K(d) = 380 nM). The follow-up cleavage of the domain II eliminates both the receptor and the enzymatic activity of tTG. Our data illuminate the coordinated interplay involving the MT1-MMP/MMP-2 protease tandem in the regulation of the cell receptors and explain the underlying biochemical mechanisms of the extensive tTG proteolysis that exists at the normal tissue/tumor boundary. Our findings also suggest that neoplasms, which express functionally active MT1-MMP and, therefore, activate soluble MMP-2, can contribute to the degradation of tTG expressed in neighboring host cells. The loss of adhesive and enzymatic activities of tTG at the interface between tumor and normal tissue will decrease cell-matrix interactions and inhibit matrix cross-linking, causing multiple pathological alterations in host cell adhesion and locomotion.     

Development of an isoenzyme-specific colorimetric assay for tissue transglutaminase 2 cross-linking activity

Tissue transglutaminase (TGase 2) belongs to the multigene transglutaminase family of Ca²⁺-dependent protein cross-linking enzymes. Based on the transamidation activity of TGase 2, a novel colorimetric assay has been developed using covalently coupled spermine to carboxy-substituted polystyrene plates and biotinylated pepT26, an excellent acyl-donor substrate, highly specific for TGase 2. The assay is based on the incorporation of the gamma-carboxamide of glutamine of pepT26 into the immobilized spermine. The amount of biotinylated pepT26 bound to the plate, as measured by the activity of streptavidin-peroxidase, is directly proportional to the TGase activity. The colorimetric procedure showed a good correlation (r = 0.995) with the commonly used radiometric filter paper method for TGase2, and provides linear dose-response curves over a wide range of hrTGase2 concentrations (2.5-40 μU/ml). In addition, the assay shows higher sensitivity when compared with our previous TG-colorimetric test (more than 50-fold increase) and other existing assays. PepT26 displays strong reactivity with TGase 2, and no reactivity with TGases 1, 3, and FXIII. The procedure constitutes a rapid, TG2-specific, sensitive, and nonisotopic method for the measurement of TGase 2 activity in as low as 4 ng of hrTGase 2 and purified guinea pig liver transglutaminase, and 1.25 μg of guinea pig liver extracts.     

Cyclin B1 is an efficacy-predicting biomarker for Chk1 inhibitors

Chk1 is the major mediator of cell-cycle checkpoints in response to various forms of genotoxic stress. Although it was previously speculated that checkpoint abrogation due to Chk1 inhibition may potentiate the efficacy of DNA-damaging agents through induction of mitotic catastrophe, there has not been direct evidence proving this process. Here, through both molecular marker and morphological analysis, we directly demonstrate that specific downregulation of Chk1 expression by Chk1 siRNA potentiates the cytotoxicities of topoisomerase inhibitors through the induction of premature chromosomal condensation and mitotic catastrophe. More importantly, we discovered that the cellular cyclin B1 level is the major determinant of the potentiation. We show that downregulation of cyclin B1 leads to impairment of the induction of mitotic catastrophe and correspondingly a reduction of the potentiation ability of either Chk1 siRNA or a small molecule Chk1 inhibitor. More significantly, we have extended the study by examining a panel of 10 cancer cell-lines with different tissue origins for their endogenous levels of cyclin B1 and the ability of a Chk1 inhibitor to sensitize the cells to DNA-damaging agents. The cellular levels of cyclin B1 positively correlate with the degrees of potentiation achieved. Of additional interest, we observed that the various colon cancer cell lines in general appear to express higher levels of cyclin B1 and also display higher sensitivity to Chk1 inhibitors, implying that Chk1 inhibitor may be more efficacious in treating colon cancers. In summary, we propose that cyclin B1 is a biomarker predictive of the efficacy of Chk1 inhibitors across different types of cancers. Unlike previously established efficacy-predictive biomarkers that are usually the direct targets of the therapeutic agents, cyclin B1 represents a non-drug-target biomarker that is based on the mechanism of action of the target inhibitor. This finding may be potentially very useful for the stratification of patients for Chk1 inhibitor clinical trials and hence, maximize its chance of success.     

The transglutaminase activating metalloprotease inhibitor from Streptomyces mobaraensis is a glutamine and lysine donor substrate of the intrinsic transglutaminase

Transglutaminase (TGase) from Streptomyces mobaraensis is an extra-cellular enzyme that cross-links proteins to high molecular weight aggregates. Screening for intrinsic substrates now revealed the dual Streptomyces subtilisin inhibitor-like inhibitor Streptomyces subtilisin and transglutaminase activating metalloprotease (TAMEP) inhibitor (SSTI), equally directed against subtilisin and the TGase activating metalloprotease TAMEP, is both a glutamine and a lysine donor protein. Reactivity of glutamines is lost during culture, most likely by TGase mediated deamidation, and, accordingly, cross-linking only occurred if SSTI from early cultures was used. Interestingly, release of buried endo-glutamines by the lipoamino acid N-lauroylsarcosine could restore SSTI reactivity. Formation of lipoamino acids by Streptomycetes suggests such compounds could also modulate in vivo TGase mediated SSTI cross-linking.     

A sensitive fluorometric assay for tissue transglutaminase

We have devised a highly sensitive fluorometric well plate assay for tissue transglutaminase that is suitable for multiple kinetic analyses/high-throughput screening of chemical inventories for inhibitors of this enzyme. The procedure measures the rate of fluorescence enhancement (lambda(exc) 260 nm, lambda(em) 538 nm) when 1-N-(carbobenzoxy-l-glutaminylglycyl)-5-N-(5'N'N'-dimethylaminonaphthalenesulfonyl)diamidopentane (glutaminyl substrate) is cross-linked to dansyl cadaverine (amine substrate). The assay procedure can be used to measure the activity of as little as 60 microU of purified guinea pig liver tissue transglutaminase (4.2 ng or 54 fmol of enzyme).     

Evidence for transglutaminase activity in plant tissue

An extract prepared from the apical meristematic region of etiolated pea seedlings was able to catalyze the incorporation of putrescine into trichloroacetic acid precipitable material. The enzyme was found to be soluble and followed a typical Michaelis-Menten kinetics when N-N-dimethyl casein was used as a substrate. Its activity was promoted by Ca²⁺ and inhibited by Cu²⁺ and dl-dithiothreitol. Other polyamines competed with putrescine as substrates and cadaverine was the most potent inhibitor of putrescine incorporation. Plant transglutaminase is capable of recognizing specific sites in substrates described for animal transglutaminase, like insulin, fibrinogen, pepsin, and thrombin. However, it can also use as substrates cellulase and creatine kinase which have not been described for transglutaminase from other sources.     

A direct fluorometric assay for tissue transglutaminase

Herein we report the design of a direct and continuous fluorometric assay for determining tissue transglutaminase (TGase) activity. The progress of the TGase-catalyzed reaction of 4-(N-carbobenzoxy-l-phenylalanylamino)-butyric acid coumarin-7-yl ester was monitored as an increase of fluorescence (lambda(exc) 330 nm, lambda(em) 460 nm) due to the release of 7-hydroxycoumarin. Using this assay, we determined the K(m) of two acceptor substrates, N-acetyl-L-lysine methyl ester and aminoacetonitrile. We also determined the K(m) of 4-(N-carbobenzoxy-L-phenylalanylamino)-butyric acid coumarin-7-yl ester for its TGase-mediated hydrolysis and for its enzymatic reaction with the acyl acceptor substrates N-acetyl-L-lysine methyl ester and aminoacetonitrile. We ascertained that the fluorescent substrate was selective toward tissue TGase by testing it with different enzymes, namely microbial transglutaminase (mTGase), Factor XIIIa, papain, and gamma-glutamyl transpeptidase. 4-(N-carbobenzoxyglycinylamino)-butyric acid coumarin-7-yl ester, lacking the benzyl side chain, was also found to be an efficient fluorogenic substrate of tissue TGase. Finally, we have shown that this method is applicable to 96-well microtiter plate format.     

Extracellular transglutaminase 2 is catalytically inactive, but is transiently activated upon tissue injury

Transglutaminase 2 (TG2) is a multifunctional mammalian protein with transamidase and signaling properties. Using selective TG2 inhibitors and tagged nucleophilic amine substrates, we show that the majority of extracellular TG2 is inactive under normal physiological conditions in cell culture and in vivo. However, abundant TG2 activity was detected around the wound in a standard cultured fibroblast scratch assay. To demonstrate wounding-induced activation of TG2 in vivo, the toll-like receptor 3 ligand, polyinosinic-polycytidylic acid (poly(I:C)), was injected in mice to trigger small intestinal injury. Although no TG2 activity was detected in vehicle-treated mice, acute poly(I:C) injury resulted in rapid TG2 activation in the small intestinal mucosa. Our findings provide a new basis for understanding the role of TG2 in physiology and disease.     

On the substrate specificity of cathepsins B1 and B2 including a new fluorogenic substrate for cathepsin B1.

Cathepsin B1 from bovine spleen exhibited its greatest rates of hydrolysis on peptide β-naphthylamide (βNA) derivatives containing paired basic residues, i.e., Cbz-Arg-Arg-βNA, t-Boc-Lys-Lys-βNA, and t-Boc-Lys-Arg-βNA. Internal peptide bonds were not attacked. At its pH 6.5 optimum, cathepsin B1 hydrolyzed Cbz-Arg-Arg-βNA (K_m 0.18 mM) 64 times faster than Bz-DL-Arg-βNA (K_m 3.3 mM or 1.6 mM for the L isomer) and was therefore chosen to replace the latter as a more soluble and sensitive substrate for the assay of cathepsin B1. Although cathepsin B2 had no action on the β-naphthylamide substrates, it did manifest carboxypeptidase activity by attacking COOH-terminal residues exposed by the action of cathepsin B1. At its pH 5.0 optimum, cathepsin B2 behaved as a SH-dependent, non-specific carboxypeptidase by releasing COOH-terminal amino acids from a variety of Cbz-Gly-X substrates and polypeptides such as glucagon, Val-Leu-Ser-Glu-Gly, and penta-lysine.