Beta B1 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 beta B1 crystallin and the almost complete disappearance of beta B3 and beta A3. 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 beta-crystallin crosslinking by transglutaminase. This notion was confirmed by using beta B1- and beta Bp-specific antisera. Both sera reacted with the 57-kDa dimer; the beta Bp-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 beta B1- and beta Bp-crystallin chains, and N-terminally truncated derivatives thereof, it was shown that in the beta B1/beta Bp dimer, glutamine residue -9 of beta Bp crosslinks to one of the lysine residues in the N-terminal extension of beta B1.     

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 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.     

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.     

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.     

Phosphorylated cystatin alpha is a natural substrate of epidermal transglutaminase for formation of skin cornified envelope.

Both keratohyalin granules (KHG) and cornified envelopes were stained histochemically in an indirect immunofluorescent study by antiphosphorylated cystatin alpha antibody, indicating that phosphorylated cystatin alpha is a component of the cornified envelope proteins. When phosphorylated cystatin alpha (P-cystatin alpha) was incubated with epidermal transglutaminase (TGase) and Ca2+ ions, polymerized protein was produced by formation of epsilon-(gamma-glutamyl)lysine cross-linking peptide bonds between lysine residues of cystatin alpha and glutamine residues of suitable protein(s) in the enzyme preparation. However, phosphorylated and non-phosphorylated cystatins were polymerized to similar extents by the TGase. Immunofluorescent and immunoelectron microscopic observations revealed that P-cystatin alpha could be detected in vivo in the KHG and cornified envelopes. Treatment of sphingosine, a specific inhibitor of protein kinase C, markedly suppressed the incorporation of cystatin alpha into KHG. Thus phosphorylation of cystatin alpha by protein kinase C may play an important role in targeting cystatin alpha into KHG.     

Acid-induced dissociation of alpha A- and alpha B-crystallin homopolymers.

Homopolymers were constructed from the alpha A and alpha B polypeptides isolated from the lens protein alpha-crystallin. As the pH is lowered from 7.0 to 3.4, these homopolymers dissociate to smaller species with molecular masses ranging from 80 to 250 kDa for the alpha A and around 140 kDa for the alpha B dissociation products. The pKa for this dissociation was 3.8 +/- 0.2 for alpha A and 4.1 +/- 0.1 for alpha B homopolymers. Further decreases in pH, to 2.5, resulted in the presence of only denatured alpha B polypeptides, whereas the alpha A dissociation products remained intact. Fractionation of the acid dissociation products from the alpha A homopolymer at pH 2.5 yielded stable species with molecular masses of 220 +/- 30, 160 +/- 20, and 90 +/- 10 kDa. The majority of the population at acid pH consisted of the 160 kDa species. Conformational analysis of these species revealed that most of the secondary structure of the original alpha A homopolymer was retained but that the tertiary structure was perturbed. Fluorescence quenching and energy transfer measurements suggested that the molecule had undergone acid expansion, with the greatest perturbation observed in the smallest particles. The results from this work suggest that alpha A homopolymers are heterogeneous populations of aggregates of a "monomeric" molecule with a molecular mass of 160 kDa. This "monomeric" molecule may be formed from the association of two tetrameric units.     

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.     

The in vivo phosphorylation sites of bovine alpha B-crystallin

Phosphate content determinations established that in alpha B-crystallin two phosphate groups can be present in vivo in bovine lenses. Comparison of tryptic digests of phosphorylated and unphosphorylated alpha B chains, revealed the location of the two phosphorylation sites in tryptic peptides T2 and T3. Thermolytic digestion and gas-phase sequencing demonstrated that Ser-19 and Ser-45 are the in vivo phosphorylation sites of bovine alpha B-crystallin. This pattern of phosphorylation differs from the previously reported in vitro obtained results.     

alpha B-crystallin is a cytoplasmic interaction partner of the kidney-specific cadherin-16

The Ca^2 +-dependent membrane-spanning classical cadherins bind directly to cytosolic catenins. This cadherin-catenin interaction is known to be critical for the fundamental role of cadherins in cell-cell adhesion. The small subfamily of the 7D-cadherins, however, cannot interact with catenins due to their highly truncated cytoplasmic tail. Thus far, no cytoplasmic interaction partner for the 7D-cadherins has been described. With the use of the cytoplasmic domain of the Ksp (kidney-specific)-cadherin, which belongs to the family of 7D-cadherins, as bait in affinity chromatography with human kidney lysates, the small heat-shock protein αB-crystallin was identified by matrix-assisted laser desorption/ionization-time-of-flight analysis as a cytosolic binding partner of Ksp-cadherin. This interaction was verified by co-immunoprecipitation analysis. With the use of overlapping peptides representing the entire αB-crystallin molecule, the N-terminal part of αB-crystallin, which does not possess chaperone activity, was identified as responsible for the binding to Ksp-cadherin. This interaction was found to be specific since only the cytoplasmic domain of Ksp-cadherin, but not LI (liver-intestine)-cadherin (another member of the 7D-cadherin family), interacted with αB-crystallin. In the human kidney, both αB-crystallin and Ksp-cadherin co-localize to cells of the collecting duct. They also co-localize with the actin cytoskeleton and co-precipitate with the latter. These findings suggest that the interaction of Ksp-cadherin with αB-crystallin is important for the connection of Ksp-cadherin to the cytoskeleton and thus for maintaining tissue integrity in the kidney.     

The lysine analog L-oxalysine is an inhibitor of RNA synthesis.

1. The lysine analog L-4-oxalysine was found to be a potent inhibitor of RNA synthesis in Candida albicans. 2. The compound was a weak inhibitor of protein synthesis and DNA synthesis was not affected. 3. The inhibition of RNA synthesis was reversed by L-lysine but not D-lysine. 4. The decrease in the level of newly synthesized RNA in cells treated with L-oxalysine was due to inhibition of de novo synthesis rather than to degradation of RNA.     

Alpha 2-antiplasmin's carboxy-terminal lysine residue is a major site of interaction with plasmin

alpha 2-Antiplasmin (AP) inhibits plasmin in a two-step reaction in which AP reversibly binds to lysine-binding sites of plasmin and, then, more slowly complexes covalently with the enzyme's active site. Here, we show that the C-terminal lysine residue of AP has a key role in binding of the inhibitor to plasmin. A synthetic peptide corresponding to the C-terminal 26 amino acid residues of AP blocked association of AP with plasmin, but this activity of the peptide was lost when its C-terminal lysine residue was removed with carboxypeptidase B. The essential role of this lysine residue was shown more directly by treating AP with carboxypeptidase B and observing that AP lost its ability to inhibit plasmin rapidly.     

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.     

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.     

The carboxy-terminal domain of Gs alpha is necessary for anchorage of the activated form in the plasma membrane

GTP-binding proteins which participate in signal transduction share a common heterotrimeric structure of the alpha beta gamma-type. In the activated state, the alpha subunit dissociates from the beta gamma complex but remains anchored in the membrane. The alpha subunits of several GTP-binding proteins, such as Go and Gi, are myristoylated at the amino terminus (Buss, J. E., S. M. Mumby, P. J. Casey, A. G. Gilman, and B. M. Sefton. 1987. Proc. Natl. Acad. Sci. USA. 84:7493-7497). This hydrophobic modification is crucial for their membrane attachment. The absence of fatty acid on the alpha subunit of Gs (Gs alpha), the protein involved in adenylate cyclase activation, suggests a different mode of anchorage. To characterize the anchoring domain of Gs alpha, we used a reconstitution model in which posttranslational addition of in vitro-translated Gs alpha to cyc- membranes (obtained from a mutant of S49 cell line which does not express Gs alpha) restores the coupling between the beta-adrenergic receptor and adenylate cyclase. The consequence of deletions generated by proteolytic removal of amino acid sequences or introduced by genetic removal of coding sequences was determined by analyzing membrane association of the proteolyzed or mutated alpha chains. Proteolytic removal of a 9-kD amino-terminal domain or genetic deletion of 28 amino-terminal amino acids did not modify the anchorage of Gs alpha whereas proteolytic removal of a 1-kD carboxyterminal domain abolished membrane interaction. Thus, in contrast to the myristoylated alpha subunits which are tethered through their amino terminus, the carboxy-terminal residues of Gs alpha are required for association of this protein with the membrane.     

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.     

The small heat-shock protein alpha B-crystallin promotes FBX4-dependent ubiquitination

AlphaB-crystallin is a small heat-shock protein in which three serine residues (positions 19, 45, and 59) can be phosphorylated under various conditions. We describe here the interaction of alphaB-crystallin with FBX4, an F-box-containing protein that is a component of the ubiquitin-protein isopeptide ligase SCF (SKP1/CUL1/F-box). The interaction with FBX4 was enhanced by mimicking phosphorylation of alphaB-crystallin at both Ser-19 and Ser-45 (S19D/S45D), but not at other combinations. Ser-19 and Ser-45 are preferentially phosphorylated during the mitotic phase of the cell cycle. Also alphaB-crystallin R120G, a mutant found to co-segregate with a desmin-related myopathy, displayed increased interaction with FBX4. Both alphaB-crystallin S19D/S45D and R120G specifically translocated FBX4 to the detergent-insoluble fraction and stimulated the ubiquitination of one or a few yet unknown proteins. These findings implicate the involvement of alphaB-crystallin in the ubiquitin/proteasome pathway in a phosphorylation- and cell cycle-dependent manner and may provide new insights into the alphaB-crystallin-induced aggregation in desmin-related myopathy.