Tetrodotoxin-binding protein in the haemolymph of shore crab Hemigrapsus sanguineus

• Project/Area Number: 11660201
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Fisheries chemistry
• Research Institution: Tokyo University of Fisheries
• Principal Investigator: NAGASHIMA Yuji Tokyo University of Fisheries, Department of Food Science an Technology, Associate Professor, 水産学部, 助教授 (40180484)
• Project Period (FY): 1999 – 2000
• Project Status: Completed (Fiscal Year 2000)
• Budget Amount: ¥3,500,000 (Direct Cost: ¥3,500,000); Fiscal Year 2000: ¥1,200,000 (Direct Cost: ¥1,200,000); Fiscal Year 1999: ¥2,300,000 (Direct Cost: ¥2,300,000)
• Keywords: tetrodotoxin-binding protein / tetrodotoxin / shore crab / haemolymph / neutralizing activity / metal ions / イソガニ / タンパク質 / 精製

Research Abstract

A shore crab Hemigrapsus sanguineus is reported to be resistant to tetrodotoxin (TTX) administrated intrahemocoelically, although it contains no detectable amount of TTX.This research project was carried out to clarify the mechanism of high resistance of the crab H.sanguineus to TTX.Analysis by gel filtration on Sepharose 6B revealed that the haemolymph contained TTX-binding proteins with mol.wt.of >2000k and 4OOk. One of the TTX-binding proteins (mol.wt.of 40Ok) was purified, monitoring the neutralizing effect against the lethal activity of TTX to mice, by ultrafiltration using a 500k mol.wt.cut-off filter membrane, lectin affinity chromatography on a ConA-Sepharose column, and gel filtration HPLC on a TSKgel G3000 column. The protein thus obtained gave only one band in native-PAGE, confirming its homogeneity. The mol.wt.of the TTX-binding protein was estimated to be about 82k under non-reducing conditions and about 72k and 82k under reducing conditions by SDS-PAGE, indicating that th e TTX-binding protein was composed of at least two subunits which appeared to have no neutralizing effect to TTX.The TTX-binding protein was an acidic gycoprotein with pI 3.5, abundant in Asp and Glu but absent in Trp, and contained 6% reducing sugar and 12% of amino-sugar.
The TTX-binding protein selectively bound to TTX, with a neutralizing ability to TTX of 6.7 mouse unit/mg protein. However, its neutralizing activity was almost lost by treatments with enzymes (trypsin, protease XIV, amylase and amyloglucosidase) and chemical detergents (1% SDS, 1% dithiothreitol, 8M urea and 6M guanidine hydrochloride), suggesting the involvement of both proteineous and sugar moieties in the binding to TTX and the importance of the steric conformation of the TTX-binding protein. Moreover, the neutralizing activity was significantly inhibited by pre-incubation of the TTX-binding protein with metal ions (Cu^<2+>, Cr^<6+> and Zn^<2+>). It is possible that the TTX-binding protein functions as a metal carrier protein in the haemolymph of the non-toxic shore crab H.sanguineus.