Basseri & Ratcliffe 2004.pdf

7/23/2019 Basseri & Ratcliffe 2004.pdf

1/8

Medical Joum.1 f thIs Rpub rn

IN VTRO INHIBITI ON OF ATTACHMET OF T H

MRICAN TRYPANOSOME, RYPASOMA

GLION HE SALIVARY LAND F I TS

VETOR, RHODIUS PROLUS, US TEPAASITE EXTACT AN PARTI AL PURFCATIN

OF AN ATTACHMENT INHIBTOR PO TE IN

" ASSE! Ph* AND NA AF **

From he *Department 0/Medicl Enoolo School 0/Public Helth

Tehn Universi 0/Medicl Sciences, ? Box 4-44 ehrn, Irn, nd he

*iomedicl nd Physiologicl Reserch Group Shool oiologcl Sciences niersi o/Wle

Swne Singleon Prk Swnse SA2 8P? Unied Kingdom.

ABSTACT

The life cycle of the American trypanosome, Typansma range, in its nvrt

brate host Rhdnus prus, is completed by invasion into th insct salivary glans.

As a prrquisit ther is an initial rcognition othe savary gland rceptors for th

adhsion/invasion by range. Threfore the prpose of this stdy was to nsti

gat th putatve recptor molculs on thr th salivary glan or parasits surfacs

which mediate the adhson/inason Initially the possibe aggltination actiity of

range pimastigotes against bloo cell was examind Then in orer to inestigate

the role of any rceptor igands on the srac o range in ahesionnasion of the

parasit GlcNc as preferntially s to isoate a ligand protin. 60ka proteinbond to the GlcNAc comn and was isolated om range (Tr-60) an the purity

of ths proten was conre by rers phase HLC Tr-60 also xhibtd a potnt

parasit nhibitory effect ndicating that this protin most probaly a glycoprotein may

act as a lgan receptor vv, the developmnt an invasion of rnge into th

saiar gand was not interfere with by the presence of a sugar inhibitor of th R

pus hemolymph lectin

JR V N2 Keyords: nosom rngeli Rhodnius prolius Svr gldsronstchn

V 8Nb 2S 83Ag 04

INODUCION

rangeli eeds esset perod o deeopmetbeore so to the sr gd d the ogepmstgote orms re resposbe or so hedheso hbto o angeli oto polixus s

r gds usg sugrs reeed tht some crbohdrtebdg protes o the surce o the trposome or gd cted s gds medtg the prstedheso Thereore, ths stud, sugr bdg prote ws used to pur some gd protes.

spdc: H.R Baseri, Deparment of Medcal Etoo-ogy Schoo o Pubc Halth Teran Unv o Mdcal ci-ce PO ox 1455-6446. a, Ia Eail: HYPRLNK

"aloHa_bar@hoaco Haidbahoaco1

Prste dheso/so s e to be preced b specc receptorgd terctos be-twee moetes o the ce surce o the prstesd moecues o ector tssues' Such suce receptors re begg to be chrtered or Try-


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nibion of Aacn onR

paooma crzi ths prste, three feccestges re chrctered b sotg gcostedprotes from ther cesurfce d hbto of r uzi so ce cuture b tbodesstrog suggested tht t est oe of these

popeptdes coud be the receptor for ruzi th e host ce 1 A gcoprote o the surfce of ruzi s oed dheso/so of the ruzito the mmm ce bod hs gcoprote wthmoecur weght 82D, s expressed thetrpomstgotes of ruzi d t s so essetfor phsc moemet d tur trsmsso of uzi.8

I ddto, the edece for the so of the frc trposome, raooma bruei, wth the ertebrte host woud suggest tht the he somemes of recogsg d dherg to oe or moregds the ector Proccc moecues (gcopro

tes o the surfce of the epmstgote forms of theprste) ow the trposome to bd to ect tpemoecues wth the ector

ects p roe ttchmet of some prstes tothe host ces e.g ect ws foud Etamoebahitoltia whch medtes dheso of trophootesto mooers of hum ces he other possbe fuc-to of ects protoos cude bdg to redbood ces b Plamodiumfaliparum cetocetercto wth Crptopor idi um parv2Q d determto of the prtcur ste of fecto wth Eimeriaspeces Chtterjee et al (2003) foud ecte

compoet wth broder specct o the surfce of Lamazoe promstgotes Schotteus (992) so re-ported the presece of ect ctt promstgotesof 18 strs of eshm but ot mstgotes d,subsequet, Sobodo et al. ( 1997) detected hemggutto ctt 16 strs of eght eshm speces I ddto, the reported tht the cubto ofstes tempertures up to 100C hd o effect ohemggutto cttes

hs stud ws coducted m UK d prtIr 200 12002 d the purpose of t ws to est-gte the roe of receptor gds o the surfce of

rageIi ttchmetso of the prste. I pr-tr, the hbtor effect of the prstes prote ex-trct to terferg wth specfc steps the so ofthe sr gds hs bee exmed

I preous stud t hs bee show tht GcNcshowed hgh hbtor effect prtcur to the sugrprecubted prste hus, GcNAbdg protes exmpe m hep to further crf the mechsmof prstes so of the host ce From ths pot ofew, the preset stud, t hs so bee tred to purfd chrctere ths receptor from the surfce of rageIi og epmstgote form

160

MA AND MTHODS

Parasites cultur

rageIi ejer 1920, str Choch ws suedbDr. G. Schub (erst ofBochum, e r

posomes were ted t 2"C Om stere cetrfuge tubes cotg I cuture medum [O68MNC, 0004M K, 0O6 N4 0.01 gu cosemg m trptose, mg m IB (er fuso broth,DFC) d 0.0mg m hem (Sgm Co orsetUK)] suppemeted wth 20% hetctted fet cfserum (FCS) (e d Youg 1988)

Insect and dissetion of salivary glands

proiu ws rered s descrbed b mbu dGrc ( 1997) d fed through prfm membre odefbrted horse bood (CS Mcroboogcs td,Botoph, Cdo, Bucghm, UK) eer O dsd occso th rbbt bood (Hr Ser b td,oughbrough, U or fresh hum bood he srgds, the sects were estheted o ce for 0 md the ther heds were hed rm wth o. forceps BS Swterd) d mmersed OmM PBS (cotg 1OmM NC d OmM sodum phosphte t p 74d380 msm) or BS OOmM rs, pH 73, 300 msm, 0mM NC) he hed d bdome were the getpued prt h s owed ccess to the sr gdducts, whch remed ttched to the hed. he gdswere the seprte d from the hed

reparation of rngeli extrctshe epmstgotes og forms of ragelifrom three

wee d short forms from oe wee od I medumwere hrested d wshed twce wth PBS (cotg0mM NC d 1OmM sodum phosphte t pH 7) bcetrfugto t 0000g IC (CetrM cetrfuge, Iterto qumet Co US) for 10 mutes hepeet ws resuspeded OO PBS to ge co-cetrto of 2 0 ces/m Becuse of hrd mem-bre d bse o preous tests the suspeso ofprstes ws hoogesed wth utrsoc dstegrto soctor (Utrsoc Dstegrtor! O wtt, MS,

UK) for 1 mute d the spu dow t OOOOg for 10mutes d th e extrcts were froe t 20 ut use

Hemglutination assay

wofod ser dutos of og d shortepmstgote for extrct were prepred stere 60 wemcroptes ( ) (Nuc, Gbco, UK) to ge duto rge of 12 to : 1024. Fe mcroters of %rbbt erthroctes PBS were dded to ech we dthe mxtures were gtted for 1 mute d the cu-bted for 2 hours t room temperture he ed potsof ggut were exmed uder stereomcroscope


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HR Bass N Raci

and y e ow caaceiic o e eyocye pe-e wen e pae wa ed a an ange A cono OOL o e eac wa eainaciaed o 20 inuea 00" A ape ee pepaed 3 ie and eaay epeaed eac ie.

Aahmen inhibiin aa ing rangeli exra

Te aiay gand of 4 and 5 ina nyp o polxu 4 gand eac ie) wee dieced a aoeand en peincuaed wi e eac o ngl o ou a oo epeae. Te aiay gand weeen quicky waed and incuaed wi e iing y-panooe ( 6 ce/L in PBS o ou a 5Eac aiay gand wa waed in PBS and e aacedypanooe wee couned y couning fo e uppe uace aea o e poeio and aneio oe oe aiay gand pependicuay o e iewing di-

ecion uing 00 quae in an eyepiece gid gaicueeauing 00 . ono conied o e ae nue of e aiay gand a ad een peincuaedwi eainaciaed ypanooe eac and uneaedaiay gand in PBS e epeien wa epeaede ie wi 2 epicaion eac

arial purain f a rangeli aahmen prein

ngl epiaigoe ong fo wee aeedo a age oue (5000 L of3 week od L cuueediu y cenifugaion a 4000g (Hig Speed 8 MSEK) o 30 inue a 4 e pee wee eupendedin OM PBS (pH 4; 380) and waed ee ie

y ceniugaion a OOOOg fo 5 inue eac and eneupenion in L PBS conaining % odiudodecyupae (SDS ypanooe wee oogenied wi an uaonicao (Uaonic Diinega-o50 wa, MSE UK and ceniuged a 0000g eupeaan wa en appied o a cNAcagaoe co-un fo poein puicaion ie iiie o e upenaan wee oaded ono a L cN Acagaoe co-un (Siga o Doe, U y adding L upenaaneac ie and incuaing o eac a R Te counwa en waed wi 0 L PBS pH 4 foowed y 20 o e ue conaining 05M and M Na eac,

and nay wi 0L o PBS pH 4 e ound po-ein wee eued uing 5L o g/L JcNAc eeued poein wa concenaed and waed wi eufe o eoe e cNAc uing a Cenicon 30 con-cenao (Aicon nc Beey SA) a 000 g a ooepeaue e ape wa n vto aacen inii-ion aay a aoe SDSpoyacyaide e Eeco-poei and Reee pae Hig Pefoance Liquidoaogapy (HPLC

SDS-larlide Gel Elerphrei

Te eued poein wa appied o SDSPAE a di

cued y Laei ( 0) weny icoie o ngl aacen poein wa concenaed ie byeezedying (appoiaey 0204 g/L and, ub-equeny in ode o denaue e poein e poeinwa ied wi 5L of 5 SDSPAE apee unde

noneducing condiion and en eaed o 0 inuea 00C e ape (20L wa en cenifuged a1 OOOOg fo 5 in o eoe dei efoe oading inoe ge Te upenaan wa appied o SDSPAE eec-opoei in a 0% poyacyaide ge o anaye eeued poein Eecopoei wa peoed a a conan oage o 200 o ca 45 in uing e ioadinipoean appaau (BioRad aoaoie LDHee epead Heoie K) Ae eecopoei, e ge wee ed wi 40% eano pu 0% aceicacid o 40 inue Deecion o e poein and waipoed y aining e ge wi 02% (w/ ie ni-

ae a decied y aa 84) inay e gewa deeoped in 5% (w/) odiu caonae conain-ing 004% (/) foain o caiaion o oecuaa, e wide ange peciion poein andad (pe-ained) ake ki (BioRad wa ued and e acuaoecua weig wa eaued y eneniu e Docu-enaion and Anayi Sye (Syngene aidge UK

igh erfrane Liquid Chragraph (LC)

HPL anayi wa caied ou uing a LDC/MionRoy appaau coniing o a pecoonio D,conaeic pup and a C4 00 o COP inegaouing a SperioDS C

anayica coun (0.34c)

a a ow ae o Lin o ceck puiy o e euedpoein fo e cAcagaoe coun e oiepae wa 50% N 50% H conaining %iuooaceic acid (TA (Siga o Doe, enicoie of e eued poein wa injeced and epuiy o e poein ouion wa deeced a a wae-eng o 220 n

RESULTS

Heaggutination assay

Te ypanooe eac owed a weak eaggu-

inin aciiy (ie < ) again ai ed ood cen cona no eagguinin aciiy wa oeed ine eac o o epiaigoe and o e eued po-ein Te ea eaen of e ong epiaigoe eaca OOC did no copeey deoy e agguinin aci-iy (ae )

Attachent inhibtion assay usng Nngeli extract.e eac of ngl epiaigoe ong o

owed a ig iniioy effec (85% educion in copaion wi e PBS cono) on e aacen of iepaaie o e uface o pol a iay gand


4/8

Ihbo of aht oR prolixus

Tab mlutntn tty f nge xtrt (n nd srt pmsts rm nd th lud prt Tr6) n rm

xtr 'm lArs lumn ns rt rytrys

Extract ourc Exp.1 Exp.2 Ep3Concntration End Concntration En Concntration En

mgmL point mmL poit mmL poit

n pmsts 65 3-4 195 3-4 28 3-4

ntrl .5 23* 195 1 2.8 2*

tnttd

Tr6 ND 1 83

Srt pmsts 2.5 228 2

BSA 2% 2% 2

*T ntrl ws nttd y htn t 1C r 23 mns ND nt dn

Tabl n r tthmn ntn ssy An xt f T rnl ws dd n lm NA

Ars lmn NAndn prn (Tr ws ud sn m/ lNA n m BS

T utd prn Tr6 s wl s rnl xr wr usd tr th dnus Sly lnds nd

wshd T slry nds wr n rtd wh th ln prsts fr ur nd t numbr

thd rypnsms n sry lnd ws ntd undr l mrsp

*T mnmm nd mxmm prtn nnrtn usd r ttmn f t lnds

**Th numr f td prsts s sn ntly lss


5/8

HR. Bsi and N Ratclie

250

0

150] 1,z

50

On .xf Boiled orgutc

ol T-60

ig l hm h ssy Th f

angei h p lu 60 fm GNAs

lum us u R. pl"w sl ls Th

sl ls h h h l pss f hu h um f h psms h sly

ls u u lh msp Cls ss

f l l p, lu p BS p

.

* Th um f h pss s sly lss


6/8

Ihbo of ngeahme oR pxus

ahein inhibitin by blking the reeptr n thealivary glan urfae Thi pre wa pei t theextrat the epiatigte lng fr a n uh inhi-bitin urre with the hrt fr f the paraiteThee reult iniate that nge lng epiatigte

pe the apprpriate ligan fr bining t poxu alivary glan, whih eiate the initial at-tahent f the paraite In fat, in eah paraite tage,pei leule aiate with the urfae ebraneare t prbably invlve in interatin with theht previuly ha been eribe, in the inver-tebrate phae f nge nly lng epiatigte anattah t the urfae f poxu alivary glan an,thi pre an be blke by preinubatin f para-ite r alivary glan with peifi ugar

Several funtin have been aribe t ugarbin-ing prtein n the urfae f prtzan, I ne f the

ain rle being the interatin between thee leulea reeptr with ht ell urfae arbhyrate, uringparaite attahentinvain. ne f the aniatefr the attahent leule ay be letin whih areknwn t eiate elltell interatin Thee l-eule have been reprte fr everal paraiti prt-zan uh a Lehn peie pratigte anaatigte, Poupu J hoonoeu' an Enoeb ho

In the preent tuy, the extrat f lng epiatigtef nge hwe lw agglutinin ativity againt rab-bit bl el that the attahent leule aywell be letinlike in thi paraite. The effet f arbhy-rate inhibitr n attahent f e prtzan their ht ell ugget the preene f letin r letin-like leular ativity in the Prtza Several authrhave tuie urfae prtein reeptr in prtzan -hein. r exaple, the 82kDa glyprtein n the ur-fae trypatigte f u whih i invlve inaalian ell invain. ungery 85) al reprtethat pu erzite ha letinlike prtein ntheir urfae that bun t re bl ell arbhyratereeptr an the erzite invain in vitr ul bepeially blke y GlNA GalN an NeuNAReently, three erzite urfae prtein have been

ientie that are pei fr GNA bining an ulbe invlve in the ining f erzite t theglynjugate n t he urfae f the re bl ell

It ha been hwn the preene f ix ugar n theurfae f poxu alivary glan an al theugar were eplye fr the attahent inhibitin aay in vitr by authr The reult hwe ngeahein wa blke trngly by GNA iniatingthe preene f pei ligan n the paraite an alivary glan. Therefre, n the hypthetial pibilitythat the attahent prtein ay bin t a ugar afnitylun, in the preent tuy, GN wa preferentially

64

hen t purify ligan prtein fr nge ing GlNagar anity lun The ilate rteinha apparent leular a at 60 Da by SDAGan it purity w nre with P ig. Thiprtein even at l nentratin 0.08 g), we

attahent inhi itry effet ig ) uggeting tat nge reeptr peially interat with glyylatealivary glan rfae leule uh a GlN i-etie whih are reent n the alivary glan I n ai-tin, the reult hwe that the leule auing theinhibitry effet fr bth the trypane extrt anthe Tr60 were t pletely heatenitive

WGAlabell letin ha a high anity fr GNAexhibite trng n pei labelling f the alivaryglan baal ebrane an ell layer Thi nteratinay nr th previu ning, iniating the highinhibitry effet f preinubating paraite in GN

an ubequent experient, eribe here, theinteratin f ti ugar with prtein reeptr n theurfae f e Thi interatin wa nt affeteby extratin f araite uing lvent, iniating thatthe bining t r60 wa eiate by glyprteinrather than glylipi. Generally, thee reult trnglyhw the preene f arbhyrate n the ellurfaef poxu alivary glan, whi are line t gly-prtein by Nglyylatin.

Several authr have reprte the rle fglyylate prtein in invain f paraiti prtz-an int the ht ell. r exaple, Barreaue 5)reprte the preene f arbhyratebining prteinn the urfae f the alivary glan f Aee eyperving a reetr fr invain f the przite fPou gneu The rle f arbhyrate-eiate aherene ha al been hwn in a anne-bining prtein f Anhoeb an a galteGalNbining prtein f Enoeb9

inally, in th preent tuy, the 60 Da prtein ilate fr e epiatigte lng fr an reg-nize GlN pei reeptr preent n R prlixu ali-vary glan an are invlve in the paraite ahein.In aitin, an ttept t n the equene f Tr60uing Ean hitry faile prbably ue t ia-

tin f the Nterinu f the prtein

COCLUSIO

xperient eribe in thi tuy, hw the blk-ing f the attahent f nge lng epiatigte nvo t the urfae f poxu alivary glan uingthe extrat f trypane A the preene f lwagglutinin ativity in the paraite extrat ay iniatethe epiatigte lng fr pee letinlie l-eule GlN, whih hwe the highet inhibitrypteny with repet t blking the trypane a


7/8

H Basser and atcle

hesin n the slvry gd suc s preerentillyused t puri gd prtein g 60 kDprteibind lcAc islted m g (60shwed ptent rsite dhesin inhiitry eect in-dicti tht this rtein y ct s lignd receptr ithe itercti bteen he trypsme nd the glndcel sce. e presen ce SDS sd iumddecylsulphte i th e buer did nt ect the iterctin this glclted prtein with the sliry glndcells indictig tt the Tr60 culd be glycprteinThe purity s cirmed by reverse phse HPLC

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8/8

Ihbto ofT Attchmet oxus

25. Sou G loy uka W Wad Da M

Domaly ad Eiia ad

o aa odo oa o aaoloy 806)

46 I 1994

6 Soodoa M a Vo: Do o a

y iania omao ad amao aToa 681) 33197

7 akl Yo ml mdd ul mdum

o ow o 'anaoma angi aao o

66

Royal So o Mdal y 8) 247,1988.28 aa Gul : l olaao ad

ado a ol ao oozoa: ol a a o

nao itoltica Gala Mb ad

o 2(6): 64364000

a Ma M ol 0, S R, uG dato ad aal ao a l

o u o oozo o CpsidI1

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