Sporadic inclusion body myositis correlates with increased expression and cross-linking by transglutaminases 1 and 2

Sporadic inclusion body myositis (SIBM) is characterized by vacuolar degeneration of muscle fibers and intrafiber clusters of paired helical filaments with abnormal amyloid deposition. Because of their potential involvement in other degenerative disorders, we have examined the expression of transglutaminases (TGases) in normal and SIBM tissues. We report that at least two different enzymes, the ubiquitous TGase 2 as well as the TGase 1 enzyme, are present in muscle tissues. However, in comparison with normal tissue, the expression of TGases 1 and 2 was increased 2.5- and 4-fold in SIBM, accompanied by about a 20-fold higher total TGase activity. By immunohistochemical staining, in normal muscle, TGase 2 expression was restricted to some endomysial connective tissue elements, whereas TGase 1 and beta-amyloid proteins were not detectable. In SIBM muscle, both TGases 1 and 2 as well as amyloid proteins were brightly expressed and co-localized in the vacuolated muscle fibers, but none of these proteins colocalized with inflammatory cell markers. Next, we isolated high molecular weight insoluble proteins from SIBM muscle tissue and showed that they were cross-linked by about 6 residues/1000 residues of the isopeptide bond. Furthermore, by amino acid sequencing of solubilized tryptic peptides, they contain amyloid and skeletal muscle proteins. Together, these findings suggest that elevated expression of TGases 1 and 2 participate in the formation of insoluble amyloid deposits in SIBM tissue and in this way may contribute to progressive and debilitating muscle disease.     

Transglutaminase 5 cross-links loricrin, involucrin, and small proline-rich proteins in vitro.

Transglutaminases (TGases) are seven enzymes, cross-linking proteins by gamma-glutamil-epsilon-lysine bonds, four of which are expressed in the skin. A new member of the TGase family, TGase 5, has been identified recently, and in the present study we evaluated its role in keratinocyte differentiation in vitro. In addition to the previously described isoforms, full-length TGase 5 and Delta3 (deletion of exon 3), we identified two new splicing variants, Delta11 and Delta3Delta11 (deletion of exons 11 or 3, 11). We expressed full-length TGase 5, Delta3, Delta11, and Delta3Delta11 isoforms in the keratinocyte and baculovirus systems. The results indicate that both full-length TGase 5 and Delta11 are active, whereas Delta3 and Delta3Delta11 have very low activity. Expression studies show that full-length TGase 5 is induced during the early stages of keratinocyte differentiation and is differently regulated in comparison with the other epidermal TGases. Kinetic and in vitro cross-linking experiments indicate that full-length TGase 5 is very efficient in using specific epidermal substrates (loricrin, involucrin, and SPR3). In keratinocyte expression system, TGase 5 isoforms are retained in an intermediate filament-enriched fraction, suggesting its association with insoluble proteins. Indeed, TGase 5 co-localize with vimentin and it is able to cross-link vimentin in vitro.     

Transglutaminase cross-linking properties of the small proline-rich 1 family of cornified cell envelope proteins. Integration with loricrin

Small proline-rich 1 (SPR1) proteins are important for barrier function in stratified squamous epithelia. To explore their properties, we expressed in bacteria a recombinant human SPR1 protein and isolated native SPR1 proteins from cultured mouse keratinocytes. By circular dichroism, they possess no alpha or beta structure but have some organized structure associated with their central peptide repeat domain. The transglutaminase (TGase) 1 and 3 enzymes use the SPR1 proteins as complete substrates in vitro but in different ways: head domain A sequences at the amino terminus were used preferentially for cross-linking by TGase 3, whereas those in head domain B sequences were used for cross-linking by TGase 1. The TGase 2 enzyme cross-linked SPR1 proteins poorly. Together with our data base of 141 examples of in vivo cross-links between SPRs and loricrin, this means that both TGase 1 and 3 are required for cross-linking SPR1 proteins in epithelia in vivo. Double in vitro cross-linking experiments suggest that oligomerization of SPR1 into large polymers can occur only by further TGase 1 cross-linking of an initial TGase 3 reaction. Accordingly, we propose that TGase 3 first cross-links loricrin and SPRs together to form small interchain oligomers, which are then permanently affixed to the developing CE by further cross-linking by the TGase 1 enzyme. This is consistent with the known consequences of diminished barrier function in TGase 1 deficiency models.     

Characterization of recombinant mouse epidermal-type transglutaminase (TGase 3): regulation of its activity by proteolysis and guanine nucleotides.

Epidermal-type TGase (TGase 3) is involved in the formation of the cornified cell envelope by cross-linking a variety of structural proteins in the epidermis. Unknown proteases activate this enzyme from the zymogen form by limited proteolysis during epidermal differentiation. It has been difficult to isolate sufficient quantities of native enzymes from tissues for biochemical studies of the properties of TGase 3. In this paper, we circumvented these problems by expressing recombinant full-length mouse TGase 3 in a baculovirus system, and purifying it to homogeneity by successive chromatography and HPLC. Treatment of the purified recombinant protein with dispase, a bacterial protease known to activate zymogens, produced activated TGase 3. The migration of TGase 3 zymogen in SDS-polyacrylamide gel electrophoresis was anomalous when the proTGase 3 was pre-incubated with calcium ion. GTP inhibited the enzymatic activity of recombinant TGase 3. Calpain, a calcium-dependent neutral protease, was a candidate protease, but had no effect on the activation of TGase 3 zymogen.     

Transglutaminase 5 is acetylated at the N-terminal end

Summary. Transglutaminases (TGases) are calcium-dependent enzymes that catalyse cross-linking between proteins by acyl transfer reaction; they are involved in many biological processes including coagulation, differentiation, and tissue repair. Transglutaminase 5 was originally cloned from keratinocytes, and a partial biochemical characterisation showed its involvement in skin differentiation, in parallel to TGase 1 and TGase 3. Here, we demonstrate, by electrospray tandem mass spectrometry that TGase 5 is acetylated at the N-terminal end. Moreover, in situ measurement of TGase activity shows that endogenous TGase 5 is active upon treatment with phorbol acetate, and the enzyme co-localises with vimentin intermediate filaments.     

Epidermal transglutaminase (TGase 3) is required for proper hair development, but not the formation of the epidermal barrier

Transglutaminases (TGase), a family of cross-linking enzymes present in most cell types, are important in events as diverse as cell-signaling and matrix stabilization. Transglutaminase 1 is crucial in developing the epidermal barrier, however the skin also contains other family members, in particular TGase 3. This isoform is highly expressed in the cornified layer, where it is believed to stabilize the epidermis and its reduction is implicated in psoriasis. To understand the importance of TGase 3 in vivo we have generated and analyzed mice lacking this protein. Surprisingly, these animals display no obvious defect in skin development, no overt changes in barrier function or ability to heal wounds. In contrast, hair lacking TGase 3 is thinner, has major alterations in the cuticle cells and hair protein cross-linking is markedly decreased. Apparently, while TGase 3 is of unique functional importance in hair, in the epidermis loss of TGase 3 can be compensated for by other family members.     

Ca2+: a stabilizing component of the transglutaminase activity of Galphah (transglutaminase II)

Galphah (transglutaminase type II; tissue transglutaminase) is a bifunctional enzyme with transglutaminase (TGase) and guanosine triphosphatase (GTPase) activities. The GTPase function of Galphah is involved in hormonal signaling and cell growth while the TGase function plays an important role in apoptosis and in cross-linking extracellular and intracellular proteins. To analyze the regulation of these dual enzymatic activities we examined their calcium-dependence and thermal stability in enzymes from several cardiac sources (mouse heart, and normal, ischemic and dilated cardiomyopathic human hearts). The GTP binding activity of Galphah was markedly inhibited by Ca2+ whereas the TGase activity was strongly stimulated, suggesting that Ca2+ acts as a regulator, switching Galphah from a GTPase to a TGase. The TGase function of Galphah of both mouse and human hearts was more thermostable in the presence of Ca2+.     

Cross linking of polyglutamine domains catalyzed by tissue transglutaminase is greatly favored with pathological-length repeats: does transglutaminase activity play a role in (CAG)(n)/Q(n)-expansion diseases?

Protein aggregates are a hallmark of Huntington's disease (HD) and other inherited neurodegenerative diseases caused by an elongated (CAG)(n) repeat in the genome and to a corresponding increase in the size of the Q(n) domain in the expressed protein. When the protein associated with HD (huntingtin) contains <35 Q repeats disease does not occur. However, an n>/=40 leads to disease. Some investigators have proposed that aggregates in the nuclei of affected cells are toxic, but other workers have suggested that the aggregates may be neutral or even protective. Whether or not they are toxic, an understanding of the processes whereby the aggregates develop may shed light on the neuropathological processes involved in the (CAG)(n)/Q(n)-expansion disorders. Q(n) domains have a tendency to non-covalently self align as 'polar zippers' rendering them less soluble, but evidence that such polar zippers occur in the aggregates in intact HD brain has so far been limited. The human brain contains at least three Ca(2+)-dependent enzymes (transglutaminases, TGases) that catalyze protein cross-linking reactions, namely TGase 1, TGase 2 (tissue transglutaminase, tTGase) and TGase 3. Q(n) aggregates have been found by several groups to be excellent substrates of tTGase. Moreover, the activity toward the Q(n) domains increases greatly as n is increased to 40 or beyond. tTGase mRNA and total TGase activity are elevated in HD brain. Moreover, some evidence suggests that Ca(2+) homeostasis is disrupted in HD brain. We propose that the combination of increased huntingtin (or huntingtin fragment containing the Q(n) domain) in the nucleus, increased the ability of the Q(n) domains to act as substrate, increased Ca(2+) levels and increased inherent TGase activity all contribute to increased cross-linking of proteins in HD brain. At first the proteasome machinery can recognize and degrade the cross-linked proteins, but over time the proteasome machinery may be overwhelmed and protein aggregates will accumulate.     

Changes in acetylcholinesterase and butyrylcholinesterase in Alzheimer's disease resemble embryonic development--a study of molecular forms.

The pattern of molecular forms of acetylcholinesterase (AChE, EC 3.1.1.7) and butyrylcholinesterase (BChE, EC 3.1.1.8) separated by density gradient centrifugation was investigated in the brain and cerebrospinal fluid in Alzheimer's disease (AD), in human embryonic brain and in rat brain after experimental cholinergic deafferentation of the cerebral cortex. While a selective loss of the AChE G4 form was a rather constant finding in AD, a small but significant increase of G1 for both AChE and BChE was found in the most severely affected cases. Both in normal human brain and in AD a significant relationship could be established between the AChE G4/G1 ratio in different brain regions and the activity of choline acetyltransferase (ChAT). A similar decrease of the AChE G4 form as observed in AD can be induced in rat by experimental cholinergic deafferentation of the cerebral cortex. The increase in G1 of both AChE and BChE in different brain regions in AD is quantitatively related to the local density of neuritic plaques which are histochemically reactive for both enzymes. In human embryonic brain, a high abundance of G1 and a low G4/G1 ratio for both AChE and BChE was found resembling the pattern observed in AD. Furthermore, both in embryonic brain and in AD AChE shows no substrate inhibition which is a constant feature of the enzyme in the adult human brain. It is, therefore, concluded that the degeneration of the cholinergic cortical afferentation in AD as reflected by a decrease of AChE G4 is accompanied by the process of a neuritic sprouting response involved in plaque formation which is probably associated with the expression of a developmental form of the enzyme.     

Polyamines as physiological substrates for transglutaminases

When normal human blood lymphocytes are treated with mitogen in the presence of [3H]putrescine, label is incorporated into a few cellular proteins. Labeled N-(gamma-glutamyl) putrescine, N1-(gamma-glutamyl)spermidine, and N8-(gamma-glutamyl)spermidine were identified in exhaustive proteolytic digests of the cellular protein fraction. The enzyme-mediated clotting of rat seminal plasma to which 14C-labeled spermidine and spermine are added is accompanied by incorporation of the polyamines into a number of seminal plasma proteins. Proteolytic digests of the protein fraction from this clotted seminal plasma contain labeled N1-(gamma-glutamyl)spermidine, N8-(gamma-glutamyl)spermidine, N1,N8-bis(gamma-glutamyl)spermidine, N1-(gamma-glutamyl)spermine, and N1,N12-bis(gamma-glutamyl)spermine. These findings support a proposal that polyamines serve as substrates for transglutaminases both in cells and in an extracellular fluid. They show differences in cellular and extracellular substrate properties of the polyamines and indicate cross-linking through these amines in the extracellular system, but provide no evidence for such cross-linking in the cells.     

Altered expression of claudin family proteins in Alzheimer’s disease and vascular dementia brains

Claudins (Cls) are a multigene family of transmembrane proteins with different tissue distribution, which have an essential role in the formation and sealing capacity of tight junctions (TJs). At the level of the blood–brain barrier (BBB), TJs are the main molecular structures which separate the neuronal milieu from the circulatory space, by a restriction of the paracellular flow of water, ions and larger molecules into the brain. Different studies suggested recently significant BBB alterations in both vascular and degenerative dementia types. In a previous study we found in Alzheimer’s disease (AD) and vascular dementia (VaD) brains an altered expression of occludin, a molecular partner of Cls in the TJs structure. Therefore in this study, using an immunohistochemical approach, we investigated the expression of Cl family proteins (Cl‐2, Cl‐5 and Cl‐11) in frontal cortex of aged control, AD and VaD brains. To estimate the number of Cl‐expressing cells, we applied a random systematic sampling and the unbiased optical fractionator method. We found selected neurons, astrocytes, oligodendrocytes and endothelial cells expressing Cl‐2, Cl‐5 and Cl‐11 at detectable levels in all cases studied. We report a significant increase in ratio of neurons expressing Cl‐2, Cl‐5 and Cl‐11 in both AD and VaD as compared to aged controls. The ratio of astrocytes expressing Cl‐2 and Cl‐11 was significantly higher in AD and VaD as compared to aged controls. The ratio of oligodendrocytes expressing Cl‐11 was significantly higher in AD and the ratio of oligodendrocytes expressing Cl‐2 was significantly higher in VaD as compared to aged controls. Within the cerebral cortex, Cls were selectively expressed by pyramidal neurons, which are the ones responsible for cognitive processes and affected by AD pathology. Our findings suggest a new function of Cl family proteins which might be linked to response to cellular stress.     

Characterization of a transglutaminase from scallop hemocyte and identification of its intracellular substrates

Scallop hemocytes contain a transglutaminase (TGase) that is electrophoretically different from the TGase in the adductor muscle. The optimum temperature of the hemocyte TGase was lower (about 15 degrees C), compared with the muscle TGase (35-40 degrees C). Other properties, such as the high sodium chloride (NaCl) and CaCl2 concentrations required for activation, instability in salt solutions, and the Km values against monodansylcadaverine (MDC) and succinylated casein, were similar for both enzymes. When hemocyte homogenate was incubated with MDC at 10 degrees C, MDC was incorporated into the 230 k and 100 k proteins of the hemocytes. The 100 k protein was only detected in the supernatant, the 230 k protein was insoluble, and the 210 k protein was detected in both fractions. In the absence of MDC, the 230 k, 210 k, and 100 k proteins were cross-linked by endogenous transglutaminase. The 230 k protein was most quickly cross-linked and formed huge polymers within 5 min. These results suggest that if scallop tissues are injured, hemocyte transglutaminase may be activated, initially cross-linking the insoluble hemocyte 230 k protein, followed by the 210 k and 100 k proteins, to form a cross-linked protein matrix with inter cross-linking of hemocyte sheets, to stop the bleeding.     

Serinc, an activity-regulated protein family, incorporates serine into membrane lipid synthesis

Cell membranes contain various transporter proteins, some of which are responsible for transferring amino acids across membrane. In this study, we report another class of carrier proteins, termed Serinc1-5, that incorporates a polar amino acid serine into membranes and facilitates the synthesis of two serine-derived lipids, phosphatidylserine and sphingolipids. Serinc is a unique protein family that shows no amino acid homology to other proteins but is highly conserved among eukaryotes. The members contain 11 transmembrane domains, and rat Serinc1 protein co-localizes with lipid biosynthetic enzymes in endoplasmic reticulum membranes. A Serinc protein forms an intracellular complex with key enzymes involved in serine and sphingolipid biosyntheses, and both functions, serine synthesis and membrane incorporation, are linked to each other. In the rat brain, expression of Serinc1 and Serinc2 mRNA was rapidly up-regulated by kainate-induced seizures in neuronal cell layers of the hippocampus. In contrast, myelin throughout the brain is enriched with Serinc5, which was down-regulated in the hippocampus by seizures. These results indicate a novel mechanism linking neural activity and lipid biosynthesis.     

Evidence for autocatalytic cross-linking of hydroxyproline-rich glycoproteins during extracellular matrix assembly in Volvox

The alga Volvox carteri is one of the simplest multicellular organisms, yet it has a surprisingly complex extracellular matrix (ECM), making Volvox suitable as a model system in which to study ECM self-assembly. Here, we analyze the primary structures and post-translational modifications of two main ECM components synthesized in response to sexual induction as well as wounding. These proteins are members of the pherophorin family with as yet unknown properties. They contain polyhydroxyproline spacers as long as 500 and 2750 residues. Even the highly purified proteins retain the capacity to self-assemble and cross-link, producing an insoluble fibrous network in an apparently autocatalytic reaction. This pherophorin-based network is located within the deep zone of the ECM. A molecular genetic search for additional members of the pherophorin family indicates that at least nine different pherophorin species can be expected to serve as precursors for ECM substructures. Therefore, the highly diversified members of the pherophorin family represent region-specific morphological building blocks for ECM assembly and cross-linking.     

Differential Proteomics Analysis of Specific Carbonylated Proteins in the Temporal Cortex of Aged Rats: The Deterioration of Antioxidant System

Oxidative stress plays a pivotal role in normal brain aging and various neurodegenerative diseases, including Alzheimer’s disease (AD). Irreversible protein carbonylation, a widely used marker for oxidative stress, rises during aging. The temporal cortex is essential for learning and memory and particularly susceptible to oxidative stress during aging and in AD patients. In this study, we used 2-DE, MALDI-TOF/TOF MS, and Western blotting to analyze the differentially carbonylated proteins in the rat temporal cortex between 1-month-old and 24-month-old. We showed that the carbonyl levels of ten protein spots corresponding to six gene products: SOD1, SOD2, peroxiredoxin 1, peptidylprolyl isomerase A, cofilin 1, and adenylate kinase 1, significantly increased in the temporal cortex of aged rats. These proteins are associated with antioxidant defense, the cytoskeleton, and energy metabolism. Several oxidized proteins identified in aged rat brain are known to be involved in neurodegenerative disorders as well. Our findings indicate that these carbonylated proteins may be implicated in the decline of normal brain aging process and provide insights into the mechanisms underlying age-associated dysfunction of temporal cortex.     

Activity-independent cell adhesion to tissue-type transglutaminase is mediated by alpha4beta1 integrin

Transglutaminases (TGases) are enzymes which catalyze cross-link formation between glutamine residues and lysine residues in substrate proteins. We have previously reported that one of the TGases, blood coagulation factor XIIIa (FXIIIa), is capable of mediating adhesion of various cells. In this paper, we report for the first time that tissue-type transglutaminase (TGc) also has cell adhesion activity. TGc-coated plastic surface promoted adhesion and spreading of cells in a TGc concentration-dependent manner. However, there are some obvious differences between cell adhesion mediated by TGc and FXIIIa. As was reported previously, the adhesion to FXIIIa is dependent on its TGase activity. In contrast, the TGc-mediated cell adhesion is independent of its TGase activity: 1) The modification of the active center cysteine with iodoacetamide blocked the enzyme activity without any effect on cell adhesion; 2) the addition of Mg2+ did not induce the enzyme activity, but it was as effective as Ca2+ for cell adhesion; 3) the addition of NH4+ inhibited the enzyme activity but did not affect the cell adhesion significantly. The integrins involved in these cell adhesions are quite different. In the case of FXIIIa, alpha vbeta3 and alpha5beta1 integrins are involved and consequently the RGD peptide substantially inhibited the adhesion. On the other hand, the cell adhesion to TGc is mediated by alpha4beta1 integrin but not alpha5beta1; a CS-1 peptide, which represents the binding site of fibronectin to alpha4beta1 integrin, completely inhibited the cell adhesion to TGc. It is possible that TGc and FXIIIa may mediate cell adhesion under different physiological and pathological situations.     

iTRAQ analysis of complex proteome alterations in 3xTgAD Alzheimer's mice: understanding the interface between physiology and disease

Alzheimer's disease (AD) is characterized by progressive cognitive impairment associated with accumulation of amyloid beta-peptide, synaptic degeneration and the death of neurons in the hippocampus, and temporal, parietal and frontal lobes of the cerebral cortex. Analysis of postmortem brain tissue from AD patients can provide information on molecular alterations present at the end of the disease process, but cannot discriminate between changes that are specifically involved in AD versus those that are simply a consequence of neuronal degeneration. Animal models of AD provide the opportunity to elucidate the molecular changes that occur in brain cells as the disease process is initiated and progresses. To this end, we used the 3xTgAD mouse model of AD to gain insight into the complex alterations in proteins that occur in the hippocampus and cortex in AD. The 3xTgAD mice express mutant presenilin-1, amyloid precursor protein and tau, and exhibit AD-like amyloid and tau pathology in the hippocampus and cortex, and associated cognitive impairment. Using the iTRAQ stable-isotope-based quantitative proteomic technique, we performed an in-depth proteomic analysis of hippocampal and cortical tissue from 16 month old 3xTgAD and non-transgenic control mice. We found that the most important groups of significantly altered proteins included those involved in synaptic plasticity, neurite outgrowth and microtubule dynamics. Our findings have elucidated some of the complex proteome changes that occur in a mouse model of AD, which could potentially illuminate novel therapeutic avenues for the treatment of AD and other neurodegenerative disorders.