Molecular characterization of a novel nuclear transglutaminase that is expressed during starfish embryogenesis

Abstract

We report the constitution and molecular characterization of a novel transglutaminase (EC 2.3.2.13) that starts to accumulate specifically in the nucleus in the starfish (Asterina pectinifera) embryo after progression through the early blastula stage. The cDNA for the nuclear transglutaminase was cloned and the cDNA-deduced sequence defines a single open reading frame encoding a protein with 737 amino acids and a predicted molecular mass of 83 kDa. A comparison of this transglutaminase with other members of the gene family revealed an overall sequence identity of 33-41%. A special sequence feature of this transglutaminase, which is not found in other transglutaminases, is the presence of nuclear localization signal-like sequences in the N-terminal region.Microinjection of hybrid constructs that encode the N-terminal segment fused to reporter proteins into the germinal vesicle of an oocyte produced chimeric proteins by transcription-coupled translation. It was found that the N-terminal segment alone was sufficient to effect nuclear accumulation of an otherwise cytoplasmic protein. These results suggest that the nuclear accumulation of the transglutaminase may play an important role in nuclear remodeling during early starfish embryogenesis.Keywords: transglutaminase; nucleus; starfish; embryo; cloning.The class of enzymes that are commonly referred to as transglutaminases (TG) (EC 2.3. Recent findings have shown that, apart from their protein modifying capabilities, tissue TG is also able to function as a component of the signal-transducing G protein complex [7]. The cDNA of G ha , involved in the transmission of adrenergic stimuli, is identical to that of tissue TG of human endothelial cells [7]. Tissue TG is localized mainly in the cytosol, but detectable tissue TG expression has been reported in the nucleus [8][9][10]. However, TG activity in the nucleus and the mechanisms of its translocation is not well understood, and nucleus-specific TG has not been reported.It is accepted that many proteins are able to cross nuclear membranes and accumulate against gradients to concentrate in the nucleus [11,12]. The nuclear translocation of proteins via the nuclear pore complex is dependent on a nuclear localization signal in the protein, which is rich in basic amino acids and may be bipartite [13][14][15]. The functional assays of such nuclear localization signals are usually based on the ability of a signal to confer nuclear localization to an otherwise non-nuclear protein.The present paper describes the occurrence of a novel TG that is localized exclusively in the nucleus of starfish (Asterina pectinifera) embryonic cells and is designated nuclear TG (nTG). The amino-acid sequence derived from the cDNA sequence contains putative nuclear localization signals [15] in the N-terminal region. We demonstrate here that the N-terminal region promotes the nuclear accumulation of an otherwise cytoplasmic protein, namely pyruvate kinase (PK), in the A. pectinifera oocyte system. This finding suggests that nuclear localization sig...

Cell-mediated immunity in arthropods: Hematopoiesis, coagulation, melanization and opsonization

“…Instead, horseshoe crabs use the TGase cross-linking reaction during the final stage of coagulation . TGases have been found in other invertebrates but their functions are different or unknown (Singer et al, 1992;Sugino et al, 2002). In horseshoe crabs, hemolymph is extremely sensitive to minute amounts of bacterial LPS, which induces conversion of coagulogen (a soluble protein) into insoluble coagulin.…”

cDNA cloning, identification, tissue localisation, and transcription profile of a transglutaminase from white shrimp, Litopenaeus vannamei, after infection by Vibrio alginolyticus

“…The enzyme is also involved in maintaining a toughened skin epidermis and hair follicle epithelia [5], in forming the fertilization plug in female rodents [6], in signal transduction, and in apoptosis [2,7]. In invertebrates, TGs were found in various species including the crayfish [8], horseshoe crab [9][10][11], grasshopper [12], starfish [13], fruitfly [14], tiger shrimp [15][16][17], and malaria mosquito [18]. Among invertebrates, in fruitfly and mosquito genomes, only one locus encoding TG with a functional domain organisation has been identified.…”

Immune gene mining by pyrosequencing in the rockshell, Thais clavigera

“…Therefore, we decided to check their transcript levels in both sexes of rockshell, because blood coagulation often represents the first line of defense in several invertebrate immunity systems. To date, TG has been characterized in different marine invertebrate species, such as horseshoe crab [39], starfish [40], tiger shrimp [41], Chinese shrimp [42], and white leg shrimp [43]. In the present study, we found the TG gene from the rockshell EST database, and analyzed their different expression patterns in several rockshell tissues of male and female.…”