- Email: [email protected]
Pufification and characterization of the trypsin inhibitor produced by plerocercoids of spirometra erinaceieuropaei
Poster Sessions I Parasitology Imernotionoi 47 (SuppI.) (1998) 283-389
342
P-0822
PURIFICATION AND CHARACTERIZATION OF THE TRYPSIN INHIBITOR PRODUCED BY PLEROCERCOIDSOF SPlROMETRAERNACEEUROPAN
Hirai K, Azechi S, Miura K, Wang H, Tademoto S, Fukumoto S -Department of Medical Zoology, Fatuity of Medicine, Tottori University, Yonago, Japan The trypsin inhibitor was purified from the aqueous extract of the plerocercoids of Spirometra erinaceieuropaei, which were hormo>erilzeb’m .tne-&rriMT i~s+tCt,dH~.b cm-&idt~ 1 .O% triton X-l 00 and then centrifuged at 105000 g for 90 min at 4’C. This inhibitor was purified using gel filtration on a column co?TDYQ?m HW-55 and in turn arion exchange chromatcoQr;aDhytiti MonoP cdumn am3 at k& gA ?&ration with Superdex 75. The trypsin inhibitory activity was measured by the method with casein as substrate. Brieny, the worm extract was preincubated with 2~ of bovine pancreatic trypsin in 0.1 M phosphate buffer pH 7.4 at 35’ C for 10 min and was followed by incubation for exactly 20 min after addition of 0.5 ml of ihe 1.0% (w/v) casein solution as substrate. The reaajon was termjnareb by adtit)on of J.5 ti of 5% Prich)oroacetic acid. 7he reaction mixture was centrifugeb ‘ror 2D nim aI 3DDD 9, 7be exxfInc*mn 0) supernatant fluid was measured at 280 nm. The trypsin inhibitor isolated from the extract of plerocer@d& was estimated as mdecJar wtiight of 27kDa by SDS-PAGE. Surprisingly for it this trypsin inhibitor was cross-reacted against anti-human growth hormone monoclonal antibody and anti-27.5 kDa protein monoclonal antibody prepared from S. mansonoides plerocercoids. Accordim_Qy.‘ttils lriiilljitor was’nom6iogous_to.~tne _c#t&n protease purified as plerocercoid growth factor.
P-0823
F!STABL.ISHMENT OF A MONOCLONAL A.Nl-BODY DIRECTEDTO A GLYCOSPHINGOLIPJD SEGLx ISOLATEDFROM ERINACEIEUROPAE SPIROMETRA
M*, &oiima fE**, Kawakami Y*, Nakamura T***,. Nakamura K**, Ucbida A*, Murata Y*, Tamai Y** *Environmental Biology, Azabu University, ** Biochemistry ***Parasitology, titasato University School of and Medicine, Kanagawa 228, Japan
Yanagisawa
We previously isolated spirometo series glycosphingolipids having a unique carbohydrate core structure [Gabl+tCaIof Spirometra 3)Gk&Cer] from the plerocercoids erinaceieuropaei. To elucidate the bia1agica1 functions of monoclonal ipirometo series glycolipids, a mouse IgM antibody, AK97, was established. The specificity of AK97 dnIoma>Dpraph?Ywas &&etineb ‘DY %im )qer immunostaining and enzyme-linked tmmunoadsorbent assay. AK97 was found to be directed to the non-reducing terminal trisaccharide sequence of SEGLx, ~G~@I”(Fuc~I-~)G~~@~-], and also showed cross-reactivity with Lex antigen with the structure, [Gal~~-4(Fucal-3)GkNAc#cNAcBI-]. By epitopic immuno&is(achcmica~ St&&g, AK97 staiaed almast wicole tissues of S. etinaceieuropaei plerocercoids, with the The staine& pre&ominant\y. bil% tegument immuna&is&n&emi& t?nd@ of t&e staining lability to organic solvent and the results of Western blot analysis of the plerocerwid glycoproteins evidenced that the antigens in the tapewe wc SEGIS; %& WSEGLS;, &c-s 4%viq the above epitopic structure. Considering that the tapeworm directly contacts with its host tissue through the tegument, the membrane surface of which is exposed to external environments, it is suspected that SEGLx and GalSEGLx on the tegument play functionally important roles in the hostparasite interactions.
P-0824
GLYCOSPHINGOLIPIDS PSEUDOPHYLLIDEAN TAPEWORMS
lriko H*, Yanagisawa M*, my’, Y*, Kojima H**, Natcunum K**,
IN
UchidaA*,
Murata
Tamai Y** *&p&m~%dEnvi %Xmenti BjdEgy, A& UtiYe&y arrd **Department of Biochemistry, Kitasato University School of Medicine, Japan aLL-_utliq&.Ri &lr &z .qXzz&? rtr Lk rirv&tnr’ lir JLr mediation of host-parasite interactions, as are functioning in bacterial and viral infection. In this context, we studied giycosphingolipids of m&5 to e&?&&5 W&-Q@ tiaz?remi& me&m&s of @+Wz&ii We pr&~ly r&s&~? mu+i5 e;l%cnrplhirgofi@ii, SEGLx and GalSEGLx from the tapeworm Spirometra erkcei plemrtids: they were &uacCerized by a carbohydrate structure
Gall_4(Fucl-3)Glc-. We proposed a term “spirometosides” for glycotipids having this carbohydrate structure. Recently, we successively &&i&d a mouse monaztonal 1gM antibody directed to SEGLx, and termed AK!97. TLC-immunostaining using AK97 EV&& hrgt: fzzt%iviv of AK97 to glvaoipiak fii
Pseudoplryllideantapeworms, S. en&& #+&W&zeH& By iri?riiY&?&~~
and D@izy&J&otMbm snadq, rcsing ‘4Kfl a
positive fluorescence was observed in almost whole tissues of these tapeworms, showing the overall distribution of “spirometosides” in &!zervcwm tissues. On & o&r &d, we fmmt &e absentre of tiese @y&i@& in Cyc&.&yWeatz ~~F-SKHRW. Thus. qimtnetosides can be implied to have a taxonomical significance, being characteristic of Pseudophyllidean tapeworms.
P-0825
m OF TEMPERATURE ON FATTY ACID COMPOSITION OF SPZROMHR.4 ERLNAC.EIEUROPAEI PLEROCERCOID
Fukushima ‘T, Gao T, Isobe A, Hojo N, Shiwaku K, Yamane Y - Dept. of Envvonmental Medicine, Shimane Medical University, Izumo. Japan k~boductirro - In general, Diphyllobothriid larva in fish which has lower body temperature with ambient lower composition of arachidonic acid develops to adult worm in humane. However, Spirometra erinrc&u?sp.& plemeercvids cause sparganoeis in humans. When the plerocercoid change the host from one with lower temperature to other with higher temperature and from higher arachidonic acid circumstance to lower one, the drastic ambient changea to the larvae should be important for clarifying the cuee of the eparganoeie. We observed the effects of temperature and host fatty acids on the fatty acid composition of the plerocercoid. and discuesed thoee rolea on sparganoeis. Met&f&? - Plwxemoids of S. erinaceikmpaei were collected from striped snakes. Fifty of the pie xdxrcaide were divided inta two pupa and the each group was incubated in 2 ml of host snake eerum for 24 h at 18 C or at 37’C, respectively. Another 100 plemcercojdswere divided into four groups and were incubated in 2 ml of steriliaed .S&G&girA rariline ax&G& 5 & *z=&&z&- & & 2.0 .&& BSA or of &%ili~ed physiological aaline alone at 10 ‘C or at 37 C for 0, 0.5, 1, 3, 6 h, respectively. The extraction of fatty acida in phoapholipid and triglyceride fractions, the derivatization and the gae ~~~&&ana@G~ were pertbrmed 88 the previous methodC13. ~cu~bloll- After 24 h incubation at 37 ‘C in host snake serum, w 6 eerie8 fatty acids, especially arachidonic acid in phoepholipid frection of the pk+rocezr+d decnzaaed aomp~~& titb those of the plerocercoid incubated at 18T in epite of an abundant arachidonic acid circumstance. A&r incubation with 5 mM arnchidonic acid at lo’% or at 37 ‘C,. in J&q&&id &w$i&\ L&wls$ .N\ A?.~ & sh_? a-sb.S& acid oompasicion wae observedat both f.empemturee. The amwut af erachidonic acid in triglyceride in the incubated plerooercoide at 37 t: increased rapidly after incubation and the maximum amount of it was observed aL about 2 h. thereafter, it decreaeedgradually. Whereas thnt amount ‘in tiitjlyceride in the incubated plerocereoide at 10 ‘C increased elowly and kept growth continuously from incubation beginning to 6 h. These results might suggest when plerocercoidn change the host from one with lower temperature to other with higher temperature, they first actively absorb and modify the exogenous aracbidonie acida, after 2-3 h. the absorbed arachidonic acids could be metabolised to others, such aa prc&xlandma[21 which should be imoortant for causing larva miprans.
-lfi?fi?*s
[llrJ&l&ima T. et .¶I Intematianal daun$ for PualJitolo#y 1996: 26 15-21. [21FukuabimaT. et al. Pamsitokqy Research 1993; 79: 624428.
342
P-0822
PURIFICATION AND CHARACTERIZATION OF THE TRYPSIN INHIBITOR PRODUCED BY PLEROCERCOIDSOF SPlROMETRAERNACEEUROPAN
Hirai K, Azechi S, Miura K, Wang H, Tademoto S, Fukumoto S -Department of Medical Zoology, Fatuity of Medicine, Tottori University, Yonago, Japan The trypsin inhibitor was purified from the aqueous extract of the plerocercoids of Spirometra erinaceieuropaei, which were hormo>erilzeb’m .tne-&rriMT i~s+tCt,dH~.b cm-&idt~ 1 .O% triton X-l 00 and then centrifuged at 105000 g for 90 min at 4’C. This inhibitor was purified using gel filtration on a column co?TDYQ?m HW-55 and in turn arion exchange chromatcoQr;aDhytiti MonoP cdumn am3 at k& gA ?&ration with Superdex 75. The trypsin inhibitory activity was measured by the method with casein as substrate. Brieny, the worm extract was preincubated with 2~ of bovine pancreatic trypsin in 0.1 M phosphate buffer pH 7.4 at 35’ C for 10 min and was followed by incubation for exactly 20 min after addition of 0.5 ml of ihe 1.0% (w/v) casein solution as substrate. The reaajon was termjnareb by adtit)on of J.5 ti of 5% Prich)oroacetic acid. 7he reaction mixture was centrifugeb ‘ror 2D nim aI 3DDD 9, 7be exxfInc*mn 0) supernatant fluid was measured at 280 nm. The trypsin inhibitor isolated from the extract of plerocer@d& was estimated as mdecJar wtiight of 27kDa by SDS-PAGE. Surprisingly for it this trypsin inhibitor was cross-reacted against anti-human growth hormone monoclonal antibody and anti-27.5 kDa protein monoclonal antibody prepared from S. mansonoides plerocercoids. Accordim_Qy.‘ttils lriiilljitor was’nom6iogous_to.~tne _c#t&n protease purified as plerocercoid growth factor.
P-0823
F!STABL.ISHMENT OF A MONOCLONAL A.Nl-BODY DIRECTEDTO A GLYCOSPHINGOLIPJD SEGLx ISOLATEDFROM ERINACEIEUROPAE SPIROMETRA
M*, &oiima fE**, Kawakami Y*, Nakamura T***,. Nakamura K**, Ucbida A*, Murata Y*, Tamai Y** *Environmental Biology, Azabu University, ** Biochemistry ***Parasitology, titasato University School of and Medicine, Kanagawa 228, Japan
Yanagisawa
We previously isolated spirometo series glycosphingolipids having a unique carbohydrate core structure [Gabl+tCaIof Spirometra 3)Gk&Cer] from the plerocercoids erinaceieuropaei. To elucidate the bia1agica1 functions of monoclonal ipirometo series glycolipids, a mouse IgM antibody, AK97, was established. The specificity of AK97 dnIoma>Dpraph?Ywas &&etineb ‘DY %im )qer immunostaining and enzyme-linked tmmunoadsorbent assay. AK97 was found to be directed to the non-reducing terminal trisaccharide sequence of SEGLx, ~G~@I”(Fuc~I-~)G~~@~-], and also showed cross-reactivity with Lex antigen with the structure, [Gal~~-4(Fucal-3)GkNAc#cNAcBI-]. By epitopic immuno&is(achcmica~ St&&g, AK97 staiaed almast wicole tissues of S. etinaceieuropaei plerocercoids, with the The staine& pre&ominant\y. bil% tegument immuna&is&n&emi& t?nd@ of t&e staining lability to organic solvent and the results of Western blot analysis of the plerocerwid glycoproteins evidenced that the antigens in the tapewe wc SEGIS; %& WSEGLS;, &c-s 4%viq the above epitopic structure. Considering that the tapeworm directly contacts with its host tissue through the tegument, the membrane surface of which is exposed to external environments, it is suspected that SEGLx and GalSEGLx on the tegument play functionally important roles in the hostparasite interactions.
P-0824
GLYCOSPHINGOLIPIDS PSEUDOPHYLLIDEAN TAPEWORMS
lriko H*, Yanagisawa M*, my’, Y*, Kojima H**, Natcunum K**,
IN
UchidaA*,
Murata
Tamai Y** *&p&m~%dEnvi %Xmenti BjdEgy, A& UtiYe&y arrd **Department of Biochemistry, Kitasato University School of Medicine, Japan aLL-_utliq&.Ri &lr &z .qXzz&? rtr Lk rirv&tnr’ lir JLr mediation of host-parasite interactions, as are functioning in bacterial and viral infection. In this context, we studied giycosphingolipids of m&5 to e&?&&5 W&-Q@ tiaz?remi& me&m&s of @+Wz&ii We pr&~ly r&s&~? mu+i5 e;l%cnrplhirgofi@ii, SEGLx and GalSEGLx from the tapeworm Spirometra erkcei plemrtids: they were &uacCerized by a carbohydrate structure
Gall_4(Fucl-3)Glc-. We proposed a term “spirometosides” for glycotipids having this carbohydrate structure. Recently, we successively &&i&d a mouse monaztonal 1gM antibody directed to SEGLx, and termed AK!97. TLC-immunostaining using AK97 EV&& hrgt: fzzt%iviv of AK97 to glvaoipiak fii
Pseudoplryllideantapeworms, S. en&& #+&W&zeH& By iri?riiY&?&~~
and D@izy&J&otMbm snadq, rcsing ‘4Kfl a
positive fluorescence was observed in almost whole tissues of these tapeworms, showing the overall distribution of “spirometosides” in &!zervcwm tissues. On & o&r &d, we fmmt &e absentre of tiese @y&i@& in Cyc&.&yWeatz ~~F-SKHRW. Thus. qimtnetosides can be implied to have a taxonomical significance, being characteristic of Pseudophyllidean tapeworms.
P-0825
m OF TEMPERATURE ON FATTY ACID COMPOSITION OF SPZROMHR.4 ERLNAC.EIEUROPAEI PLEROCERCOID
Fukushima ‘T, Gao T, Isobe A, Hojo N, Shiwaku K, Yamane Y - Dept. of Envvonmental Medicine, Shimane Medical University, Izumo. Japan k~boductirro - In general, Diphyllobothriid larva in fish which has lower body temperature with ambient lower composition of arachidonic acid develops to adult worm in humane. However, Spirometra erinrc&u?sp.& plemeercvids cause sparganoeis in humans. When the plerocercoid change the host from one with lower temperature to other with higher temperature and from higher arachidonic acid circumstance to lower one, the drastic ambient changea to the larvae should be important for clarifying the cuee of the eparganoeie. We observed the effects of temperature and host fatty acids on the fatty acid composition of the plerocercoid. and discuesed thoee rolea on sparganoeis. Met&f&? - Plwxemoids of S. erinaceikmpaei were collected from striped snakes. Fifty of the pie xdxrcaide were divided inta two pupa and the each group was incubated in 2 ml of host snake eerum for 24 h at 18 C or at 37’C, respectively. Another 100 plemcercojdswere divided into four groups and were incubated in 2 ml of steriliaed .S&G&girA rariline ax&G& 5 & *z=&&z&- & & 2.0 .&& BSA or of &%ili~ed physiological aaline alone at 10 ‘C or at 37 C for 0, 0.5, 1, 3, 6 h, respectively. The extraction of fatty acida in phoapholipid and triglyceride fractions, the derivatization and the gae ~~~&&ana@G~ were pertbrmed 88 the previous methodC13. ~cu~bloll- After 24 h incubation at 37 ‘C in host snake serum, w 6 eerie8 fatty acids, especially arachidonic acid in phoepholipid frection of the pk+rocezr+d decnzaaed aomp~~& titb those of the plerocercoid incubated at 18T in epite of an abundant arachidonic acid circumstance. A&r incubation with 5 mM arnchidonic acid at lo’% or at 37 ‘C,. in J&q&&id &w$i&\ L&wls$ .N\ A?.~ & sh_? a-sb.S& acid oompasicion wae observedat both f.empemturee. The amwut af erachidonic acid in triglyceride in the incubated plerooercoide at 37 t: increased rapidly after incubation and the maximum amount of it was observed aL about 2 h. thereafter, it decreaeedgradually. Whereas thnt amount ‘in tiitjlyceride in the incubated plerocereoide at 10 ‘C increased elowly and kept growth continuously from incubation beginning to 6 h. These results might suggest when plerocercoidn change the host from one with lower temperature to other with higher temperature, they first actively absorb and modify the exogenous aracbidonie acida, after 2-3 h. the absorbed arachidonic acids could be metabolised to others, such aa prc&xlandma[21 which should be imoortant for causing larva miprans.
-lfi?fi?*s
[llrJ&l&ima T. et .¶I Intematianal daun$ for PualJitolo#y 1996: 26 15-21. [21FukuabimaT. et al. Pamsitokqy Research 1993; 79: 624428.