Isolation of a cDNA Encoding a Novel Member of the Transglutaminase Gene Family from Human Keratinocytes

116

Transglutaminases (TGases) are seven enzymes, cross-linking proteins by ␥-glutamil-⑀-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 ⌬3 (deletion of exon 3), we identified two new splicing variants, ⌬11 and ⌬3⌬11 (deletion of exons 11 or 3, 11). We expressed full-length TGase 5, ⌬3, ⌬11, and ⌬3⌬11 isoforms in the keratinocyte and baculovirus systems. The results indicate that both full-length TGase 5 and ⌬11 are active, whereas ⌬3 and ⌬3⌬11 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 crosslinking 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.

“…TGase 5 has, in fact, also been detected in other cell lines (dermal fibroblast, osteosarcoma, eritroleukemia (8)) and in other tissues (data not shown).…”

Section: Identification Of Two New Splicing Variants Expression Inmentioning

confidence: 83%

mentioning

confidence: 99%

See 1 more Smart Citation

Transglutaminase 5 Cross-links Loricrin, Involucrin, and Small Proline-rich Proteins in Vitro

Candi

Oddi

Terrinoni

et al. 2001

116

“…Seven members of the TGase family have been identified in the human genome so far, of which four (TGases 1, 2, 3, and X) are expressed in the epidermis (30,31), although to date only TGases 1 and 3 have verified roles in CE assembly (32,33). TGase 1 is expressed as a 106-kDa monomeric protein, which is constitutively N-myristoylated and S-palmitoylated on its amino-terminal 10-kDa domain, thereby directing the enzyme to plasma membranes (34 -36).…”

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confidence: 99%

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

Nemes¹,

Demény²,

Marekov³

et al. 2000

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 -hydroxyceramides onto CE proteins. Using involucrin and an epidermal -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.Assembly of an effective epidermal barrier structure is an essential adaptation to terrestrial life. In mammals the outermost bulwark of this barrier is the cornified layer of the epidermis, composed of flattened corneocytes mortared together by orderly lipid laminae. During terminal differentiation, individual corneocytes acquire a specialized cell peripheral structure termed the cornified cell envelope (CE), 1 which is responsible for maintenance of mechanical and chemical protection and indirectly contributes to water permeability barrier (see Refs. 1 and 2, for reviews). The CE is composed of two parts. The ϳ10 nm thick protein envelope is formed by covalent cross-linking of several structural proteins by sulfhydryl oxidases and transglutaminases (TGases). This highly insoluble protein meshwork is coated by the lipid envelope, a ϳ5 nm thick layer of -hydroxyceramides with uniquely long (C 28 -C 36 ) fatty acyl moieties (3). These are covalently attached by ester bonds through their -hydroxyl group to selected glutamines to envoplakin, periplakin, and involucrin components of the protein envelope (4).Terminal differentiation of keratinocytes is accompanied by vigorous lipid metabolism and synthesis of keratinization-specific lipids in the granular layer. Newly synthesized lipids are temporarily stored in cytoplasmic lamellar bodies, in which they are arranged as stacks of tetralaminar sheets. The lamellar body lipids consist largely of free fatty acids, (glucosyl)ceramides, cholesterol, and its acyl or sulfate esters (3). In the uppermost granular layer the lamellar bodies fuse with the cell membrane, and release their contents which assume broad, multilamellar lipid sheets between corneocytes. This process approxima...

“…Therefore, based on the known properties of the two enzymes, we suggest that the activity increase may be due in significant part to abnormal proteolytic activation of TGase 1 in SIBM tissue. However, we cannot exclude the possibility that other known or as yet unknown TGases may also contribute to this increase (48). Nevertheless, further detailed studies on the role and biochemistry of TGase 1 in normal and diseased muscle tissue are warranted.…”

Section: Discussionmentioning

confidence: 89%

Sporadic Inclusion Body Myositis Correlates with Increased Expression and Cross-linking by Transglutaminases 1 and 2

Choi

Park

Kim

et al. 2000

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 ␤-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.