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.     

A specific colorimetric assay for measuring transglutaminase 1 and factor XIII activities

Transglutaminase (TGase) is an enzyme that catalyzes both isopeptide cross-linking and incorporation of primary amines into proteins. Eight TGases have been identified in humans, and each of these TGases has a unique tissue distribution and physiological significance. Although several assays for TGase enzymatic activity have been reported, it has been difficult to establish an assay for discriminating each of these different TGase activities. Using a random peptide library, we recently identified the preferred substrate sequences for three major TGases: TGase 1, TGase 2, and factor XIII. In this study, we use these substrates in specific tests for measuring the activities of TGase 1 and factor XIII.     

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.     

Immunological Detection of Proteolytically Activated Epidermal-type Transglutaminase (TGase 3) Using Cleavage-site-specific Antibody

Transglutaminase 3 (TGase 3), involved in the cross-linking of structural proteins in the epidermis, is activated by limited proteolysis of zymogen into two fragments during keratinocyte differentiation. Using recombinant TGase 3, the N-terminus sequence of the proteolyzed fragment was analyzed. Antibody against the synthetic peptide corresponding to the cleavage site specifically detected the fragment in the mouse forestomach extract.     

Immunological detection of proteolytically activated epidermal-type transglutaminase (TGase 3) using cleavage-site-specific antibody

Transglutaminase 3 (TGase 3), involved in the cross-linking of structural proteins in the epidermis, is activated by limited proteolysis of zymogen into two fragments during keratinocyte differentiation. Using recombinant TGase 3, the N-terminus sequence of the proteolyzed fragment was analyzed. Antibody against the synthetic peptide corresponding to the cleavage site specifically detected the fragment in the mouse forestomach extract.     

Cell type-specific activation of intracellular transglutaminase 2 by oxidative stress or ultraviolet irradiation: implications of transglutaminase 2 in age-related cataractogenesis

Transglutaminase (TGase) 2 is a ubiquitously expressed enzyme that modifies proteins by cross-linking or polyamination. An aberrant activity of TGase 2 has implicated its possible roles in a variety of diseases including age-related cataracts. However, the molecular mechanism by which TGase 2 is activated has not been elucidated. In this report, we showed that oxidative stress or UV irradiation elevates in situ TGase 2 activity. Neither the expression level nor the in vitro activity of TGase 2 appeared to correlate with the observed elevation of in situ TGase 2 activity. Screening a number of cell lines revealed that the level of TGase 2 activation depends on the cell type and also the environmental stress, suggesting that unrecognized cellular factor(s) may specifically regulate in situ TGase 2 activity. Concomitantly, we observed that human lens epithelial cells (HLE-B3) exhibited about 3-fold increase in in situ TGase 2 activity in response to the stresses. The activated TGase 2 catalyzed the formation of water-insoluble dimers or polymers of alphaB-crystallin, betaB(2)-crystallin, and vimentin in HLE-B3 cells, providing evidence that TGase 2 may play a role in cataractogenesis. Thus, our findings indicate that in situ TGase 2 activity must be evaluated instead of in vitro activity to study the regulation mechanism and function of TGase 2 in biological and pathological processes.     

The calcium sensing receptor and its alternatively spliced form in murine epidermal differentiation

We have recently reported that human keratinocytes express both the full-length calcium sensing receptor (CaR) and an alternatively spliced form lacking exon 5, which were suggested to be involved in calcium induced keratinocyte differentiation. To understand further the role of these CaRs, we analyzed the structure of mouse CaRs, and investigated their role using a mouse model in which only the full-length CaR was disrupted. Our results show that both the full-length and the alternatively spliced variant lacking exon 5 encoding 77 amino acids of the extracellular domain were expressed in mouse epidermis. The deletion of the full-length CaR increased the production of the alternatively spliced form of CaR in mutant mice. The keratinocytes derived from these mutant mice did not respond to extracellular calcium, suggesting that the full-length CaR is required to mediate calcium signaling in the keratinocytes. The loss of the full-length CaR altered the morphologic appearance of the epidermis and resulted in a reduction of the mRNA and protein levels of the keratinocyte differentiation marker, loricrin. These results indicate that CaR is important in epidermal differentiation, and that the alternatively spliced form does not fully compensate for loss of the full-length CaR.     

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.     

Ordered structure acquisition by the N- and C-terminal domains of the small proline-rich 3 protein

The cell envelope (CE) is a vital structure for barrier function in terminally differentiated dead stratified squamous epithelia. It is assembled by transglutaminase (TGase) cross-linking of several proteins, including hSPR3 in certain specialized epithelia normally subjected to mechanical trauma. Biochemical studies show that hSPR3 serves as a complete substrate for TGase1, TGase2, and TGase3. Multiple adjacent glutamines and lysines of only head-and-tail domain sequences are used by each enzyme for cross-linking. Structural data suggest that the hSPR3 central repeats, as well as hSPR1 and hSPR 2, are highly flexible and mobile; thus, the TGases might not be able to recognize the residues localized on the repeats as adequate substrate. To investigate this hypothesis further and to complete the structural investigation of hSPR3, we performed circular dichroism (CD) studies on peptides corresponding to the N- and C-terminal domain. CD spectra have also been carried out in the presence of different concentrations of the structure-promoting agent cosolvent trifluoroethanol (TFE), which mimics a partial hydrophobic environment found in vivo in or next to the membrane. In fact, this agent increases the dielectric constant of water proportionally, depending on its concentration, and confers structuring properties to the solution, to peptides and proteins that have a structuring propensity. The results indicate that in both the N-terminal and C-terminal, peptides acquire a more ordered structure as a function of the TFE concentration in water. This ability of both N- and C-terminal domain to acquire a more stable ordered conformation might be relevant for SPR3 to act as substrate of TGases. Indeed, only the N- and C-terminus is cross-linked by TGase1 and 3.     

Transglutaminase crosslinking and structural studies of the human small proline rich 3 protein.

The cell envelope (CE) is a vital structure for barrier function in terminally differentiated dead stratified squamous epithelia. It is assembled by transglutaminase (TGase) cross-linking of several proteins, including SPR3 in certain specialized epithelia normally subjected to mechanical trauma. We have expressed recombinant human SPR3 in order to study its cross-linking properties. It serves as a complete substrate for, and is cross-linked at similar efficiencies by, the three enzymes (TGases 1, 2 and 3) that are widely expressed in many epithelia. Multiple adjacent glutamines (4, 5, 16, 17, 18, 19 and 167) and lysines (6, 21, 164, 166 and 168) of only head and tail domain sequences are used for cross-linking. However, each enzyme preferentially uses certain residues on the head domain. Moreover, our in vitro data suggest a defined temporal order of cross-linking of SPR3 in vivo: It is first cross-linked by TGase 3 into short intra- and inter-chain oligomers which are later further cross-linked to the CE by TGase 1. To investigate the absence of cross-linking in the central domain (e.g. lysine in position 2 of each of the 16 repeats) we performed structural studies on recombinant SPR3 and on a synthetic peptide containing three repeats of the central domain. 2D H-1 NMR spectroscopy, TOCSY and ROESY, shows strong and medium intensity NOEs connectivities along the amino acid sequence with one weak long range NOE contact between Thr and Cys of subsequent repeats. Distance geometry computation on the basis of intensities of NOEs found generated 50 compatible structures grouped in three main families differing by the number of H-bonds. These measurements were repeated at different concentrations of trifluoroethanol (TFE)-water mixture, an alpha-helical promoting solvent, in order to check the stability of the conformations determined; no changes were observed up to 50% TFE in solution. Also temperature changes did not produce any variation in the ROESY spectrum in the same condition as above. The NMR and circular dichroism data strongly indicate the presence of an ordered (not alpha-helix nor beta-sheet) highly flexible structure in the eight amino acids repetitive units of SPR3, confirming the prediction of one possible beta-turn per each repeating unit. Thus, biochemical and biophysical data, strongly support SPR3 to function as a flexible cross-bridging protein to provide tensile strength or rigidity to the CE of the stratified squamous epithelia in which it is expressed.     

Progressive stages of "transdifferentiation" from epidermal to mesenchymal phenotype induced by MyoD1 transfection, 5-aza-2''- deoxycytidine treatment, and selection for reduced cell attachment in the human keratinocyte line HaCaT

The ability of the myogenic determination gene (MyoD1) to convert differentiating human keratinocytes (HaCaT cell-line) to the myogenic pathway and the effect of MyoD1 on the epidermal phenotype was studied in culture and in surface transplants on nude mice. MyoD1 transfection induced the synthesis of myosin, desmin, and vimentin without substantially altering the epidermal differentiation properties (morphology, keratin profile) in vitro nor epidermal morphogenesis (formation of a complex stratified squamous epithelium) in surface transplants, demonstrating the stability of the keratinocyte phenotype. 5-Aza-CdR treatment of these MyoD1-transfected cells had little effect on the cultured cells but a morphologically unstructured epithelium was formed with no indications of typical cell layers including cornification. Since prevention of epidermal strata in transplants was not accompanied by blocked epidermal differentiation markers (keratins K1 and K10, involucrin, and filaggrin), the dissociation of morphogenesis and expression of these markers argues for independently controlled processes. A subpopulation of less adhesive cells, isolated from the 5-aza-CdR treated MyoD1-transfectants, had lost most epithelial characteristics in culture (epidermal keratins, desmosomal proteins, and surface-glycoprotein Gp90) and had shifted to a mesenchymal/myogenic phenotype (fibroblastic morphology, transactivation of Myf3 and myogenin, expression of myosin, desmin, vimentin, and Gp130). Moreover, the cells had lost the ability to stratify and remained as a monolayer of flat elongated cells in transplants. These subsequent changes from a fully differentiated keratinocyte to a mesenchymal/myogenic phenotype strongly argue for a complex "transdifferentiation" process which occurred in the original monoclonal human epidermal HaCaT cells.     

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.     

Cholesterol 3-sulfate interferes with cornified envelope assembly by diverting transglutaminase 1 activity from the formation of cross-links and esters to the hydrolysis of glutamine

The loss of transglutaminase 1 enzyme (TGase 1) activity causes lamellar ichthyosis. Recessive X-linked ichthyosis (XI) results from accumulation of excess cholesterol 3-sulfate (CSO(4)) in the epidermis but the pathomechanism how elevated epidermal CSO(4) causes ichthyosis is largely unknown. Here we provide evidence that XI is also a consequence of TGase 1 dysfunction. TGase 1 is a key component of barrier formation in keratinocytes: it participates in the cross-linking of cell envelope (CE) structural proteins, and also forms the lipid bound envelope by esterification of long chain omega-hydroxyceramides onto CE proteins. Using involucrin and an epidermal omega-hydroxyceramide analog as substrates, kinetic analyses revealed that at membrane concentrations above 4 mol %, CSO(4) caused a marked and dose-dependent inhibitory effect on isopeptide and ester bond formation. Sequencing of tryptic peptides from TGase 1-reacted involucrin showed a large increase in deamidation of substrate glutamines. We hypothesize that supraphysiological levels of CSO(4) in keratinocyte membranes distort the structure of TGase 1 and facilitate the access of water into its active site causing hydrolysis of substrate glutamine residues. Our findings provide further evidence for the pivotal role of the TGase 1 enzyme in CE formation.     

Screening for the preferred substrate sequence of transglutaminase using a phage-displayed peptide library: identification of peptide substrates for TGASE 2 and Factor XIIIA

Mammalian transglutaminase (TGase) catalyzes covalent cross-linking of peptide-bound lysine residues or incorporation of primary amines to limited glutamine residues in substrate proteins. Using an unbiased M13 phage display random peptide library, we developed a screening system to elucidate primary structures surrounding reactive glutamine residue(s) that are preferred by TGase. Screening was performed by selecting phage clones expressing peptides that incorporated biotin-labeled primary amine by the catalytic reactions of TGase 2 and activated Factor XIII (Factor XIIIa). We identified several amino acid sequences that were preferred as glutamine donor substrates, most of which have a marked tendency for individual TGases: TGase 2, QxPphiD(P), QxPphi, and QxxphiDP; Factor XIIIa, QxxphixWP (where x and phi represent a non-conserved and a hydrophobic amino acid, respectively). We further confirmed that the sequences were favored for transamidation using modified glutathione S-transferase (GST) for recombinant peptide-GST fusion proteins. Most of the fusion proteins exhibited a considerable increase in incorporation of primary amines over that of modified GST alone. Furthermore, we identified the amino acid sequences that demonstrated higher specificity and inhibitory activity in the cross-linking reactions by TGase 2 and Factor XIIIa.     

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.     

Human EGF Receptor (HER) Family and Heregulin Members Are Differentially Expressed in Epidermal Keratinocytes and Modulate Differentiation

Human EGF receptor (HER), also designated HER1, is an activatable tyrosine kinase receptor. HER1 activation regulates cell growth and differentiation in epidermal keratinocytes. Expression of other HER family members was investigated in human keratinocytes cultured under autocrine conditions. HER2 and HER3 are expressed and upregulated by confluence, concurrent with induction of epidermal differentiation. HER4 is not expressed by keratinocytes. Maximum expression of the cognate ligand, heregulin, is observed in subconfluent keratinocytes, and the expression of both heregulin alpha and beta isoforms is downregulated with confluence. Recombinant heregulin isoforms have no effect on colony formation and keratinocyte proliferation, but heregulin beta activates tyrosine phosphorylation of HER2 and HER3, with no effect on HER1, in confluent differentiating keratinocyte cultures. Also, heregulin beta increases HER2/HER3 heterodimerization under those conditions. Treatment of confluent cultures by heregulin beta correlates with cell signaling and inhibition of epidermal differentiation. Together, HER2, HER3, and heregulin constitute a potential autocrine-paracrine system involved in epidermal homeostasis and repair, as well as in hyperproliferative pathologies.