Biochemical characterization and cloning of transglutaminases responsible for hemolymph clotting in Penaeus monodon and Marsupenaeus japonicus.
To investigate the shrimp blood clotting enzyme, a transglutaminase in the hemocytes of Penaeus monodon (abbreviated as TGH) was purified. TGH is an abundant homodimeric cytosolic protein with 84.2 kDa subunits. It clotted shrimp plasma and incorporated fluorescent dansylcadaverine into succinyl casein upon activation by CaCl(2) in vitro. IC(50) for the activation was 3 mM, which is below the shrimp plasma Ca(2+) level. Showing similar properties as other type II transglutaminase, TGH was particularly unstable after activation. MALDI-TOF/TOF mass-analyses of tryptic peptides of P. monodon TGH confirmed its identity to STG I (AY074924) previously cloned. A possible allele of the other isozyme STG II (AY771615) has also been cloned from the P. monodon cDNA and designated as PmTG. The predicted PmTG protein sequence is 58% similar to that of STG I and 99.2% to that of STG II. Likewise, a novel enzyme Mj-TGH was purified and cloned from Marsupenaeus japonicus hemocytes. Results of sequence alignment and phylogenetic analyses of these transglutaminases suggest that STG I and Mj-TGH are 83% identical and orthologous to each other, while PmTG/STG II and a previously cloned M. japonicus transglutaminase (AB162767) are their paralogs. Protein of the latter two could not be isolated, their regulated expression was discussed.
Yeh
Maw-Sheng
MS
Department of Food and Nutrition, Hung Kuang University, Sha Lu, Taiwan.
Kao
Ling-Rong
LR
Huang
Chang-Jen
CJ
Tsai
Inn-Ho
IH
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001777:Blood Coagulation / Q000502:physiology; D002118:Calcium / Q000737:chemistry; D002384:Catalysis; D002413:Cations, Divalent / Q000737:chemistry; D003001:Cloning, Molecular; D004795:Enzyme Stability; D015971:Gene Expression Regulation, Enzymologic; D006434:Hemocytes / Q000737:chemistry / Q000201:enzymology; D006458:Hemolymph / Q000201:enzymology / Q000502:physiology*; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D008970:Molecular Weight; D033561:Penaeidae / Q000201:enzymology* / Q000235:genetics; D010449:Peptide Mapping; D010802:Phylogeny; D017386:Sequence Homology, Amino Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D011503:Transglutaminases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
D002413:Cations, Divalent; D011503:Transglutaminases; D002118:Calcium