SYNTHETI C PEPTI DES FROM THE CI RCUMSPOROZOI TE PROTEI NS OF PLASMODI UM FALCI PARUM AND PLASMODI UM KNOWLESI RECOGNI ZE THE HUMAN HEPATOMA CELL LI NE HepG2- A16 IN VI TRO By STEPHEN B. ALEY, MI CHELLE D. BATES, J AMES P. TAM, * AND MI CHAEL R . HOLLI NGDALE Fr om t he Mal ari a Depart ment , Bi omedi cal Resear ch I nst i t ut e, Rockvi l l e, Mar yl and 20852, and t he * Depar t ment of Bi ochem i st r y, The Rockef el l er Uni ver si t y, New Yor k 10021 Mal ari a spor ozoi t es, upon i nj ect i on by an i nf ect ed anophel i ne mosqui t o, ar e r api dl y and speci f i cal l y l ocal i zed in t he hepat ocyt es of t he mammal i an host . I nhi bi t i on of i nvasi on by mAbs or speci f i c ser a suggest s t hat t he ci r cumspor ozoi t e ( CS) ' pr ot ei n pl ays a maj or r ol e i n t hi s pr ocess ( 1- 5) , but t he speci f i c mec hani s ms i nvol ved ar e not under st ood . We have i nvest i gat ed ( 6) t he possi bi l i t y t hat t he N1 conser ved r egi on of t he CS pr ot ei ns f r omPl asmodi um f al ci par um and Pl asmodi um knowl esi is i nvol ved in spor ozoi t e/ hepat ocyt e r ecogni t i on . Usi ng synt het i c pep- t i des based on t hi s r egi on and t he cl oned human hepat oma cel l l i ne, HepG2- A16, as a t ar get cel l f or i nvasi on by P. f al ci par um spor ozoi t es ( 3) , we pr esent evi dence t hat P. f al ci par um spor ozoi t es coul d use cel l r ecogni t i on of or by t hi s por t i on of t he CS pr ot ei n as a par t of t he hepat ocyt e i nvasi on pr ocess . Chem i cal cr ossl i nki ng of r adi ol abel ed N 1 pept i de to t he HepG2- A16 cel l l i ne i dent i f i ed t wo hepat ocyt e pr ot ei ns i nvol ved in pept i de r ecogni t i on . Vol ume 164 December 1986 1915- 1922 Mat eri al s and Met hods Pept i des wer e synt hesi zed by t he st epwi se sol i d- phase met hod ( 7) on a mul t i det achabl e r esi n ( 8) ( p- acyl oxybenzhydr yl am i ne- copol yst yr ene- 1 %-di vi nyl benzene) . t - but yl oxycar - bonyl ( Boc) - am i no aci ds with benzyl al cohol - der i ved si de chai n- pr ot ect i ng gr oups wer e at t ached on p- acyl oxybenzhydr yl am i ne r esi n (0 .4 mmol / g) and t he pept i des wer e as s em- bl ed on a 990 M synt hesi zer ( Bec k man I nst rument s, I nc ., Ful l er t on, CA) . A doubl e- coupl i ng pr ot ocol vi a di cycl ohexyl acar bodi i m i de was used to gi ve a coupl i ng ef f i ci ency of 99 . 7% compl et i on per st ep . Cl eavage of pept i des was ef f ect ed by t he degr adat i ve depr o- t ect i on met hod ( 8) . The im i dazol e- DNP pr ot ect i ng gr oup was r emoved by 1 Mt hi ophenol in di met hyl f ormam i de and t he benzyl pr ot ect i ng gr oups by a m i xt ur e of r eagent CF, SO, H/ CF, C0 2 H/ CH- , SCH3/ p- cr esol ( 10 : 50 : 30 : 10, vol / vol ) at 10° C f or 4h . The cr ude and unpr ot ect ed pept i de st i l l at t ached to t he r esi n was t hen r el eased as pept i de- p- hydr oxybenzhydr yl am i ne by met hyl am i nol ysi s in CH3 NH2 / et hanol / t et r ahydr of ur ane ( 10 : 45 : 45, vol / vol ) at 20° C f or 16 h . The pept i de- p- hydr oxybenzhydr yl am i ne was pur i f i ed on a r ever se- phase C- 18 col umn (2.5 X 30 c m) usi ng aqueous CF 3 CO2H (0 .05%) and acet oni t r i l e ( 80%) gr adi ent . In s ome Thi s wor k was suppor t ed by Agency f or I nt er nat i onal Development cont r act s DPE- 0453- C- 00- 3051- 00 and DPE- 0453- A- 00- 5012- 00 . ' Abbr evi at i ons used in t hi s paper : CS, ci r cumspor ozoi t e; DSS, di succi nyl suber i m i dat e ; ISI, i nhi - bi t i on of spor ozoi t e i nvasi on ; MHBI , met hyl - p- hydr oxybenzi m i dat e ; Pf N 1, P . f al ci par um N 1 pept i de . J . Ex p . MED . ©The Rockef el l er Uni ver si t y Pr ess - 0022- 1007/ 86/ 12/ 1915/ 08 $1 . 00 1915 Downloaded from http://rupress.org/jem/article-pdf/164/6/1915/1096436/1915.pdf by guest on 23 October 2022