Limulus hemocyte transglutaminase. Its purification and characterization, and identification of the intracellular substrates.
To investigate further the molecular events of the intracellular coagulation cascade in limulus hemocytes, a transglutaminase (TGase), which may be involved in the formation of a stabilized gel, was purified and characterized. Through the purification procedures consisting of six steps, 1.6 mg of TGase with a specific activity of 940 amine incorporation unit/mg was obtained from 32.4 g of Tachypleus tridentatus hemocytes. The purified TGase gave a single band on SDS-polyacrylamide gel electrophoresis with a molecular mass of 86 kDa, and demonstrated mammalian-type II TGase-like enzymatic properties. The TGase activity was Ca(2+)-dependent and was inhibited by primary amines, EDTA, and SH-reagents. Moreover, two major potential substrates for TGase were identified in the hemocyte lysate by using dansylcadaverine (DCA) incorporation in the presence of 10 mM CaCl2 and 10 mM dithiothreitol. Of these protein substrates, an 80-kDa protein contained a large number of proline residues, amounting to about 22% of the total amino acids. On the other hand, an 8.6-kDa protein abundantly present in the hemocytes was characterized as a Cys-rich protein consisting of 81 amino acid residues and a calculated molecular mass of 8,671. The entire amino acid sequence of this protein was established. Also, the 8.6-kDa protein was readily cross-linked intermolecularly by TGase, forming multimers as large as pentamers. We speculate that like plasma factor XIIIa, limulus TGase and its two protein substrates in the hemocytes may play an important role in the defense of this animal against invading microorganisms.
Tokunaga
F
F
Department of Biology, Faculty of Science, Kyushu University, Fukuoka, Japan.
Yamada
M
M
Miyata
T
T
Ding
Y L
YL
Hiranaga-Kawabata
M
M
Muta
T
T
Iwanaga
S
S
Ichinose
A
A
Davie
E W
EW
D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004229:Dithiothreitol / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D006458:Hemolymph / Q000166:cytology / Q000201:enzymology; D006737:Horseshoe Crabs / Q000201:enzymology*; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011506:Proteins / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity; D011503:Transglutaminases / Q000302:isolation & purification* / Q000378:metabolism*
D011506:Proteins; D011503:Transglutaminases; D002118:Calcium; D004229:Dithiothreitol