Ca hepsin L1, he Majo P o ease In ol ed in
Li e Fluke (Fasciola hepa ica) Vi ulence
PROPEPTIDE CLEAVAGE SITES AND AUTOACTIVATION OF THE ZYMOGEN SECRETED FROM
GASTRODERMAL CELLS*
Recei ed o publica ion, Augus 11, 2003, and in e ised o m, Janua y 29, 2004
Published, JBC Pape s in P ess, Janua y 30, 2004, DOI 10.1074/jbc.M308831200
Pe e R. Collins‡§, Colin M. S ack‡¶储, Sand a M. O’Neill‡, Sean Doyle¶, Thecla Ryan‡,
Ge a d P. B ennan**, Angela Mousley**‡‡, Michael S ewa **, Aa on G. Maule**,
John P. Dal on‡§§¶¶, and Sheila Donnelly‡储储
F om he ‡School o Bio echnology, Dublin Ci y Uni e si y, Dublin 9, Republic o I eland, he ¶Depa men o Biology,
Na ional Uni e si y o I eland, Maynoo h, Co. Kilda e, Republic o I eland, he **Pa asi ology Resea ch G oup,
Queen’s Uni e si y Bel as , Medical Biology Cen e, 97 Lisbu n Road, Bel as BT9 7BL, Uni ed Kingdom, and
he §§Ins i u e o he Bio echnology o In ec ious Diseases (IBID), Uni e si y o Technology, Sydney (UTS),
Wes bou ne S ee , Go e Hill, Sydney, New Sou h Wales 2065, Aus alia
The sec e ion and ac i a ion o he majo ca hepsin
L1 cys eine p o ease in ol ed in he i ulence o he
helmin h pa hogen Fasciola hepa ica was in es iga ed.
Only he ully p ocessed and ac i e ma u e enzyme can
be de ec ed in medium in which adul F. hepa ica a e
cul u ed. Howe e , immunocy ochemical s udies e-
ealed ha he inac i e p oca hepsin L1 is packaged in
sec e o y esicles o epi helial cells ha line he pa a-
si e gu . These obse a ions sugges ha p ocessing and
ac i a ion o p oca hepsin L1 occu s ollowing sec e ion
om hese cells in o he acidic gu lumen. Exp ession o
he 37-kDa p oca hepsin L1 in Pichia pas o is showed
ha an in e molecula p ocessing e en wi hin a con-
se ed GXNXFXD mo i in he p opep ide gene a es an
ac i e 30-kDa in e media e o m. Fu he ac i a ion o
he enzyme was ini ia ed by dec easing he pH o 5.0 and
in ol ed he p og essi e p ocessing o he 37 and 30-
kDa o ms o o he in e media es and inally o a ully
ma u e 24.5 kDa ca hepsin L wi h an addi ional 1 o 2
amino acids. An ac i e si e mu an p oca hepsin L, con-
s uc ed by eplacing he Cys
26
wi h Gly
26
, ailed o au-
op ocess. Howe e , [Gly
26
]p oca hepsin L was p o-
cessed by exogenous wild- ype ca hepsin L o a ma u e
enzyme plus 10 amino acids a ached o he N e minus.
This exogenous p ocessing occu ed wi hou he o ma-
ion o a 30-kDa in e media e o m. The esul s indica e
ha ac i a ion o p oca hepsin L1 by emo al o he
p opep ide can occu by di e en pa hways, and ha
his akes place wi hin he pa asi e gu whe e he p o-
ease unc ions in ood diges ion and om whe e i is
libe a ed as an ac i e enzyme o addi ional ex aco po-
eal oles.
Fasciola hepa ica is a helmin h pa asi e ha causes li e
luke disease in ca le and sheep wo ldwide and has ecen ly
eme ged as an impo an pa hogen o humans (1). Ca hepsin
L1, a majo cys eine p o ease sec e ed by he pa asi e plays a
pi o al ole in a ious aspec s o i s pa hogenici y. Fo exam-
ple, he enzyme akes pa in nu ien acquisi ion by ca abo-
lizing hos p o eins o abso bable pep ides (2), acili a es he
mig a ion o he pa asi e h ough he hos in es ine and li e
by clea ing in e s i ial ma ix p o eins such as ib onec in,
laminin, and na i e collagen (3), and is implica ed in he inac-
i a ion o hos immune de enses by clea ing immunoglobulins
(4, 5). Fu he , ca hepsin L1 has ecen ly been shown o sup-
p ess Th1 immune esponses in in ec ed labo a o y animals
making hem suscep ible o concu en bac e ial in ec ions (6,
7, 8). Acco dingly, he p o ease has been ecognized as an
impo an a ge a which pa asi e in e en ion s a egies
should be di ec ed (9). In his ega d, we ha e shown ha he
induc ion o an i-ca hepsin L immune esponses p io o a
challenge in ec ion o F. hepa ica la ae elici s high le els o
p o ec ion in ca le agains disease (10, 11, 12).
Phylogene ic s udies ha e shown ha he F. hepa ica ca hep-
sin L1 belongs o an enzyme lineage ha e en ually ga e ise
o he mammalian ca hepsin Ls, Ks, and Ss (2). The isola ion o
a cDNA encoding F. hepa ica ca hepsin L e ealed ha he
enzyme, like i s mammalian homologs, is syn hesized as an
inac i e p ep oenzyme consis ing o a p epep ide, a p opep ide
and ma u e enzyme egion (13). By analogy wi h he mamma-
lian p o eases we assume ha he p epep ide is emo ed ol-
lowing ansloca ion in o he endoplasmic e iculum and ha
he N- e minal p opep ide ex ension is in ol ed in se e al
unc ions including in acellula a ge ing o he enzyme (14–
16), co ec olding o he ma u e enzyme by ac ing as an
in amolecula chape one (17–19) and p e en ion o uncon-
olled p o eolysis by binding o he enzyme subs a e cle in a
e e se, non-p oduc i e di ec ion (14). As has been demon-
s a ed o he p opep ide o human ca hepsin L and o he
papain-like cys eine p o eases (14, 17, 19, 20) he ee p opep-
ide o he F. hepa ica p oca hepsin L is a speci ic and po en
inhibi o o he cogna e ma u e enzyme a neu al pH bu does
no bind o he enzyme a pH 5.5–3.5 (21).
Human lysosomal p oca hepsin L is s able a high pH be-
cause he p opep ide p o ec s he p o ein om he dena u ing
e ec s o he alkali (15). Remo al o he p opep ide o gene a e
he ully ac i e ma u e enzyme occu s a he lowe lysosomal
* The cos s o publica ion o his a icle we e de ayed in pa by he
paymen o page cha ges. This a icle mus he e o e be he eby ma ked
“ad e isemen ” in acco dance wi h 18 U.S.C. Sec ion 1734 solely o
indica e his ac .
§ Suppo ed by a g an ecei ed om En e p ise I eland and Ildana
Bio ech.
储Funded by he Heal h Resea ch Boa d (HRB), I eland.
‡‡ Funded by a join No h-Sou h Coope a ion g an om he HRB
(I eland) and he Resea ch and De elopmen O ice (No he n I eland).
¶¶ To whom co espondence should be add essed: Ins i u e o he
Bio echnology o In ec ious Diseases (IBID), Uni e si y o Technology,
Sydney (UTS), Wes bou ne S ee , Go e Hill, Sydney, NSW 2065, Aus-
alia. Tel.: 61-2-9514-4142; Fax: 61-2-9514-4201; E-mail: john.dal on@
u s.edu.au.
储储 Funded by The Wellcome T us .
THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 279, No. 17, Issue o Ap il 23, pp. 17038–17046, 2004
© 2004 by The Ame ican Socie y o Biochemis y and Molecula Biology, Inc. P in ed in U.S.A.
This pape is a ailable on line a h p://www.jbc.o g17038
pH o 5.5 (14, 15, 22, 23). I has been widely epo ed ha
clea age o he p o egion o cys eine p o eases can occu au o-
ca aly ically in i o unde acidic condi ions (15, 24, 25, 26). As
he s abili y o he p opep ide-p o ease complex is dependen
on elec os a ic in e ac ions, educ ion o he en i onmen al
pH, weakens he bond be ween he p opep ide and he ca aly ic
si e. As a consequence, he p oenzyme possibly adop s a loose
con o ma ion, in which he p opep ide is bound less igh ly
in o he ac i e si e making i mo e suscep ible o p o eolysis
(16, 27).
The p ecise mechanism o p o eoly ic con e sion om p oen-
zyme o ma u e enzyme is s ill ac i ely deba ed. The h ee-
dimensional s uc u e o p oca hepsin L (14) and p oca hepsin
B (28, 29, 30) show ha he N e minus o he ma u e enzyme
is qui e emo ed om he ac i e si e hus making i di icul o
isualize an au oca aly ic clea age e en . Mo eo e , ci cula
dich oism s udies e eal ha ac i a ion does no in ol e sig-
ni ican con o ma ional changes in he s uc u e o p oca hep-
sin L (15) o p oca hepsin B (16). The e o e, he ini ial e en in
au oca alysis may in ol e an ac i e p oenzyme, possibly c e-
a ed by he educed pH, ha clea es ano he p oenzyme in he
icini y o he N e minus and se s o a chain eac ion (15, 16).
Howe e , mo e ecen s udies on p oca hepsin B and p oca-
hepsin S iden i ied au op o eoly ic in e media es o p ocessing
when cys a in was included in in i o ac i a ion eac ions,
which suppo ed he iew ha he segmen o he p opep ide
ha binds he ac i e si e cle is suscep ible o clea age (31).
Acco dingly, i was sugges ed ha an ini ial slow in amolec-
ula clea age e en wi hin his segmen o he p opep ide ig-
ge s a mo e apid cascade o in e molecula clea ages a he N
e minus (31).
Ea lie s udies on he p ocessing o yeas -exp essed ecom-
binan papain iden i ied a conse ed hep apep ide (Gly-Xaa-
Asn-Xaa-Phe-Xaa-Asp) mo i loca ed be ween esidues ⫺42
and ⫺36 in he p opep ide ha may be a si e o ini ial clea age
in he pH-dependen au oac i a ion o he enzyme (32). I was
sugges ed ha he lowe ing o he pH pe u bed he nega i e
cha ge o Asp
⫺36
esul ing in a con o ma ional change ha
swi ched on he p ocessing e en s by allowing p o eolysis o
occu a he Ala
⫺37
/Asp
⫺36
bond. Following his p ima y clea -
age u he emo al o he emaining amino acids o he
p opep ide may esul om he p o eoly ic ac i i y o he in-
e media e species, esul ing in ully ac i e ma u e p o ease
(32).
Gi en he impo ance o ca hepsin L p o eases in he i u-
lence o F. hepa ica and o he helmin h pa hogens (2) i is
impo an o unde s and he mechanisms o hei syn hesis,
p ocessing and ac i a ion. We ha e p e iously shown ha ca-
hepsin L1 is sec e ed by his pa asi e and he e o e unc ions
ex acellula ly (4, 33) simila o he mammalian ca hepsin Ls
in ol ed in he p ocessing o he ho mone hy oglobulin (34–
36). In he p esen s udy we ha e employed con ocal lase and
elec on immunocy ochemis y o show ha he enzyme is
s o ed in sec e o y esicles o he gas ode mal epi helial cells
in i s inac i e p oenzyme o m. We in es iga e he au oac i a-
ion o F. hepa ica ca hepsin L1 using he wild- ype p oenzyme
exp essed in Pichia pas o is. In e molecula p ocessing was
s udied by gene a ing an inac i e p oca hepsin L mu an ,
whe e he ca aly ic Cys esidue a posi ion 26 was subs i u ed
wi h a Gly, which was incapable o au oac i a ion. Ou esul s
show ha P. pas o is-exp essed ca hepsin L can au oac i a e
a low pH o an ac i e in e media e o m by an ini ial clea age
wi hin he conse ed hep apep ide (Gly-Xaa-Asn-Xaa-Phe-
Xaa-Asp), as p e iously desc ibed o papain (32). Fu he ac-
i a ion o he enzyme occu s by clea age in he icini y o he
N e minus. In con as , in e molecula p ocessing o he mu-
an inac i e p oca hepsin L o a ma u e p oduc by exogenous
ma u e ca hepsin L did no in ol e he o ma ion o he abo e
in e media e bu was ini ia ed by a di ec clea age close o
he N e minus. These da a p o ide a mechanism by which
F. hepa ica syn hesizes and sec e es a ully ac i a ed p o ease
ha is essen ial o i s exis ence as a pa asi e.
EXPERIMENTAL PROCEDURES
Ma e ials—Z-Phe-A g-NHMec
1
and Z-Phe-Ala-CHN
2
we e ob ained
om Bachem (S . Helens, UK). DTT and EDTA we e ob ained om
Sigma (Do se , Poole, UK). P es ained molecula weigh ma ke s and
he A II and SnaBI es ic ion enzymes we e ob ained om New
England Biolabs (UK) L d. (Hi chin, UK). P ime s we e ob ained om
Sigma-Genosys (Pampis o d, UK). The pPIC9K ec o and Pichia
pas o is s ain GS115 we e ob ained om In i ogen Co p. (San
Diego, CA). Ni-NTA aga ose and columns we e ob ained om Qiagen
(C awley, UK).
In Vi o Cul i a ion o Pa asi es—Adul F. hepa ica we e ob ained
om in ec ed ca le a a local aba oi and cul u ed in i o in RPMI
1640 con aining 30 mMHEPES, 1% glucose and 25 mg/ml gen amycin
as desc ibed by Dal on and He e nan (37). The medium was collec ed
a e 6 h, clea ed by cen i uga ion a 14,000 ⫻g o 30 min a 4 °C and
s o ed a ⫺20 °C.
P epa a ion o An ip opep ide and An ima u e Ca hepsin L1 An i-
se a—Na i e ma u e ca hepsin L1 (nFheCL1) was pu i ied om exc e-
o y-sec e o y (ES) p oduc s o adul F. hepa ica by gel pe mea ion and
ion exchange ch oma og aphy and an ise um p epa ed in abbi s as p e-
iously desc ibed (4, 38). Recombinan ca hepsin L1 p opep ide was gen-
e a ed and pu i ied as desc ibed by Roche e al. (21) and an ise um
p epa ed by immunizing New Zealand Whi e abbi s i e imes wi h 20
␮
g o p o ein o mula ed in F eund’s Comple e and Incomple e Adju an .
Immuno luo escence and Immunoelec on Mic oscopy—Adul F. he-
pa ica we e eco e ed om in ec ed ca le a a local aba oi , washed,
and anspo ed o he labo a o y in mammal saline (0.9% NaCl) a
37 °C. Pa asi es we e insed in mammal saline and allowed o egu -
gi a e hei gu con en s be o e being la - ixed in 4% pa a o maldehyde
(PFA) in PBS (pH 7.2) o 4 h. They we e washed in an ibody diluen
(ABD: PBS wi h, 0.1% bo ine se um albumin; 0.3% T i on X-100; 0.1%
sodium azide) o 24 h be o e being incuba ed o 48 h a 4 °Cin
an ise um p epa ed agains pu i ied ma u e ca hepsin L1 (dilu ed
1:3000) and subsequen ly washed in ABD (24 h, 4 °C). Swine an i-
abbi e ame hyl hodamine iso hiocyana e (TRITC; 1:100; Dako L d.)
was used o isualize bound p ima y an ibody be o e he wo ms we e
washed in ABD (24 h, 4 °C) and moun ed on glass mic oscope slides in
PBS/glyce ol (1:9) con aining 2.5% 2,4-diazabicylco 2.2.2 oc ane. Spec-
imens we e iewed using a Leica TCS-NT con ocal scanning lase
mic oscope.
Fo elec on mic oscopy wo ms we e washed in mammal saline and
ixed o 1hin2%double-dis illed glu a aldehyde (GTA) (Aga Scien-
i ic) in 0.1 Msodium cacodyla e bu e (pH 7.2) con aining 3% suc ose
a 4 °C. Following ho ough washing in bu e , specimens we e dehy-
d a ed h ough g aded e hanol o p opylene oxide, in il a ed and em-
bedded in Aga 100 esin (Aga Scien i ic). Ul a hin sec ions (80–90
nm) we e cu on a Reiche Ul acu E ul amic o ome, collec ed on ba e
200-mesh nickel g ids and d ied a oom empe a u e. Fo immunogold
labeling sec ions we e e ched wi h 10% hyd ogen pe oxide o 5 min and
insed ho oughly wi h 20 mMT is-HCl bu e (pH 8.2) con aining 0.1%
bo ine se um albumin and Tween 20 (1:40 dilu ion). G ids we e incu-
ba ed in no mal goa se um (1:20 dilu ion) o 30 min and hen ans-
e ed o p ima y an ibody dilu ed o 1:20,000 wi h 0.1% bo ine se um
albumin/T is-HCl bu e o 12–18 h. G ids we e hen washed in bo ine
se um albumin/T is-HCl and ans e ed o a 20
␮
l d op o 10 nm
gold-conjuga ed goa an i- abbi IgG (Bio Cell In e na ional) o 2ha
oom empe a u e. Following ano he bu e wash, g ids we e ligh ly
ixed wi h 2% double-dis illed GTA o 3 min, and inally washed wi h
bu e and insed wi h dis illed wa e . G ids we e double s ained wi h
u anyl ace a e (5 min) and lead ci a e (3 min) and examined in a FEI
(Philips) CM100 ansmission elec on mic oscope, ope a ing a 100 keV.
Con ols consis ed o (i) incuba ion o whole-moun s/sec ions wi h
seconda y an ibody in he absence o p ima y an ibody and (ii) incuba-
ion wi h p eimmune se um ollowed by he seconda y an ise um.
1
The abb e ia ions used a e: Z-Phe-A g-NHMec, benzyloxyca bonyl-L-
phenylalanyl-L-a ginine 4-me hylcouma inyl-7-amide; Z-Phe-Ala-CHN
2
,
benzyloxyca bonyl-L-phenylalanyl-L-alanine-diazome hylke one; DTT, di-
hio h ei ol; PBS, phospha e-bu e ed saline; NTA, ni ilo iace ic acid.
P ocessing o F. hepa ica Ca hepsin L1 17039
Cons uc ion o Exp ession Vec o s Encoding cDNA o Wild- ype
P oca hepsin L1 ( Fhep oCL1) and Gly
26
P oca hepsin L1 ( mu Fhep o-
CL1) and T ans o ma ion in o Pichia pas o is—The F. hepa ica p oca-
hepsin L (Fhep oCL1) was ampli ied by PCR om he pAAH5 Saccha-
omyces ce e isiae exp ession ec o in o which he ull-leng h cDNA
had been p e iously cloned in ou labo a o y (13). P ime s we e used
(see p ime s A and D below) o inco po a e a SnaBI es ic ion si e a
he 5⬘-end o he gene and an A II es ic ion si e and His
6
ag
sequence a he 3⬘-end. The 980-bp agmen was inse ed in o he
A II/SnaB1 si e o P. pas o is exp ession ec o pPIC9K (In i ogen).
Mu an s we e gene a ed om his cons uc by a PCR-based si e-
di ec ed mu agenesis me hod known as gene splicing by o e lap ex ension
(SOEing) (39) using he pPIC9K-Fhep oCL1 DNA as a empla e. The
cons uc ion o he inac i e Fhep oCL1 mu an in ol ed changing he
ac i e si e cys eine (Cys
26
) esidue o a glycine in a wo-s ep PCR p ocess.
The p ime s used we e as ollows: p ime A, 5⬘-GCGGCTACGTATCGA-
ATGATGATTTGTGGCAT-3⬘; p ime B, 5⬘-GAATGCCCAACCGGAGCC-
AC-3⬘; p ime C, 5⬘-GTGGCTCCGGTTGGGCATTC-3⬘; p ime D, 5⬘-GC-
GCCTAGGTCAGTGGTGGTGGTGGTGGTGGGCCC-3⬘.
The unde lined nucleo ides indica e he eplacemen s in oduced.
Each eac ion used one lanking p ime ha hyb idized a one end o
he a ge sequence (p ime A o D) and one o e lapping in e nal
p ime ha hyb idizes a he si e o he mu a ion and con ains he
misma ched base (p ime B o C). In he i s ound o ampli ica ion wo
sec ions o he cDNA we e ampli ied using p ime combina ions A⫹B
and C⫹D. These wo PCR p oduc s, wi h an o e lap o 21 bp a one end
o each agmen , we e hen combined in a second PCR o ampli y he
en i e mu Fhep oCL1 cDNA. P ime s o his eac ion we e he wo
ou side p ime s used in each o he wo i s ound eac ions (p ime s A
and D). Fo all PCRs, high- ideli y Taq polyme ase was used (25 cycles
a 94 °C o 30 s, 55 °C o 1 min, and 72 °C o 2 min). The mu Fhep-
oCL1 cDNA was hen inse ed in o he A II/SnaB1 si e o exp ession
ec o pPIC9K. Bo h he wild- ype ( Fhep oCL1) and mu an ( mu F-
hep oCL1) plasmid inse we e sequenced o e i y he p esence o
co ec gene sequence and mu a ion.
The FheCLI o mu FheCL1 plasmids we e linea ized by diges ion
wi h SalI and in oduced o P. pas o is GSII5 cells by sphe oplas ing
(40). T ans o man s we e selec ed o hei abili y o g ow on his idine-
de icien aga pla es and on aga pla es con aining minimal media and
me hanol. Inse ion o FheCLI o mu FheCL1 in o P. pas o is was
con i med by PCR using p ime s speci ic o he yeas genome (41).
Exp ession and Pu i ica ion o P oca hepsin L1 and Gly
26
P oca hep-
sin L1—Yeas ans o man s we e cul u ed in 250 ml o BMGY b o h,
bu e ed o pH 6.0, in 1 li e o ba led lasks a 30 °C un il an OD
600
o
2–6 was eached. Cells we e ha es ed by cen i uga ion a 2000 pm
o 5 min, and p o ein exp ession induced by esuspending in 50 ml o
BMMY b o h, bu e ed a pH 6.0, 7.0, o 8.0, con aining 1% me hanol
(41). The cul u es we e g own a 30 °C wi h shaking a 225 pm o 3
days, 1 ml samples we e emo ed daily and hen il e -s e ilized me h-
anol was added o main ain a inal concen a ion o 1%. To assess he
e ec o he ca hepsin L cys eine inhibi o Z-Phe-Ala-diazome hyl-
ke one (-CHN
2
) on he p oduc ion o p oca hepsin L1 by yeas he
inhibi o was added a he ime o induc ion o a inal concen a ion o
25
␮
Mand hen wice daily a he same concen a ion o e he subse-
quen 3 days.
Recombinan p o eins we e pu i ied om yeas medium by a ini y
ch oma og aphy using Ni-NTA-aga ose (Qiagen). B ie ly, a column p e-
pa ed wi h 1 ml o esin was equilib a ed by passing h ough 10 ml o
50 mMsodium phospha e bu e , pH 8.0, con aining 300 mMNaCl and
10 mMimidazole. 10 ml o yeas media supe na an was mixed wi h 40
ml o he same bu e and applied o he column. The column was
washed wi h 15 ml o 50 mMsodium phospha e bu e , pH 8.0, con ain-
ing 300 mMNaCl and 20 mMimidazole, and bound p o ein elu ed using
50 mMsodium phospha e bu e , pH 7.0, con aining 300 mMNaCl and
250 mMimidazole. Pu i ied ecombinan p o eases we e dialysed
agains PBS and s o ed a ⫺20 °C. Samples o yeas medium supe na-
an s (10
␮
l) and pu i ied p o eins (⬃1–5
␮
g) we e analyzed by 12%
SDS-polyac ylamide gel elec opho esis (SDS-PAGE) and immunoblo -
ing (see below).
Fluo ome ic Subs a e Assay o Ca hepsin L1 Ac i i y—Ca hepsin
L1 ac i i y was de e mined by he luo ome ic subs a e assay de-
sc ibed by Dowd e al. (38). Yeas cul u e supe na an s o pu i ied
ecombinan p o ease we e assayed in a o al olume o 1 ml o sub-
s a e/bu e mix (2.5 mMEDTA, 2 mMDTT, 0.1 Msodium phospha e
bu e , pH 6.0, and 10
␮
MZ-Phe-A g-NHMec). The eac ion was incu-
ba ed a 37 °C and s opped a e 30 min by he addi ion o 200
␮
lo 10%
ace ic acid. Fluo escence was eco ded a an exci a ion wa eleng h o
370 nm and an emission wa eleng h o 440 nm. The ac i i y o he
samples we e calcula ed om a s anda d cu e o NHMec anging om
0 o10
␮
M, and p esen ed as nanomoles o NHMec min
⫺1
ml
⫺1
.
In Vi o P ocessing o P oca hepsin L1 and Gly
26
P oca hepsin L1—
Pu i ied ecombinan p oca hepsin L1 (⬃5
␮
g) was incuba ed in ac i-
a ion bu e (0.1 Msodium ci a e bu e , pH 5.0, 1 mMDTT, and 1.25
mMEDTA) a 37 °C. Samples we e aken a a ious ime poin s up o
2 h and he p o eoly ic clea age o he p opep ide isualized by 12%
SDS-PAGE. Aliquo s we e also assayed o enzyme ac i i y wi h he
speci ic subs a e Z-Phe-A g-NHMec as desc ibed abo e.
Exogenous p ocessing o Gly
26
p oca hepsin L1 was ca ied ou by
mixing 5
␮
g o he pu i ied mu an enzyme wi h 0.5
␮
g o wild- ype
p oca hepsin L ha had been ac i a ed as desc ibed abo e. P ocessing
o he mu an p o ein o e a pe iod o 3ha 37°C was analyzed by 12%
SDS-PAGE. To con i m ha [Gly
26
]p oca hepsin L1 was exp essed as a
co ec ly olded p o ein a simila expe imen was pe o med in pa allel
wi h mu an enzyme p epa a ions ha had been un olded by hea ing a
95 °C o 3 min.
Iden i ica ion o Clea age P oduc s by N- e minal Sequencing—Fol-
lowing 12% SDS-PAGE, p o eins we e ans e ed o poly inylidene
di luo ide memb ane using a semid y ans e cell a 15V o 20 min.
The memb ane was washed wi h dH
2
O and s ained wi h 0.025% Coo-
massie B illian Blue R-250 in 40% me hanol (40). P o ein bands o
in e es we e subjec ed o N- e minal sequencing a Genosphe e Bio-
echnologies (Pa is, F ance).
Sequence Analysis—The F. hepa ica ca hepsin L1 p o ein sequence
was aligned wi h se e al ela ed ca hepsin sequences using Clus alX
1.81. P o ein sequences used included Ca ica papaya,Fasciola hepa ica
ca hepsin L1, F. hepa ica ca hepsin L2, F. gigan ica ca hepsin L1,
Schis osoma mansoni ca hepsin L2, Caeno habdi is elegans CPL-1,
mouse ca hepsin L, a ca hepsin L, and human ca hepsin L.
2
Se-
quences we e numbe ed acco ding o he papain numbe ing used by
Ve ne e al. (32) whe e he p opep ide esidues a e eco ded as a
nega i e beginning om he clea age si e be ween p opep ide and
ma u e enzyme.
SDS-PAGE and Immunoblo ing—P o ein samples we e sepa a ed
by 12% SDS-PAGE and gels s ained wi h a 0.1% w/ solu ion o Coo-
massie B illian Blue R-250 in 40% me hanol/10% ace ic acid (42).
Immunoblo s we e p epa ed by ans e ing p o eins o ni ocellulose
memb anes, p esoaked in a ans e bu e (50 mMT is, 384 mMglycine,
20% me hanol), and hen blocking hese o 1 h a oom empe a u e
wi h 5% milk in PBS/0.1% Tween 20. The ni ocellulose memb anes
we e p obed wi h polyclonal abbi an ima u e ca hepsin L1 se um,
abbi an i- ecombinan p opep ide and p eimmunized con ol abbi
se um dilu ed 1/1,000 in 1% milk/PBS/0.1% Tween-20 o 45 min a
oom empe a u e. They we e hen washed h ee imes o 5 min each
wi h 1% milk/PBS/0.1% Tween-20, ollowed by incuba ion o 45 min a
oom empe a u e wi h a 1/10,000 dilu ion o seconda y an ibody (goa
an i- abbi IgG-pe oxidase conjuga e) p epa ed in 1% milk/PBS/0.1%
Tween-20. Blo s we e washed as be o e and bound an ibody isualized
wi h 3,3⬘-diaminobenzidine (DAB, Sigma).
RESULTS AND DISCUSSION
Sec e ion o Ca hepsin L P o eases by F. hepa ica Pa asi es—
When adul F. hepa ica a e main ained in i o hey sec e e
wo majo p o eases ha we e p e iously cha ac e ized as 27.5
ca hepsin L1 and 29 kDa ca hepsin L2 in ou labo a o y (4, 38).
N- e minal sequencing e ealed a sequence AVPDK o bo h
enzymes ha co ela ed wi h he N e minus o ully p ocessed
ma u e ca hepsin L enzymes. In addi ion, immunoblo ing ex-
pe imen s demons a ed ha while hey a e bo h eac i e wi h
se a p epa ed agains pu i ied ma u e ca hepsin L1, nei he
eac s wi h se a p epa ed speci ically agains a ecombinan
p opep ide o ca hepsin L1 (Fig. 1, see also Fig. 3B). Isola ion o
cDNAs encoding he wo enzymes showed ha hey exhibi a
high le el o iden i y (77%) a he amino acid le el ha explains
hei c oss- eac i i y wi h an ise um p epa ed agains ei he
enzyme (13, 38).
The wo p o eases a e membe s o a phylogene ic lineage
2
P o ein sequences and accession numbe s om GenBank
TM
a e as
ollows: Ca ica papaya papain (P00784), F. hepa ica ca hepsin L1
(U62288), F. hepa ica ca hepsin L2 (U62289), F. gigan ica ca hepsin L1
(AF112566), S. mansoni ca hepsin L2 (Z32529), C. elegans CPL-1
(NP_507199), mouse ca hepsin L (P06797), a ca hepsin L (KHRTL),
and human ca hepsin L (M20496).
P ocessing o F. hepa ica Ca hepsin L117040
ha includes he mammalian ca hepsin Ls and ha e subs a e
speci ici ies ha a e ypical o his g oup, i.e. a p e e ence o
posi i ely cha ged esidues, such as a ginine, in he P1 posi ion
and hyd ophobic esidues, such as phenylalanine and leucine,
in he P2 posi ion (14). In con as o mammalian ca hepsin Ls,
howe e , he enzymes exhibi ac i i y o e a wide pH ange
(4.0–8.5) and a e ema kably s able a neu al pH; hey e ain
mos o hei ac i i y ollowing a 24-h incuba ion a pH 7.0 and
37 °C whe eas human ca hepsin L is comple ely inac i a ed in
less han 20 min unde he same condi ions (43). These en-
hanced s abili y p ope ies we e sugges ed o be impo an in
hos p o ein diges ion in he pa asi e gu and in acili a ing he
mig a ion o he pa asi e h ough he hos in es ine and li e
(33).
Immunolocaliza ion and in si u hyb idiza ion s udies p e i-
ously ca ied ou by ou labo a o ies had shown ha he ca-
hepsin L p o eases a e syn hesized wi hin he gas ode mis
ha lines he pa asi e gu (4, 33). The gas ode mis is com-
posed o epi helial cells ha a e columna in shape and con ain
nume ous dense esicles si ua ed a he apical end (see Fig.
2A). Al hough sec e o y in unc ion, hese esicles ha e been
desc ibed as lysosomal-like and o almos hi y yea s ha e
been suspec ed o con ain p o eases (44–47). Con ocal lase
immunocy ochemis y using an ise um p epa ed agains he
na i e ma u e ca hepsin L localized his p o ease o he gas-
ode mal epi helial cells (Fig. 2B) h oughou he gu o adul
F. hepa ica. The pa e n o immuno eac i e labeling appea ed
punc a e and was mo e dense owa d he apical end o he
gas ode mal cells indica ing ha he ca hepsin L p o ease was
associa ed wi h he sec e o y esicles (Fig. 2C). Elec on mi-
c oscopy e ealed ha he enzyme is indeed s o ed in sec e o y
esicles wi hin hese cells (Fig. 2, D–F). The sec e o y esicles
we e s ongly immuno eac i e wi h an an ise um p epa ed
agains he ecombinan -exp essed p opep ide (Fig. 2E) and o
he ma u e po ion o ca hepsin L1 (Fig. 2F) indica ing ha he
enzymes a e p esen in hese esicles as he inac i e p o o ms,
p oca hepsin L. (The speci ici y o he se a o he p opep ide
and ma u e egions o he p oca hepsin L1 is demons a ed in
Fig. 3B.)
The abo e obse a ions p o ide impo an insigh s in o he
egula ion o p o ease ac i i y wi hin he gas ode mis o hese
pa asi es. Fi s , in ag eemen wi h he obse a ion by Hal on
and co-wo ke s (44, 46, 47) i appea s ha he unc ion o he
esicles is o package p o eoly ic enzymes o deli e y in o he
gu lumen. Second, he accumula ion o p oca hepsin L1, he
inac i e p ecu so , in he sec e o y esicles a he han ac i e
ma u e p o ease makes good sense gi en he high abundance o
hese esicles in he epi helial cells and hence he possibili y o
p o ease leakage in o he cy oplasm (see Fig. 2A). Thi d, he
da a sugges ha ac i a ion o he p oca hepsin L1 akes place
ollowing sec e ion o he p o eases in o he gu lumen. Elec-
on mic oscopy has shown ha he con en s o he sec e o y
esicles a e ex uded in o he gu lumen whe e hey mix wi h
he inges ed blood and issue meal be ween he long ex uding
lamellae (Re s. 47 and 48; see Fig. 2A). Enzyme ac i a ion is
likely o ake place he e as he pH is es ima ed o be ⬃5.5 o
sligh ly lowe (47, 48). Once ac i a ed, he p o ease would be in
di ec con ac wi h he bloodmeal and could immedia ely begin
i s unc ion in p o ein ca abolism. Since hese helmin h pa a-
FIG.1.SDS-PAGE and immunoblo analysis o F. hepa ica ex-
c e o y-sec e o y (ES) p oduc s. ES p oduc s ob ained om me-
dium in which F. hepa ica we e cul u ed was analyzed by SDS-PAGE
and immunoblo ing. Lane M, molecula size ma ke s; lane 1, Coomas-
sie Blue-s ained 12% SDS-PAGE gel o ES p oduc s; lane 2, ES p oduc s
p obed wi h an ise um p epa ed in abbi s agains na i e ma u e ca-
hepsin L1; lane 3, ES p oduc s p obed wi h an ise um p epa ed in
abbi s agains ecombinan p opep ide o p oca hepsin L1; lane 4,ES
p oduc s p obed wi h no mal abbi se um. A ows indica e posi ion o
majo sec e ed and ully p ocessed ca hepsin L1 and ca hepsin L2.
FIG.2.Immunolocaliza ion o p oca hepsin L1 in F. hepa ica
gas ode mal epi helial cells. A, schema ic ep esen a ion o gas o-
de mal epi helial cells in F. hepa ica (a e Smy h and Hal on, Re . 46).
g, Golgi; gl, gu lamellae; n, nuclei; sb, sec e o y bodies. B, con ocal
scanning lase mic og aph showing immuno eac i i y o ma u e ca-
hepsin L1 wi hin he gas ode mis o adul F. hepa ica; he nuclei
(a ows) o he gas ode mal cells a e clea ly isible. gl, gu lamellae;
lu, gu lumen. C, con ocal scanning lase mic og aph showing immu-
no eac i i y o ma u e ca hepsin L1 wi hin he gas ode mal cells o
adul F. hepa ica; he nuclei (a ows) o he gas ode mal cells a e
clea ly isible. Immuno eac i i y appea s as a punc ua e pa e n a he
apical end o he cells whe e he sec e o y esicles a e loca ed. gm, gu
muscle; lu, gu lumen. D, elec on mic og aph showing non- eac i i y o
sec e o y bodies (a ows) wi hin he epi helial cells o he gas ode mis
wi h con ol p eimmunized abbi se um. gl, gu lamellae. E, elec on
mic og aph showing he localiza ion F. hepa ica p oca hepsin L1 o
sec e o y bodies o he gas ode mal epi helial cells wi h an ise um
p epa ed agains he ecombinan p opep ide o p oca hepsin L1. gl,
gu lamellae. F, elec on mic og aph showing he localiza ion o F. he-
pa ica ca hepsin L1 o sec e o y bodies o he gas ode mal epi helial
cells wi h an ise um p epa ed agains pu i ied ma u e po ion o ca-
hepsin L1. No e ha he labeling is con ined o he con en s o he
sec e o y bodies.
P ocessing o F. hepa ica Ca hepsin L1 17041
si es ha e a blind-ending gu , emo al o undiges ed p oduc s
and accumula ed hema in is achie ed by simply oiding he
con en s app oxima ely e e y 3 h (46, 47). In his way ac i e
ma u e ca hepsin L would be egula ly libe a ed om he
pa asi e in o he su ounding in es inal o li e issues whe e
i could pe o m i s unc ion in issue deg ada ion and hus aid
he mo emen o he pa asi e h ough hese issues (47, 49).
The sec e ion o F. hepa ica p oca hepsin con as s wi h he
in acellula a icking o mammalian p oca hepsin L h ough
he Golgi appa a us o acidic lysosomes whe e he enzyme is
p ocessed o he ma u e o m by emo al and deg ada ion o
he p opep ide (14, 15). Howe e , a low le el o p oca hepsin L
is also sec e ed om no mal cells and is hough o play an
ex acellula p ocessing unc ion (50), while high le els o se-
c e ion has been co ela ed wi h he me as a ic ac i i y o
ans o med cells (51, 52). A compa ison may also be made wi h
human ca hepsin K, which is sec e ed by os eoclas s and unc-
ions in bone eso p ion (25, 53, 54). Following adhe ence o
os eoclas s o he bone su ace an ex acellula compa men
known as an abso p ion pi is c ea ed be ween cell and bone
in o which ca hepsin K is sec e ed. The pH wi hin he pi is
sligh ly acidic and is conside ed impo an no only o he
dissolu ion o he bone bu also o he ac i a ion o p oca hep-
sin K o i s ac i e p o eoly ic o m (53, 54). Ca hepsin B, L, and
K ha e also been shown o be sec e ed in o he ex acellula
lumen o hy oid ollicles whe e hey play a ole in he p o eo-
ly ic p ocessing o co alen ly c oss-linked soluble hy oglobulin
o libe a e he hy oid ho mone hy oxine (T
4
) (34–36).
Exp ession and P ocessing o Wild- ype P oca hepsin L1—To
s udy he mechanism by which he F. hepa ica p oca hepsin L1
is p ocessed o an ac i e ma u e enzyme he ecombinan
p oenzyme was exp essed in he yeas P. pas o is. The ans-
o med yeas cells we e induced by 1% me hanol in BMMY
medium bu e ed a pH 6.0, 7.0, o 8.0 and hen he ecombi-
nan p o ein sec e ed in o he medium analyzed by SDS-PAGE.
Two majo p o eins mig a ing a 37 and 30 kDa we e isualized
(Fig. 3A). These componen s mig a e highe han he ma u e
ca hepsin L1 and ca hepsin L2 sec e ed by he adul F. hepa -
ica pa asi es in 12% SDS-PAGE (Fig. 3B). Immunoblo ing
expe imen s showed ha polyclonal an ise um p epa ed
agains he p opep ide po ion and agains he ma u e po ion
o p oca hepsin L bo h eac ed wi h he P. pas o is-exp essed
37 and 30 kDa componen s con i ming ha hese a e wo o ms
o he p oca hepsin L. By con as , he ully p ocessed enzymes
sec e ed by he pa asi e a e eac i e wi h an ise um p epa ed
agains he ma u e po ion o he p oca hepsin L1 bu a e no
eac i e wi h an ise um p epa ed agains he p opep ide po -
ion (Fig. 3B).
The 37 and 30 kDa componen s we e pu i ied om he pH
8.0 yeas medium by Ni-NTA aga ose a ini y ch oma og aphy
and subjec ed o N- e minal sequence analysis. This showed
ha he 37 kDa p o ein ep esen ed he p oca hepsin L (plus 5
amino acids o he yeas
␣
- ac o sec e o y signal a he N
e minus) while he 30-kDa componen ep esen ed an in e -
media e p ocessed o m beginning a esidue Leu
⫺41
(papain
numbe ing) wi hin he p opep ide (see Fig. 5).
The amoun o he ecombinan p o eins in he yeas me-
dium inc eased o e he 3 days ollowing induc ion wi h me h-
anol; his inc ease was obse ed a pH 6.0, 7.0, and 8.0 (Fig. 3).
Howe e , he ela i e a io o he 37 and 30 kDa p o eins was
dependen on he bu e ing pH o he medium, wi h he p o-
po ion o 37 kDa inc easing wi h inc easing pH. A pH p o ile
o ac i i y o he ecombinan F. hepa ica ca hepsin L showed
ha i was simila o ha o he na i e ma u e enzyme (38, 43),
i.e. he ecombinan enzyme was ac i e o e he pH ange 4.0
o 8.5 bu was mos ac i e a pH 6.0.
3
The e o e, by inc easing
he pH o he yeas medium abo e he pH op imum o ac i i y
o he F. hepa ica ca hepsin L he le el o p ocessing o he
37-kDa o m o he 30-kDa in e media e o m was educed.
To add ess his issue u he , yeas cells we e cul u ed a pH
8.0 in he p esence and absence o he ca hepsin L-speci ic
inhibi o Z-Phe-Ala-CHN
2
(Fig. 4,Aand B). Al hough he in-
hibi o was added wice daily a a concen a ion o 25
␮
M,
which is 250- old g ea e han i s K
i
o ca hepsin L (38, 40),
samples o medium aken daily showed ha ca hepsin L ac i -
i y was no comple ely inhibi ed (i is possible ha he inhibi o
is no s able in he medium) (see Fig. 4B). Ne e heless, he
p opo ion o 37-kDa componen in cul u e supe na an s con-
aining inhibi o was g ea e han ha in cul u es wi hou
inhibi o , al hough he 30-kDa p o ein was s ill e iden . These
da a suppo he idea ha p oca hepsin L1 sec e ed om P. pas-
o is au op ocesses o an in e media e ac i e o m and ha his
au op ocessing is p e en ed by he ca hepsin L p o ease inhib-
i o Z-Phe-Ala-CHN
2
. Es ima ions o yeas cell iabili y
showed ha a he concen a ions used he inhibi o did no
a ec he g ow h o he yeas cells.
3
To examine whe he u he in e molecula p ocessing could
ake place a a pH lowe han 6.0, he 37 kDa p oca hepsin L1
and 30-kDa in e media e o ms we e i s pu i ied om pH 8.0
yeas cul u e supe na an by a ini y ch oma og aphy on Ni-
NTA aga ose and dialyzed agains PBS (pH 7.3). The pH o
3
P. R. Collins and J. P. Dal on, unpublished da a.
FIG.3.SDS-PAGE and immunoblo analysis o F. hepa ica p o-
ca hepsin L1 exp ession om P. pas o is.A, cul u es o P. pas o is
ans o med wi h a cDNA encoding p oca hepsin L1 we e induced wi h
1% me hanol. Fe men a ions we e ca ied ou a 30 °C in media bu -
e ed o pH 6.0, 7.0, and 8.0. Aliquo s (10
␮
l) emo ed a 48 and 72 h
a e induc ion we e analyzed by 12% SDS-PAGE. The 37 and 30 kDa
componen s a e indica ed. B, compa ison o P. pas o is-exp essed p o-
ca hepsin L wi h na i e F. hepa ica-sec e ed ma u e ca hepsin L1.
Aliquo s o adul F. hepa ica ES p oduc s (lanes 1,3, and 5) we e
compa ed wi h samples aken om P. pas o is medium a e induc ion
o 72 h a pH 8.0 (lanes 2,4, and 6). Lane M, molecula size ma ke s;
panel a, Coomassie Blue-s ained 12% SDS-PAGE gel; panel b, samples
p obed wi h an ise um p epa ed in abbi s agains ma u e po ion o
ca hepsin L1; panel c, samples p obed wi h an ise um p epa ed in
abbi s agains ecombinan p opep ide po ion o p oca hepsin L1.
No e, he an ip opep ide se um eac s wi h he 37 and 30 kDa P.
pas o is-exp essed componen s (indica ed wi h a ows) bu no wi h
pa asi e-p oduced ma u e ca hepsin L1 o ca hepsin L2 (indica ed by
an as e isk). Con ol p eimmunized abbi se um was no eac i e wi h
any p o eins (no shown).
P ocessing o F. hepa ica Ca hepsin L117042

samples aken om his p epa a ion we e hen educed o 5.0
by he addi ion o 0.1 Msodium ci a e con aining 1 mMDTT
and 1.25 mMEDTA, and hen incuba ed a 37 °C (Fig. 5).
Analysis o samples emo ed a a ious ime poin s o e a
pe iod o 2 h e ealed ha he 37 kDa p oca hepsin L is
p ocessed o he 30-kDa in e media e o m and hen h ough
a ious in e media es ha e en ually gi e ise o a single
pep ide mig a ing a 25.4 kDa. Co esponding wi h his p oc-
essing was he g adual 5- old inc ease in he ac i i y o ca hep-
sin L by he end o he 2-h incuba ion pe iod (da a no shown).
N- e minal sequence analysis o he 25.4 kDa p oduc e ealed
wo sequences, NRAVP and RAVPD, which a e jus 2 and 1
amino acids om he ma u e p o ein p ocessing si e, espec-
i ely (see Fig. 5). This in e molecula p ocessing was com-
ple ely inhibi ed by adding 10
␮
MZ-Phe-Ala-CHN
2
o he s a -
ing mix and he e o e can be a ibu ed o he ac ion o
ca hepsin L (da a no shown).
The clea age si e be ween Gly
⫺42
-Leu
⫺41
ha gene a ed he
30 kDa in e media e o m obse ed by SDS-PAGE analysis
(29.6 kDa om amino acid sequence) was o pa icula in e es
as i occu ed wi hin he Gly
⫺42
-Xaa-Asn-Xaa-Phe-Xaa-Asp
⫺36
mo i ha was p e iously iden i ied as pi o al in he in e mo-
lecula p ocessing o papain exp essed in he yeas S. ce e i-
siae. By andom mu agenesis s udies, and by sequence com-
pa isons o a ious papain-like p o eases, Ve ne e al. (32)
showed ha he Gly
⫺42
and Asp
⫺36
esidues wi hin his mo i
we e he mos cons ained and p oposed ha wo clea age
si es, Gly
⫺42
-Leu
⫺41
and Ala
⫺37
-Asp
⫺36
we e impo an o
p oenzyme p ocessing. The la e was conside ed mo e signi -
ican because clea age a his si e was p edic ed o pe u b he
nega i e cha ge o he Asp
⫺36
esidue (32). The Asp
⫺36
esidue
is absolu ely conse ed in non-ca hepsin B-like cys eine p o-
eases o helmin h pa asi es, plan s and mammals (including
ca hepsin L, ca hepsin S, and ca hepsin K) and c ys allo-
g aphic s udies on human ca hepsin L showed ha i pa ici-
pa es in an impo an sal b idge be ween p opep ide and ma-
u e enzyme (14).
On inspec ion o he F. hepa ica p oca hepsin L p opep ide
sequence we ound ha he Gly
⫺42
-Xaa-Asn-Xaa-Phe-Xaa-
Asp
⫺36
mo i is comple ely conse ed in his pa asi ic helmin h
and in o he s including F. gigan ica and S. mansoni, and in he
ee-li ing helmin h C. elegans (Fig. 6). Mo eo e , he Leu
⫺41
esidue is also conse ed in all o hese sequences hus p ese -
ing he Gly
⫺42
-Leu
⫺41
bond and implica ing i s impo ance in
p opep ide unc ion in hese helmin hs. Howe e , hese wo
esidues a e no conse ed in he human, mouse, and a ca-
hepsin L sequences whe e Gly
⫺42
is eplaced by Glu o Ala and
Leu
⫺41
is eplaced by Me (Fig. 6). While i is emp ing o
specula e ha he changes a hese posi ions explains why an
in e media e 30-kDa o m was no obse ed in s udies on he
in e molecula p ocessing o human p oca hepsin L (15), Mc-
Queney e al. (25) ound ha he in e molecula p ocessing o
an inac i e mu an o m o p oca hepsin K by ac i a ed ca hep-
sin K did in ol e a clea age be ween Ala
⫺42
and Me
⫺41
.As
poin ed ou by Coulombe e al. (14), while he e may be a di ec
in ol emen o he Asp
⫺36
in he Gly
⫺42
-Xaa-Asn-Xaa-Phe-
Xaa-Asp
⫺36
mo i in he pH-dependan p ocessing o p o o ms
o cys eine p o eases, clea age a o he pep ide bonds wi hin
his mo i could in luence he local cha ge s a e ha hen may
igge p ocessing.
Exp ession and Exogenous P ocessing o Gly
26
P oca hepsin
L1—To u he s udy he in e molecula p ocessing o F. he-
pa ica p oca hepsin L1 we exp essed an inac i e o m o he
p o ein by eplacing he ac i e si e Cys
26
wi h Gly
26
. Exp es-
sion o his ecombinan mu an in medium bu e ed a pH 6.0
FIG.4.P oduc ion o p oca hepsin L1 by ans o med P. pas-
o is in he p esence and absence o Z-Phe-Ala-diazome hyl-
ke one. A, ans o med P. pas o is we e cul u ed a 30 °C and pH 8.0
in he p esence (⫹) o absence (⫺) o he cys eine p o ease inhibi o
Z-Phe-Ala-CHN
2
(25
␮
M). Aliquo s o cul u e supe na an , emo ed
a e 48 and 72 h, we e analyzed by SDS-PAGE. The 37 and 30 kDa
bands a e indica ed. B, ecombinan yeas cul u ed in he p esence
(do ed line) and absence (solid line)o 25
␮
MZ-Phe-Ala-CHN
2
. Ca hep-
sin L ac i i y in he cul u e supe na an was measu ed wi h he lu-
o ogenic subs a e Z-Phe-A g-NHMec. Ac i i y uni s a e p esen ed as
nmol o NHMec eleased min
⫺1
ml
⫺1
.
FIG.5.Au oca aly ic p ocessing o p oca hepsin L1 a pH 5.0.
Pu i ied p oca hepsin L1 (5.0
␮
g) was incuba ed a 37 °C o 2hin0.1
Msodium ci a e bu e , pH 5.0, con aining 2 mMDTT and 2.5 mM
EDTA. Samples emo ed a a ious ime poin s we e analyzed by
SDS-PAGE. The p edominan p o eins o 37, 30, and 24.5 kDa we e
subjec ed o N- e minal sequencing. The schema ic indica es he amino
acid sequences su ounding he in e molecula p ocessing si es (a -
owed) ha gene a ed hese componen s and hei loca ion wi hin he
p opep ide o p oca hepsin L1. The box indica es he conse ed mo i
GXNXFXD.
P ocessing o F. hepa ica Ca hepsin L1 17043
and 8.0 showed ha i mig a es as a single band a 37 kDa in
SDS-PAGE (Fig. 7). N- e minal sequencing con i med ha he
37 kDa p o ein ep esen ed he comple e p oca hepsin L1, and
enzyme assays showed ha i lacks ac i i y agains he sub-
s a e Z-Phe-A g-NHMec.
3
The p oduc ion o he mu an p o-
ca hepsin L1 is almos 4- old g ea e han ha o he combined
37 and 30 kDa componen s obse ed in pa allel cul u es o
wild- ype p oca hepsin L1 (see Fig. 7). Fu he mo e, he mu-
an enzyme did no p ocess o lowe molecula size o ms when
incuba ed a pH 5.0.
3
Addi ion o ecombinan wild- ype ca hepsin L1, which was
ac i a ed as desc ibed abo e, o he mu an p oca hepsin L, a
pH 5.0, esul ed in he p og essi e appea ance o a mino band
a ⬃35 kDa o e an incuba ion pe iod o 3 h and a second majo
band a 24.5 kDa ha co-mig a es wi h ully ac i e ca hepsin L
( he in ensi y o he 24.5-band inc eases o e he eac ion pe-
iod, Fig. 8). N- e minal sequencing a emp s we e unsuccess-
ul o he 35 kDa p o ein bu a sequence o HGVPY (wi h less
han 10% GVPYE) was ob ained o he 24.5 kDa p o ein; he
clea age si e is he e o e loca ed 10 amino acids p io o he N
e minus o ma u e ca hepsin L1. Expe imen s in which ac i-
a ed wild- ype ca hepsin L1 was added o hea -dena u ed
mu an p oca hepsin L showed ha he la e was sensi i e o
comple e deg ada ion by ac i e ca hepsin L1 and con i ms ha
he exp essed mu an is co ec ly olded (in ensi y o he 24.5
does no inc ease, compa e Fig. 8, Aand B).
An o e iew o he clea age si es obse ed by au oac i a ion
by he wild- ype enzyme and by in e molecula p ocessing o
mu an enzyme by exogenously added wild- ype ca hepsin L1
is shown in Fig. 9. The clea age si es de e mined o he na i e
ma u e ca hepsin sec e ed by F. hepa ica pa asi es in i o a e
also indica ed. I is clea ha au oac i a ion and in e molecu-
la ac i a ion occu by clea ages a di e en si es and hus
p ocessing o p oca hepsin L1 o an ac i e enzyme can be
achie ed by mo e han one pa hway. O pa icula no e is he
absence o a clea age si e wi hin he Gly
⫺42
-Xaa-Asn-Xaa-Phe-
Xaa-Asp
⫺36
mo i ha would gene a e he 30-kDa in e medi-
a e componen by exogenous ac i a ion. The dominance o his
clea age si e in yeas -exp essed wild- ype ca hepsin L and he
s ic conse a ion o he ou ele an esidues sugges s an
impo an ole o his mo i in he p ocessing o F. hepa ica
p oca hepsin L1. The impo ance o his mo i in he p ocessing
o papain was demons a ed by Ve ne e al. (32) using a ious
mu an s in Gly
⫺42
-Xaa-Asn-Xaa-Phe-Xaa-Asp
⫺36
sequence;
ce ain esidue eplacemen s ga e ise o some non- unc ional
inco ec ly olded enzymes, while o he s cause he accumula-
ion o inac i e o pa ly ac i e p ecu so s.
Fu he in e molecula p ocessing and ac i a ion o he 30-
kDa in e media e componen and di ec ac i a ion o p oca-
hepsin L1 by exogenous enzyme ook place in he C- e minal
po ion o he p opep ide. Elucida ion o he h ee-dimensional
s uc u e o human p oca hepsin L (14) and ca hepsin B (29,
30) e ealed ha his pa o he p opep ide is less s uc u ed
and is eadily accessible o p o eases. Tes amen o his ac is
he collec i e da a showing ha papain (32) ca hepsin L (14),
ca hepsin B (29, 30), ca hepsin S (31), and ca hepsin K (25) can
all be ac i a ed o ma u e enzymes ei he by cis- o ans-
clea age a a ious bonds wi hin his segmen . Ca hepsin B o
he helmin h pa asi e S. mansoni can also be ac i a ed by
FIG.6. Compa ison o he GXNXFXD mo i be ween papain
and ca hepsin L p o eases o helmin hs and mammals. The
GXNXFXD mo i sequence o papain is iden ical o ha o F. hepa ica
ca hepsin L1 and ca hepsin L2 and o he ca hepsin Ls o ela ed he
ema odes F. gigan ica and S. mansoni (iden ical esidues highligh ed
in black). The a ow indica es he posi ion o he in e molecula clea -
age si e, be ween he Gly
⫺42
-Leu
⫺41
bond (papain numbe ing), ha
gene a es he in e media e 30 kDa componen o he F. hepa ica p o-
ca hepsin L when i is exp essed in P. pas o is. The Gly
⫺42
is subs i-
u ed o Glu in he a and mouse ca hepsin L sequences, and o Ala
in he human ca hepsin L sequence (highligh ed in g ay, papain
numbe ing).
FIG.7.Sec e ion o he mu an p oca hepsin L1 om P. pas-
o is.P. pas o is cells ans o med wi h wild- ype p oca hepsin L1 (w )
o he p oca hepsin L1 ha had he ac i e si e Cys
26
eplaced by Gly
(mu ) we e cul u ed a 30 °C in media bu e ed o pH 6.0 and 8.0. A e
48 h o induc ion wi h 1% ( / ) me hanol samples o cul u e supe na-
an we e analyzed by SDS-PAGE. The 30-kDa in e media e o m o
p oca hepsin L was obse ed only in he wild- ype cul u es.
FIG.8. In e molecula p ocessing o p oca hepsin L1 Cys
26
-
Gly
26
mu an by exogenously added ac i e ca hepsin L1. A,pu
-
i ied p oca hepsin Cys
26
-Gly
26
mu an (5.0
␮
g) was incuba ed o up o
3hin0.1Msodium ci a e bu e , pH 5.0, con aining 1 mMDTT and
1.25 mMEDTA a 37 °C in he p esence o 0.5
␮
g o wild- ype ca hepsin
L1 ha was ac i a ed acco ding o p ocedu es shown in Fig. 5. The
posi ion o he ma u e wild- ype ca hepsin L a 24.5 kDa (2.5
␮
g,
loading) is indica ed in he lane labeled w wi h he a ow.B, pa allel
expe imen s we e ca ied as abo e bu in his case pu i ied p oca hep-
sin Cys
26
-Gly
26
mu an (5.0
␮
g) was un olded by hea ing a 95 °C o 3
min p io o he addi ion o ac i a ed wild- ype ca hepsin L1.
FIG.9.Amino acid sequence o he p opep ide o F. hepa ica
ca hepsin L1 wi h clea age si es ha lead o ac i a ion indi-
ca ed. The downwa d solid a ow indica es he clea age si e obse ed
when na i e ma u e ca hepsin L1 is sec e ed in i o by adul F.
hepa ica. The i s open a ow indica es he si e o clea age wi hin he
GXNXFXD mo i (boxed) ha gi es ise o he 30 kDa in e media e
componen when p oca hepsin L1 is exp essed in P. pas o is, and he
second and hi d open a ows show he si es o clea age when his is
u he ac i a ed in i o a pH 5.0 (see Fig. 5). The upwa d solid a ows
show he si es o clea age o p oca hepsin Cys
26
-Gly
26
mu an by exog-
enously added ma u e ca hepsin L1.
P ocessing o F. hepa ica Ca hepsin L117044
ans-clea age a his segmen (55, 56). Howe e , he si es o
clea age o he wild- ype and mu an p oca hepsin L1 di e ed
sugges ing ha p io clea age wi hin he Gly
⫺42
-Xaa-Asn-Xaa-
Phe-Xaa-Asp
⫺36
mo i o he wild- ype enzyme may ha e al-
e ed he accessibili y o he C e minus o p o ease ac ion. I is
also e iden ha bo h p ocessing pa hways lea e addi ional
amino acids a he N e minus o he ma u e enzyme, whe eas
ca hepsin L1 eco e ed om he medium in which pa asi es
we e cul u ed was ully p ocessed o he cha ac e is ic N- e -
minal esidue. This sugges s ha addi ional p o ease ac ion,
possibly by an exopep idase, may be equi ed o emo e hese
addi ional amino acids.
In he p esen s udy we show ha he sec e ion and ac i a-
ion o F. hepa ica ca hepsin L1 has simila i ies wi h ha o
hei mammalian coun e pa s. Mos impo an ly, we ha e
elucida ed ha ca hepsin Ls a e sec e ed in o he pa asi e
lumen om esicles syn hesized wi hin he gas ode mal epi-
helial cells and ha i is possible o hese o become ac i e by
au oca alysis wi hin he sligh ly acidic en i onmen o he gu .
The ca hepsin L p o eases a e syn hesized in copious amoun s
by F. hepa ica pa asi es (0.5–1.0
␮
g pe adul pa asi e pe
hou ) and ea ly au o adiog aphic s udies by Hanna (57) es i-
ma ed ha he u no e a e om he syn hesis o he con en s
o he sec e o y esicles o hei libe a ion in o he gu lumen
was apid (⬃1 h). The means by which hese enzymes a e
a icked o he sec e o y esicles and he possible in ol e-
men o he Gly
⫺42
-Xaa-Asn-Xaa-Phe-Xaa-Asp
⫺36
mo i (32) o
a pH-dependen in e ac ion wi h a memb ane-bound ecep o
(58) equi es in es iga ion. A ole o ca bohyd a e moie ies
may be excluded since he F. hepa ica p oca hepsin L s udied
he e con ains no po en ial N-glycosyla ion si es, and s udies
ha e indica ed ha he na i e enzyme is no glycosyla ed (38,
42), al hough homologs om o he helmin hs, such a S. man-
soni, a e glycosyla ed (59).
Mammalian ca hepsins play a numbe o impo an biologi-
cal unc ions such as p o ein u no e , an igen p ocessing and
issue emodeling (50). Howe e , hey ha e also been impli-
ca ed in a ious pa hological condi ions including umo in a-
sion and me as asis (50, 60, 61), os eopo osis (53, 54, 62), and
ch onic in lamma o y disease (63, 64). In pa asi es, cys eine
p o eases pe o m a numbe o pi o al unc ions such as eed-
ing, issue pene a ion and immunomodula ion ha allow
hem o in ec and es ablish disease in he hos (2, 56). A
numbe o s udies ha e demons a ed ha hese enzymes ep-
esen a ge s a which new chemo he apeu ic and immuno-
p ophylac ic s a egies can be di ec ed (2, 12, 56, 65). While he
goal o imp o ing ou knowledge on he ac i a ion mechanisms
o mammalian ca hepsin p o ease is o p o ide new ways o
ea ing he diseases hey cause, simila s udies on he pa a-
si e homologs would allow compa a i e analyses o he pu -
pose o designing pa asi e-speci ic ea men s. The easibili y
o his app oach was demons a ed in ou labo a o y by show-
ing ha p opep ides o F. hepa ica p oca hepsin Ls a e po en
inhibi o s o hei cogna e enzymes bu exhibi li le o no
ac i i y agains hei mammalian homologs.
Acknowledgmen s—We hank Nial La kin and John P. Dal on o he
an ip opep ide se um.
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