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Limulus hemocyte transglutaminase. Its purification and characterization, and identification of the intracellular
F Tokunaga1, M Yamada, T Miyata
1Department of Biology, Faculty of Science, Kyushu University, Fukuoka, Japan.
Insights
Researchers purified and characterized a transglutaminase (TGase) from horseshoe crab hemocytes. This enzyme and its substrates may be crucial for the animal
Area of Science:
- Biochemistry
- Molecular Biology
- Immunology
Background:
- The intracellular coagulation cascade in horseshoe crab hemocytes is not fully understood.
- Transglutaminase (TGase) is a potential enzyme involved in gel formation during this cascade.
Purpose of the Study:
- To purify and characterize a TGase from Tachypleus tridentatus hemocytes.
- To identify and characterize TGase substrates within hemocytes.
- To elucidate the role of TGase in the horseshoe crab immune defense.
Main Methods:
- Six-step purification of TGase from hemocytes.
- SDS-polyacrylamide gel electrophoresis to determine molecular mass.
- Enzymatic assays to characterize TGase activity (Ca(2+)-dependence, inhibition by various reagents).
- Dansylcadaverine incorporation to identify TGase substrates.
- Amino acid sequencing of identified protein substrates.
Main Results:
- 1.6 mg of TGase with specific activity of 940 U/mg was purified.
- Purified TGase has a molecular mass of 86 kDa and exhibits mammalian-type II TGase-like properties.
- Two major TGase substrates were identified: an 80-kDa proline-rich protein and an 8.6-kDa cysteine-rich protein.
- The 8.6-kDa protein was cross-linked by TGase into multimers up to pentamers.
Conclusions:
- Limulus hemocytes contain a Ca(2+)-dependent TGase with mammalian-type II properties.
- Identified TGase substrates, particularly the 8.6-kDa protein, are involved in TGase-mediated cross-linking.
- This TGase system likely plays a significant role in horseshoe crab defense against pathogens, analogous to Factor XIIIa in mammalian plasma.
Abstract:
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.
