Identification of mammalian-type transglutaminase in Physarum polycephalum. Evidence from the cDNA sequence and involvement of GTP in the regulation of transamidating activity.
Transglutaminase (TGase) catalyses the post-translational modification of proteins by transamidation of available glutamine residues. While several TGase genes of fish and arthropods have been cloned and appear to have similar structures to those of mammals, no homologous gene has been found in lower eukaryotes. We have cloned the acellular slime mold Physarum polycephalum TGase cDNA using RT-PCR with degenerated primers, based on the partial amino acid sequence of the purified enzyme. The cDNA contained a 2565-bp ORF encoding a 855-residue polypeptide. By Northern blotting, an mRNA of approximately 2600 bases was detected. In comparison with primary sequences of mammalian TGases, surprisingly, significant similarity was observed including catalytic triad residues (Cys, His, Asn) and a GTP-binding region. The alignment of sequences and a phylogenetic tree also demonstrated that the structure of P. polycephalum TGase is similar to that of TGases of vertebrates. Furthermore, we observed that the purified TGase had GTP-hydrolysing activity and that GTP inhibited its transamidating activity, as in the case of mammalian tissue-type TGase (TGase 2).
Department of Applied Biological Sciences, Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa, Nagoya, Japan.
Fumitaka
F
Wada
Akio
A
Nakamura
Tomohiro
T
Masutani
Koji
K
Ikura
Masatoshi
M
Maki
Kiyotaka
K
Hitomi
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D018076:DNA, Complementary / Q000235:genetics; D016054:DNA, Protozoan / Q000235:genetics; D019143:Evolution, Molecular; D019204:GTP-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism*; D006801:Humans; D008322:Mammals / Q000378:metabolism; D008969:Molecular Sequence Data; D010802:Phylogeny; D016839:Physarum polycephalum; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D015800:Protozoan Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D011503:Transglutaminases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism
D018076:DNA, Complementary; D016054:DNA, Protozoan; D015800:Protozoan Proteins; D011993:Recombinant Fusion Proteins; D006160:Guanosine Triphosphate; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D011503:Transglutaminases; D019204:GTP-Binding Proteins