INFECTION AND IMMUNITY, Jan. 2007, p. 184–192
Copyright © 2007, American Society for Microbiology. All Rights Reserved.
Vol. 75, No. 1
Proteolytic Processing of the Cryptosporidium Glycoprotein
gp40/15 by Human Furin and by a Parasite-Derived
Furin-Like Protease Activity?
Jane W. Wanyiri,1Roberta O’Connor,1Geneve Allison,1Kami Kim,2Anne Kane,3
Jiazhou Qiu,3Andrew G. Plaut,3and Honorine D. Ward1*
Division of Geographic Medicine and Infectious Diseases1and GRASP Digestive Diseases Center,3
Tufts-New England Medical Center, 750 Washington Street, Boston, Massachusetts 02111,
and Albert Einstein College of Medicine, New York, New York2
Received 13 June 2006/Returned for modification 19 July 2006/Accepted 5 October 2006
The apicomplexan parasite Cryptosporidium causes diarrheal disease worldwide. Proteolytic processing of
proteins plays a significant role in host cell invasion by apicomplexan parasites. In previous studies, we
described gp40/15, a Cryptosporidium sp. glycoprotein that is proteolytically cleaved to yield two surface
glycopeptides (gp40 and gp15), which are implicated in mediating infection of host cells. In the present study,
we showed that biosynthetically labeled gp40/15 is processed in Cryptosporidium parvum-infected HCT-8 cells.
We identified a putative furin cleavage site RSRR2 in the deduced amino acid sequence of gp40/15 from C.
parvum and from all Cryptosporidium hominis subtypes except subtype 1e. Both human furin and a protease
activity present in a C. parvum lysate cleaved recombinant C. parvum gp40/15 protein into 2 peptides, identified
as gp40 and gp15 by size and by immunoreactivity with specific antibodies. C. hominis gp40/15 subtype 1e, in
which the RSRR sequence is replaced by ISKR, has an alternative furin cleavage site (KSISKR2) and was also
cleaved by both furin and the C. parvum lysate. Site-directed mutagenesis of the C. parvum RSRR sequence to
ASRR resulted in inhibition of cleavage by furin and the C. parvum lysate. Cleavage of recombinant gp40/15
and a synthetic furin substrate by the C. parvum lysate was inhibited by serine protease inhibitors, by the
specific furin inhibitor decanoyl-Arg-Val-Lys-Arg-chloromethylketone (Dec-RVKR-cmk), and by calcium chelators,
suggesting that the parasite expresses a Ca2?dependent, furin-like protease activity. The furin inhibitor
Dec-RVKR-cmk decreased C. parvum infection of HCT-8 cells, suggesting that a furin-like protease activity may
be involved in mediating host-parasite interactions.
The apicomplexan parasite Cryptosporidium is a significant
cause of diarrheal disease worldwide (16, 30). Cryptosporidi-
osis is asymptomatic or self-limiting in immunocompetent in-
dividuals but may be severe, chronic, and life-threatening in
immunocompromised patients, such as those with AIDS. Sev-
eral outbreaks of waterborne cryptosporidiosis have been re-
ported worldwide. Because of the potential for intentional
contamination of water supplies, Cryptosporidium is listed as a
category B priority pathogen for bioterrorism by the Centers
for Disease Control and the National Institutes of Health (27).
There are 2 main Cryptosporidium species that cause human
infections. Cryptosporidium hominis primarily infects humans,
whereas Cryptosporidium parvum infects humans as well as
other animals (33). Nitazoxanide is the only drug approved in
the United States by the Food and Drug Administration for
use in immunocompetent individuals with cryptosporidiosis.
However, this drug is not effective against cryptosporidial in-
fection in immunocompromised hosts (35). Therefore, the con-
tinued search for novel therapeutic interventions is critical.
Previously, we (5) and others (25, 28, 31) cloned Cpgp40/15,
a highly polymorphic Cryptosporidium gene which encodes a
precursor glycoprotein gp40/15 (also referred to as GP60, S60,
or Cp17). This glycoprotein is proteolytically processed to yield
mature glycopeptides gp40 and gp15, which remain nonco-
valently associated following cleavage. The C-terminal gp15
peptide is anchored in the membrane via a glycophosphatidyl
inositol linkage, is localized to the surface of invasive stages
(sporozoites and merozoites), and is shed in trails during glid-
ing motility (5, 9, 28, 31). gp15 (also referred to as Cp17 or
S16) is an immunodominant protein consistently recognized by
sera from infected persons (25). Monoclonal immunoglobulin
A (IgA) antibodies to this protein are partially protective in a
murine “backpack tumor” model (5), and the presence of
serum antibodies to Cp17 (same as gp15) correlate with pro-
tection from diarrhea in infected humans (22). The N-terminal
gp40 fragment is a secreted mucin-like glycoprotein which is
present on the surface of invasive stages (most likely in asso-
ciation with gp15) and is also shed from these stages during
gliding motility (5, 28). gp40 binds to intestinal epithelial cells,
and antibodies to gp40 inhibit C. parvum infection in vitro (5).
Both gp40 and gp15 contain mucin-type O-glycans that have
exposed Gal(?1-3)GalNAc and/or GalNAc ?1-3-Ser/Thr resi-
dues (4, 31). Lectins that recognize these residues block sporo-
zoite attachment (4) and completely and irreversibly ablate
sporozoite infectivity for host cells (10), implicating these car-
bohydrates in attachment and invasion. Taken together, all of
these studies suggest that both g40 and gp15 are important in
mediating C. parvum infection of host cells.
* Corresponding author. Mailing address: Division of Geographic
Medicine and Infectious Diseases, Tufts-New England Medical Cen-
ter, 750 Washington Street, Boston, MA 02111. Phone: (617) 636-7022.
Fax: (617) 636-5292. E-mail: firstname.lastname@example.org.
?Published ahead of print on 16 October 2006.
The Cpgp40/15 gene and its products are highly polymorphic
(28), particularly in C. hominis isolates, which cause most hu-
man infections (33). At least 8 allelic subtypes have been de-
scribed among C. parvum and C. hominis isolates based on
single-nucleotide and single-amino-acid polymorphisms at this
locus. The finding of extensive polymorphism in the Cpgp40/15
locus is consistent with its gene products being surface-associ-
ated virulence determinants that may be under selective host
immune pressure and indirectly supports a role for these gly-
coproteins in mediating infection.
Proteolytic processing is a common posttranslational modi-
fication of a number of proteins involved in attachment and
invasion of apicomplexan parasites such as Plasmodium spp.
and Toxoplasma spp. (reviewed in references 3, 13, and 32).
Processing of these proteins occurs either during transport
through the secretory pathway or after secretion onto the par-
asites’ surface. In many cases, proteolytic processing of these
proteins has been shown to be essential for invasion of host
cells by these parasites, raising the possibility that the proteases
involved in processing may represent potential targets for in-
tervention. However, proteolytic processing of Cryptospo-
ridium sp. proteins has not been previously reported.
The aim of this study was to examine the processing of
Cryptosporidium sp. gp40/15 and to characterize the pro-
tease(s) responsible. Examination of the deduced amino acid
sequence of gp40/15 revealed an arginine-rich amino acid se-
quence, RSRR, located upstream of the amino terminus of
mature gp15 (Fig. 1). RSRR2 is a signature recognition site
for the serine endoprotease furin, which cleaves C-terminal to
an arginine residue that is preceded by one or more basic
residues at P2, P4, or P6 (starting at the scissile bond which is
indicated by 2 and counting toward the N terminus, substrate
residues are designated P1, P2, and so on) (26). Furin, a mem-
ber of the subtilisin-like proprotein convertase family, is a
major processing enzyme of the constitutive secretory pathway
(26, 29). In addition to its role in processing a number of
mammalian precursor proteins, furin also processes precursors
of pathogen-derived proteins, such as bacterial toxins and virus
envelope glycoproteins (26). In this study, we identified the
cleavage site of gp40/15 and showed that it is processed by
human furin and by protease activity in C. parvum lysates that
has furin-like substrate specificity. The enzyme(s) responsible
for this activity has not been identified.
MATERIALS AND METHODS
Reagents. The fluorogenic furin substrate, t-butyloxycarbonyl-Arg-Val-Arg-
Arg-7-amino-4-methylcoumarin (Boc-RVRR-AMC) was obtained from Bachem
Biosciences (King of Prussia, PA). Recombinant human furin was obtained from
New England Biolabs (Beverley, MA). The furin inhibitor decanoyl-Arg-Val-
Lys-Arg-chloromethylketone (Dec-RVKR-cmk) was obtained from Bachem
Biosciences. The peptidylchloromethylketone serine protease inhibitors Phe-
Pro-Arg-chloromethylketone (PPACK) and Glu-Gly-Arg-chloromethylketone
(GGACK) were obtained from Haematologic Technologies, Inc. (Essex Junc-
Cells. Human ileocecal adenocarcinoma cells (HCT-8) were propagated in
Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen, Carlsbad, CA) sup-
plemented with 10% fetal bovine serum (FBS), 25 mM HEPES, 4 mM L-
glutamine, penicillin (100 U/ml), and streptomycin (100 ?g/ml) (complete
DMEM) at 37°C in 5% CO2.
Parasites. C. parvum (Iowa isolate) oocysts were obtained from Bunch Grass
Farms, Deary, ID. Prior to use, oocysts were treated with 1.75% (vol/vol) sodium
hypochlorite for 10 min on ice, washed with 20 mM phosphate buffer, pH 7.2,
containing 150 mM sodium chloride (phosphate-buffered saline [PBS]). Oocysts
(109) were excysted by incubation in 1 ml of PBS at 37°C for 1 h. The mixture of
excysted oocysts and sporozoites was lysed by detergent extraction with 1% (vol/vol)
Triton X-100 on ice for 1 h. The Triton X-100 lysate was adjusted to 20 mM
2-(N-morpholino)ethanesulfonic acid (MES), pH 7.0, 1 mM CaCl2, 0.1% Triton
and the supernatant (henceforth referred to as C. parvum lysate) was used for
subsequent assays. To determine the effect of Dec-RVKR-cmk on excystation, 4 ?
105hypochlorite-treated oocysts were suspended in DMEM and incubated with
concentrations of Dec-RVKR-cmk ranging from 1 to 500 ?M for 1 h at 37°C, and
the percent excystation was calculated as follows: (number of excysted oocysts/total
number of oocysts counted) ? 100. The effect of Dec-RVKR-cmk on the viability of
excysted sporozoites was assessed by uptake or exclusion of the fluorogenic dyes
fluorescein diacetate and propidium iodide, respectively, as described previously (2).
Antibodies. 4E9 is an IgM monoclonal antibody (MAb) directed against a
carbohydrate epitope on gp40 (5). CrA1 is an IgA MAb directed against a
protein epitope on gp15 (5). Rabbit antisera against recombinant gp40 and gp15
were generated (R. M. O’Connor, J. W. Wanyiri, and H. D. Ward, unpublished
Metabolic labeling of intracellular parasite proteins. HCT-8 cells were grown
to confluence in six-well plates and infected with 4 ? 106C. parvum oocysts per well.
Twenty-four hours after infection, the culture medium was replaced with methio-
nine/cysteine-free DMEM supplemented with 10% dialyzed FBS, with or without
1.7 ?g/ml of ricin (Vector Laboratories, CA) to block host cell protein synthesis.
Infected HCT-8 cells were incubated for 1.5 h at 37°C, 5% CO2prior to the addition
to 10 min. Infected HCT-8 cells were washed once in PBS and then extracted for 30
min on ice with radioimmunoprecipitation assay (RIPA) lysis buffer (50 mM Tris
buffer, pH 7.5, 100 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, and
0.2% [wt/vol] sodium dodecyl sulfate [SDS]) containing 10 ?g/ml RNase (Ambion),
20 ?g/ml DNase 1 (Ambion), and the following protease inhibitors: 100 ?M
N-tosyl-L-lysine chloromethyl ketone (TLCK; Calbiochem), 100 ?M (4-amidino-
phenyl)-methane-sulfonyl fluoride (Sigma), 2.3 mM leupeptin (Sigma), 10 ?M trans-
epoxysuccinyl-L-leucylamido-(4-guanidino) butane (E64; Sigma), and 40 ?g/ml
acetyl-L-leucyl-L-leucyl-L-norleucinal (Calbiochem). The lysate was centrifuged at
2,000 ? g for 10 min, and the supernatant was collected for immunoprecipitation.
Immunoprecipitation. Five microliters of rabbit anti-gp40 or preimmune se-
rum was incubated with 100 ?l of the supernatant overnight at 4°C with gentle
rocking, followed by incubation with prewashed protein G-Sepharose (Amer-
sham Biosciences, Piscataway, NJ) overnight at 4°C. After being washed (15,000 ?
g for 15 s) 10 times with RIPA lysis buffer, the bound immune complexes were
eluted by boiling in SDS-polyacrylamide gel electrophoresis (PAGE) sample
buffer, separated by SDS-PAGE and transferred to a polyvinylidene difluoride
(PVDF) membrane (Millipore, Billerica, MA). The membrane was treated with
Autoflour (Atlanta, GA), dried, and exposed to film at ?70°C.
FIG. 1. Deduced amino acid sequence of the portion of gp40/15
surrounding the N terminus of gp15 from C. parvum and C. hominis
subtypes. gp40/15 sequences from C. parvum (Cp) (AF155624) and C.
hominis (Ch) subtypes 1a (AF440634), 1b (AF440626), 1c (AF440622),
1d (AF440625), 1e (AF440629), 1f (AY700389), and 1g (AY700395)
were aligned using the Clustal W algorithm of the Vector NTI v8.0
program (Invitrogen). GenBank accession numbers of the sequences
are in parentheses. Differences are indicated in boldface type, and the
putative furin consensus cleavage sequences are underlined. The glu-
tamic acid residue corresponding to the previously determined N ter-
minus of mature native C. parvum gp15 is in italics.
VOL. 75, 2007 PROCESSING OF CRYPTOSPORIDIUM GLYCOPROTEIN gp40/15 185
Production and purification of recombinant gp40/15 (rgp40/15) from C. par-
vum and C. hominis subtype 1e. The C. parvum gp40/15 coding sequence was
PCR amplified from C. parvum (GCH1 isolate) genomic DNA as described
previously (5). The C. hominis gp40/15 subtype 1e coding sequence was PCR
amplified from DNA isolated from the stool of a South Indian child infected with
C. hominis subtype 1e (obtained as a kind gift from Sitara Rao and Gagandeep
Kang, Christian Medical College Hospital, Vellore, India) using the sense primer
5?-GGTATTGAGGGTCGCGATGTTTCTGTTGAGAGC-3? and the anti-
sense primer 5?-AGAGGAGAGTTAGAGCCGATGTATCTAAATCCAAAA
GC-3?. Both sequences were cloned into the pET32Xa/LIC vector (Novagen,
Madison, WI), which contains an internal S tag, internal and C-terminal His tags,
and N-terminal thioredoxin (Trx) tag sequences. Overexpression in Escherichia
coli AD494(DE3) (C. parvum) or E. coli Origami B(DE3)(pLysS) (C. hominis
subtype 1e) (both E. coli cells strains were obtained from Novagen) was induced
with 1 mM isopropyl-?-thiogalactopyranoside (Gold Biotechnology, Inc., St.
Louis, MO) as described previously (5). Site-directed mutagenesis was per-
formed using the QuickChange 11 XL site-directed mutagenesis kit (Stratagene,
La Jolla, CA) according to the manufacturer’s protocol.
Site-directed mutations were designed to convert C. parvum gp40/15 Arg215to
Ala215. The sense primer 5?-GCGGGTCAGGCTTCATCAGCGTCAAGAAG
ATCACTCTCAG-3? and antisense primer 5?-CTGAGAGTGATCTTCTTGAC
GCTGATGAAGCCTGACCCGC-3? (the mutated nucleotides are underlined)
were designed using Primer X (http://bioinformatics.org/primerx) and synthe-
sized by Operon Biotechnologies (Huntsville, AL). Recombinant fusion proteins
were purified by nickel-nitrilotriacetic acid metal affinity chromatography using
Ni-NTA Superflow resin (QIAGEN, Valencia, CA) in accordance with the
manufacturer’s directions. Protein concentration was measured with the bicin-
choninic acid protein assay kit (Pierce Chemical Co., Rockford, IL).
In vitro cleavage of rgp40/15. One to two micrograms of rgp40/15 was incu-
bated with 1 to 2 U of furin or 30 ?l of C. parvum lysate (equivalent to 3 ?107
oocysts) in a final volume of 50 ?l of enzyme buffer at 37°C. For inhibition
experiments, 50 mM EDTA, 10 mM 4-(2-aminoethyl)benzenesulphonyl fluoride
(AEBSF), 10 mM phenylmethylsulfonyl fluoride (PMSF), 10 mM leupeptin, or
100 ?M Dec-RVKR-cmk was preincubated with 1 U of furin or 30 ?l of C.
parvum lysate (equivalent to 3 ?107oocysts) for 30 min at room temperature
before the addition of gp40/15. The reactions were stopped by the addition of
SDS-PAGE sample buffer, followed by heating at 95°C for 5 min. Cleavage
products were resolved by SDS-PAGE and detected by Coomassie blue staining
and Western blotting with horseradish peroxidase-conjugated S-protein (Nova-
gen, Madison, WI) that recognizes the S tag in the recombinant protein. Iden-
tities of the cleavage products were confirmed by Western blotting with rabbit
anti-rgp40 antiserum and anti-gp15 MAb CrA1 (5).
N-terminal amino acid sequencing. Following resolution of rgp40/15 cleavage
products by SDS-PAGE, the proteins were electrotransferred to PVDF mem-
branes and identified by Coomassie blue staining in two separate experiments.
The ?12-kDa bands from each experiment, identified as gp15 by immunoblot-
ting a parallel strip of the membrane, were excised and processed for limited
NH2-terminal sequence analysis of the first 5 amino acids by automated Edman
degradation at the Tufts University Core facility.
Enzyme assays. The activities of furin and C. parvum lysate were monitored
using the synthetic peptidyl fluorogenic substrate Boc-RVRR-AMC. The en-
zyme sample (either 1 U of furin or 1 ?l of C. parvum lysate [equivalent to 106
oocysts]) was incubated with enzyme buffer containing 1 mM Boc-RVRR-AMC
in 96-well microtiter plates at 37°C for various times. For inhibition studies, the
enzyme sample was preincubated with 0.1 to 5 mM concentrations of the fol-
lowing inhibitors at room temperature for 1 h prior to the addition of substrate:
aprotinin, E64, AEBSF, pepstatin A, PMSF, TLCK, iodoacetamide, leupeptin,
or 1,10-phenanthroline. The fluorescence due to released AMC was measured
with a spectrophotometer (1420 VICTOR multilabel counter; Perkin-Elmer Life
Sciences, Boston, MA) at an excitation wavelength of 380 nm and an emission
wavelength of 460 nm.
To determine the pH optimum for the furin-like protease activity in the C.
parvum lysate, the assay was performed in enzyme buffer with the pH adjusted in
0.5-pH unit increments ranging from 4.0 to 8.0 using either 10% glacial acetic
acid or 1 M sodium hydroxide in water as described previously (21). For deter-
mination of the Michaelis-Menten kinetic parameters Vmaxand Km, C. parvum
lysates were treated with increasing concentrations of Boc-RVRR-AMC ranging
from 1 ?M to 1 mM in 100 ?l of enzyme buffer in 96-well microtiter plates and
incubated for 60 min at 37°C. Assay values were transformed into ?M AMC
produced/min using a standard curve. Data were analyzed using GraphPad
PRISM4 software (GraphPad Software, San Diego, CA).
Effect of Dec-RVKR-cmk on C. parvum infection in vitro. Hypochlorite-steril-
ized oocysts (5 ? 104/well) in DMEM were preincubated with increasing con-
centrations of Dec-RVKR-cmk or with DMEM alone at 37°C for 30 min and
then added to confluent HCT-8 cell monolayers grown in 96-well microtiter
plates. The cells were then incubated for 24 h at 37°C in 5% CO2. Infection was
quantified using an enzyme-linked immunosorbent assay-based infection assay,
as described previously (5). In other experiments, HCT-8 cells were preincubated
with 2, 20 or 200 ?M Dec-RVKR-cmk for 30 min at 37°C and then washed 3
times with warm (37°C) complete DMEM prior to the addition of oocysts. In
parallel, oocysts were treated with the same concentrations of Dec-RVKR-cmk
for 30 min at 37°C and then washed 3 times with warm (37°C) complete DMEM
prior to incubation with the host cells.
HCT-8 cell viability assay. HCT-8 cell monolayers were treated with various
concentrations of Dec-RVKR-cmk in DMEM (without phenol red) supplemented
with 10% FBS, 25 mM HEPES, 4 mM L-glutamine, penicillin (100 U/ml), and
streptomycin (100 ?g/ml) and incubated for 24 h at 37°C in 5% CO2. Viability of the
cells was determined using a 3-[4,5-dimethylthiazol-2]-2,5-diphenyltetrazolium bro-
mide cell proliferation assay kit (Molecular Probes, Eugene, OR).
Statistical analysis. All assays were performed in duplicate or triplicate, and
the means and standard deviations were determined. All experiments were
repeated at least twice. Statistical analysis was performed using GraphPad
PRISM 4 software. Analysis of variance was used to compare differences in
Biosynthetically labeled gp40/15 is processed in C. parvum-
infected HCT-8 cells. Previous studies suggested that gp40/15
is proteolytically processed to yield gp40 and gp15 (5, 28). This
conclusion was based on reactivity of both gp40- and gp15-
specific antibodies with a ?49- to 60-kDa protein and reactivity
of ?40- to 45-kDa and ?12- to 15-kDa proteins with gp40- and
gp15-specific antibodies, respectively, in C. parvum-infected
intestinal epithelial cells. To directly demonstrate processing of
gp40/15, we biosynthetically labeled parasite proteins in in-
fected HCT-8 cells with [35S]methionine/cysteine. Ricin was
used to selectively block host cell protein synthesis (8). Labeled
proteins were immunoprecipitated with gp40-specific antisera
(gp15 contains neither methionine nor cysteine residues and,
hence, cannot be labeled). Under these conditions, we were
able to immunoprecipitate two proteins with relative molecu-
lar masses of ?49 and 40 kDa from infected but not uninfected
HCT-8 cells (Fig. 2A). The processed 40-kDa band was de-
tected after pulse-labeling for only 2 min (the earliest time at
which incorporation of radiolabel could be detected) (data not
shown), suggesting that processing occurred soon after syn-
Human furin cleaves recombinant C. parvum and C. hominis
gp40/15 in vitro. Previous studies reported that the N terminus
of the mature gp15 peptide is a glutamic acid (E) residue in the
C-terminal portion of gp40/15 (Fig. 1). Examination of the
amino acid sequence surrounding this residue identified an
arginine-rich amino acid stretch containing a putative furin
cleavage site, RSRR2 (Fig. 1). To determine if furin could
cleave C. parvum gp40/15 in vitro, we cloned and overex-
pressed recombinant gp40/15 in E. coli (5) and treated the
purified fusion protein rgp40/15 (which migrates with the ex-
pected relative molecular mass of 52 kDa on SDS-PAGE) with
recombinant human furin. This resulted in cleavage of
rgp40/15 into 2 fragments on SDS-PAGE with a relative mo-
lecular mass corresponding to the expected relative molecular
mass of ?40 kDa for rgp40 and ?12 kDa for rgp15 (Fig. 2B).
This was confirmed by Western blotting with S-protein, which
binds to the N-terminal S tag in the rgp40/15 and rgp40 fusion
proteins but does not bind to the C-terminal rgp15 fragment
(Fig. 2C). Cleavage was completely inhibited by EDTA, con-
186WANYIRI ET AL.INFECT. IMMUN.
sistent with the known requirement for Ca2?by furin (21) and
was also inhibited by the specific furin inhibitor Dec-RVKR-
cmk (11) (Fig. 2B). Western blotting using gp40- and gp15-
specific antibodies showed that the ?52-kDa and 40-kDa
bands reacted with the anti-gp40 antibody (Fig. 2D) and the
?12-kDa band reacted with CrA1, an anti-gp15 monoclonal
antibody (Fig. 2E). The immunoreactivity of the cleavage
products with gp40- and gp15-specific antisera was consistent
with that observed following cleavage of native gp40/15 (5).
Although gp40/15 is highly polymorphic in C. hominis iso-
lates, the RSRR sequence is conserved in 6 of 7 C. hominis
subtypes. However, in subtype 1e, this sequence is replaced
FIG. 2. Native gp40/15 is processed in C. parvum-infected HCT-8 cells, and recombinant gp40/15 is processed by human furin. (A) HCT-8 cells
infected with C. parvum for 24 h were biosynthetically labeled with [35S]methionine/cysteine for 10 min. Cell lysates were immunoprecipitated with
antiserum to rgp40 or preimmune serum, and the immunoprecipitated proteins were resolved by SDS-PAGE and transferred to PVDF mem-
branes, followed by autoradiography. Lane 1, total lysate; lane 2, immunoprecipitation with preimmune serum; lane 3, immunoprecipitation with
rabbit anti-rgp40 serum. (B to F) rgp40/15 from C. parvum (B, C, D, and E) or C. hominis subtype 1e (F) was incubated with human furin at 37°C
in the presence or absence of EDTA or Dec-RVKR-cmk. Cleavage products were resolved by SDS-PAGE, followed by Coomassie blue staining
(B) or Western blotting with S-protein (C and F), anti-gp40 (D), and anti-gp15 (E) antibodies. Lane 1, untreated gp40/15; lane 2, gp40/15 treated
with furin; lane 3, gp40/15 treated with furin and Dec-RVKR-cmk; lane 4, gp40/15 treated with furin and EDTA; lane 5, gp40/15 treated with
FIG. 3. Recombinant gp40/15 is processed into gp40 and gp15 by a protease activity in C. parvum lysate. rgp40/15 was treated with C. parvum
lysate for 2 h at 37°C in the presence or absence of serine protease inhibitors, EDTA, or Dec-RVKR-cmk. Cleavage products were resolved by
SDS-PAGE, followed by Coomassie blue staining (A) or Western blotting with S-protein (B and C). (A) Lane 1, untreated rgp40/15; lane 2,
rgp4015 treated with C. parvum lysate; (B) lane 1, untreated rgp40/15; lane 2, rgp40/15 treated with C. parvum lysate; lane 3, rgp40/15 treated with
C. parvum lysate and EDTA; lane 4, rgp40/15 treated with C. parvum lysate and AEBSF; lane 5, rgp40/15 treated with C. parvum lysate and PMSF;
lane 6, rgp40/15 treated with C. parvum lysate and leupeptin; (C) lane 1, untreated rgp40/15; lane 2, rgp40/5 treated with C. parvum lysate; lane
3, rgp40/5 treated with C. parvum lysate and Dec-RVKR-cmk.
VOL. 75, 2007 PROCESSING OF CRYPTOSPORIDIUM GLYCOPROTEIN gp40/15187
with ISKR. This sequence meets the requirement of an argi-
nine residue at P1 and has the preferred basic residue for furin
cleavage at P2 but lacks a basic residue in the P4 position.
However, previous studies have shown that a basic (arginine or
lysine) residue in the P2 and P6 position can compensate for an
unfavorable residue at P4 (15). The subtype 1e sequence has a
lysine residue at P6 and could thus potentially be cleaved by
furin. To determine if this was the case, we cloned and over-
expressed gp40/15 from this subtype in E. coli and treated the
recombinant fusion protein with furin. As seen in Fig. 2F, this
protein was indeed cleaved by furin, although the cleavage was
A C. parvum-derived, furin-like serine protease activity
cleaves recombinant gp40/15. To determine whether C. par-
vum expresses enzyme/s that can cleave gp40/15, we treated
purified rgp40/15 with a crude C. parvum lysate. Figure 3A
demonstrates that the C. parvum lysate partially cleaved
rgp40/15 in vitro, generating fragments whose relative molec-
ular mass on SDS-PAGE corresponded to those of rgp40 and
rgp15. To further characterize the protease activity responsible
for this cleavage, we preincubated the C. parvum lysate with
the divalent cation chelator EDTA, the serine protease inhib-
itors AEBSF, PMSF, and leupeptin or the specific furin inhib-
itor Dec-RVKR-cmk before adding the rgp40/15. EDTA in-
hibited cleavage, indicating the requirement of divalent cations
for enzymatic activity. AEBSF, PMSF, and leupeptin also in-
hibited the cleavage of rgp40/15 by the C. parvum lysate (Fig.
3B), indicating that cleavage is due to a serine-like protease
activity. Furthermore, processing was inhibited by Dec-RVKR-
cmk, suggesting that the protease activity has a furin-like spec-
ificity (Fig. 3C). The C. hominis gp40/15 subtype 1e was also
cleaved by the C. parvum lysate (data not shown).
A mutation in the RSRR cleavage site prevents processing of
gp40/15 by furin and by the C. parvum lysate. To further
investigate the sequence requirement for cleavage, we used
site-directed mutagenesis to generate a gp40/15 mutant by
changing the P4 arginine residue to alanine (R215A). gp40/15
(R215A) was not cleaved either by furin or by C. parvum lysate
(Fig. 4). These data indicate that this protease activity, like
furin, requires a basic amino acid in the P4 position for activity.
Human furin and the C. parvum furin-like protease activity
cleave rgp40/15 at similar sites. To compare the sites at which
human furin and the C. parvum furin-like protease activity
cleaved gp40/15, we carried out limited amino-terminal se-
quence analysis on the gp15 fragment obtained after cleavage
of rgp40/15 with these enzymes. The N-terminal amino acid
sequence of the gp15 fragment obtained following furin cleav-
occurred C-terminal to the P1 residue R218(Table 1). How-
ever, the N-terminal sequence of the fragment obtained by
treatment with C. parvum lysate was221SEETS225, indicating
that the parasite protease activity cleaved 2 residues down-
stream of the furin cleavage site (Table 1), or that the gp15
product underwent amino-terminal modification, perhaps by a
C. parvum aminopeptidase (1, 24).
The C. parvum protease activity is similar to that of furin in
its enzyme kinetics, inhibitor profile, pH optimum, and cal-
cium dependence. Since the C. parvum protease activity that
processed gp40/15 was similar to that of furin in its substrate
specificity, it was of interest to determine if this protease ac-
tivity exhibited similar enzyme kinetics. We first showed that
the protease activity in the C. parvum lysate could cleave Boc-
RVRR-AMC (a synthetic fluorogenic substrate that is effi-
ciently cleaved by furin) (23) in a time- and dose-dependent
manner (data not shown).
We then determined the enzyme kinetics for cleavage of the
Boc-RVRR-AMC peptide by the C. parvum lysate. The rate
was assayed at various substrate concentrations using iden-
tical amounts of lysate. Initial velocity data were then used
to establish apparent Kmand Vmaxvalues for the C. parvum
protease activity. Using these data for Lineweaver-Burke
analysis, we determined a Kmof 15.17 ? 6.4 ?M and a Vmax
of 0.013 ? 0.001 ?M/min. This Kmvalue is lower than the
Kmof 25.9 ? 0.3 ?M reported for furin (21), suggesting that
the C. parvum protease activity has an even greater affinity
To further characterize the C. parvum protease activity, we
219SLSEE223, indicating that, as expected, cleavage
FIG. 4. A mutation in the RSRR cleavage site inhibits processing
of gp40/15 by furin and by a protease activity in C. parvum lysate.
rgp40/15 and rgp40/15 (R215A) were treated with furin or C. parvum
lysate overnight at 37°C. Cleavage products were resolved by SDS-
PAGE, followed by Coomassie blue staining (A) or Western blotting
with S-protein (B). (A) Lane 1, untreated rgp40/15; lane 2, rgp40/15
treated with furin; lane 3, untreated rgp40/15 (R215A); lane 4, rgp40/15
(R215A) treated with furin; (B) lane 1, untreated rgp40/15; lane 2,
rgp40/15 treated with C. parvum lysate; lane 3, untreated rgp40/15
(R215A); lane 4, rgp40/15 (R215A) treated with C. parvum lysate.
TABLE 1. N-terminal sequence of gp15
C. parvum gp15 (GCH1 isolate)a
C. parvum Cp17 (Maine isolate)a
C. parvum Cp17 (Maine isolate)b
C. parvum gp15 (Iowa isolate)a
C. parvum S16 (naturally infected
C. parvum S16 (naturally infected
rgp15cfrom human furin cleavageb
rgp15cfrom C. parvum lysate
aDeduced amino acid sequence.
bExperimentally determined amino acid sequence.
cE. coli-expressed recombinant.
188WANYIRI ET AL.INFECT. IMMUN.
determined the effect of various classes of protease inhibitors
on the hydrolysis of Boc-RVRR-AMC by the lysate. The re-
sults are shown in Table 2. Aprotinin, an inhibitor of trypsin-
like serine proteases, had no effect on the furin-like protease
activity, whereas other serine protease inhibitors, including
AEBSF, leupeptin, TLCK, and PMSF, inhibited activity to a
varying extent. In addition, the metalloproteinase inhibitor
1,10-phenanthroline also significantly inhibited this activity.
This profile suggests that the C. parvum lysate contains metal-
lodependent serine protease activity.
To further assess the substrate specificity of the C. parvum
protease activity, we investigated the inhibitory effects of var-
ious peptidylchloromethylketones on cleavage of Boc-RVRR-
AMC by the lysate. The specific furin inhibitor Dec-RVKR-
cmk, which has a pair of basic residues at P1 and P2 combined
with an arginine at P4, was the most inhibitory, while both
PPACK and GGACK, which contain a single arginine at P1,
were poor inhibitors (Table 2). These results confirm that the
protease activity present in the C. parvum lysate has furin-like
The pH optimum for cleavage of Boc-RVRR-AMC by the
C. parvum lysate was assayed in buffers of different pH values
ranging from 4.0 to 8.0 and was found to be active over a broad
pH range. Peak activity was observed at pH 7.0, which is
identical to that of furin (21).
A direct determination of the Ca2?requirement of the C.
parvum furin-like protease activity was not possible, since the
crude lysate preparation already contains Ca2?. We therefore
determined how much EDTA was required to fully inhibit
activity. As shown in Table 3, 50 mM EDTA almost completely
(97%) eliminated hydrolysis of Boc-RVRR-AMC. The addi-
tion of 3 mM Ca2?reversed the inhibition by EDTA (Table 3).
Of the divalent cations tested (Ca2?, Mg2?, and Mn2?), only
Ca2?was effective in reversing the inhibitory effect of the
EDTA (Table 3). This is consistent with the finding that
EGTA, which specifically chelates Ca2?, also inhibits the furin-
like activity of the lysate (Table 3). Since cleavage of the
synthetic substrate Boc-RVRR-AMC and of rgp40/15 were
both blocked by the same serine protease inhibitors as well as
by Dec-RVKR-cmk and EDTA, it is very likely that the pro-
tease activity in the C. parvum lysate that cleaves Boc-RVRR-
AMC is responsible for processing gp40/15.
Dec-RVKR-cmk inhibits C. parvum infection of HCT-8 cells.
Since gp40 and gp15 are implicated in mediating C. parvum
infection, it was of interest to determine if inhibitors of pro-
teolytic cleavage of gp40/15 had any effect on infection of host
cells by the parasite in vitro. As shown in Fig. 5, the specific
furin inhibitor Dec-RVKR-cmk inhibited infection of HCT-8
cells by C. parvum in a dose-dependent manner, with ?50%
inhibition occurring at a concentration of 10 ?M. The inhibi-
tory effects of Dec-RVKR-cmk were not due to a toxic effect
on the host cells or the parasite, since the inhibitor (at the
maximal concentration used) had no effect on either host cell
or parasite viability (data not shown). In addition, inhibition of
FIG. 5. The specific furin inhibitor Dec-RVKR-cmk inhibits C. par-
vum infection of HCT-8 cells. HCT-8 cells were infected for 24 h with
oocysts that had first been pretreated with various concentrations of
Dec-RVKR-cmk (or medium alone as a control) for 30 min. Infection
was quantified using an enzyme-linked immunosorbent assay-based
infection assay. Each data point represents the mean ? standard de-
viation of results from two separate experiments, each performed in
triplicate. ?, P ? 0.01; ??, P ? 0.001.
TABLE 2. Effect of protease inhibitors on cleavage of Boc-RVRR-
AMC by C. parvum protease activity
Class of protease
Activity (% of control) at
100 ? 0 100 ? 0.0
Serine and cysteine
8 ? 0.0
48 ? 0.2
46 ? 0.4
61 ? 11
64 ? 14
59 ? 1.8
2 ? 0.1
11 ? 0.0
12 ? 0.0
12 ? 1.6
30 ? 4.0
34 ? 8.0
85 ? 13
81 ? 15
41 ? 3.4
59 ? 13
79 ? 0.2
86 ? 7.8
70 ? 1.1
88 ? 6.9
Iodoacetamide93 ? 1.8 95 ? 1.6
131 ? 4.6110 ? 7.0
aActivity determined by hydrolysis of Boc-RVRR-AMC. Data are means ?
standard deviations of results from two separate experiments, each performed in
bDoes not inhibit subtilisins.
TABLE 3. Requirement of divalent cations for cleavage of Boc-
RVRR-AMC by C. parvum protease activity
Activity (% of control)a
None (control, enzyme buffer).........................................100 ? 0
EDTA (10)......................................................................... 66 ? 1.5
EGTA (50)......................................................................... 15 ? 8.3
CaCl2(3).............................................................................110 ? 5.2
EDTA (50) ? CaCl2(3)................................................... 82 ? 3.5
EDTA (50) ? MgCl2(3).................................................. 11 ? 1.1
EDTA (50) ? MnCl2(3) ................................................. 11 ? 0.3
2 ? 2.3
aActivity determined by hydrolysis of Boc-RVRR-AMC. Data are means ?
standard deviations of results from two separate experiments, each performed in
VOL. 75, 2007PROCESSING OF CRYPTOSPORIDIUM GLYCOPROTEIN gp40/15189
infection was not due to prevention of oocyst excystation, since
excystation was not altered in the presence of the inhibitor
(data not shown). Inhibition of infection did not appear to be
due to an effect on HCT-8 cell-derived furin, since preincuba-
tion of the cells with Dec-RVKR-cmk followed by washing of
the cells prior to addition of oocysts had no significant effect on
infection (data not shown). However, when oocysts were pre-
treated with the same concentrations of the inhibitor and the
inhibitor was washed off before the cells were infected, there
was a decrease in infection, albeit not as great as when the
inhibitor was present throughout the infection (data not
shown), suggesting that the inhibition of infection was due to
an effect on the parasite-derived furin activity rather than the
host cell furin.
Proteolytic processing of surface and apical proteins of api-
complexans such as Plasmodium spp. and Toxoplasma spp.
plays a critical role in invasion of host cells by these parasites
(3, 13, 32). Here we show that the Cryptosporidium glycopro-
tein gp40/15 is proteolytically processed by human furin and by
a parasite-derived furin-like protease activity, into gp40 and
gp15, surface glycopeptides that are implicated in mediating
parasite infection of host cells.
Immunoprecipitation of biosynthetically labeled C. parvum
proteins from infected HCT-8 cells confirmed that gp40/15 is
processed during infection of host cells. Labeling was facili-
tated by using ricin to block incorporation of radiolabel into
host cell proteins. This approach has been used for selective
labeling of intracellular proteins of other apicomplexans, such
as Toxoplasma and Eimeria (8), but has not previously been
reported for Cryptosporidium spp. Detection of processed
gp40/15 as early as 2 min after pulse-labeling suggests that
cleavage is likely to occur during passage through the secretory
pathway, as is the case with a number of T. gondii and P.
falciparum proteins (12, 13).
The arginine-rich RSRR consensus in the gp40/15 protein
sequence from C. parvum and all C. hominis subtypes except C.
hominis subtype 1e matched a substrate consensus sequence
that is recognized by subtilisin-like proconvertases such as
furin (26, 29). Furin requires arginine residues at P1 and P4
and prefers lysine or arginine residues at P2, leading to our
prediction that gp40/15 would be a substrate for this enzyme.
Cleavage of rgp40/15 into products of the appropriate size and
immunoreactivity by human furin confirmed that this protein
was indeed a substrate for this enzyme. This raised the possi-
bility that gp40/15 may be cleaved by host-derived furin, con-
sistent with furin cleavage of other pathogen-derived virulence
determinants (26). However, we also found that recombinant
gp40/15 could be cleaved by protease activity present in a C.
parvum lysate. While host-derived furin is present in the intes-
tine (18), which is the site of infection, the finding that pro-
cessing occurs soon after synthesis of the precursor supports
the notion of cleavage of gp40/15 by a parasite-derived pro-
Site-directed mutagenesis of the furin cleavage consensus
site from RSRR to ASRR resulted in complete inhibition of
gp40/15 cleavage by both furin and the C. parvum lysate, dem-
onstrating that, like furin, the C. parvum protease activity re-
quires a basic residue at P4. Furin cleavage of the C. hominis
gp40/15 1e in which the RSRR sequence is replaced by ISKR,
indicates that the lack of a basic residue at P4 is compensated
by a basic residue (lysine) at P6, as seen in other studies (14).
Interestingly, although the RSRR sequence in gp40/15 is con-
served in all the other subtypes, the 1e subtype is the only one
that has a basic residue at the P6 position. Despite the fact that
gp40/15 is highly polymorphic, the finding of conserved puta-
tive furin cleavage sites among all the known subtypes suggests
that processing of this protein is functionally important.
The finding that human furin cleaved, as expected, C-termi-
nal to the P1 arginine, whereas the N terminus of the gp15
fragment derived from cleavage by the parasite protease activ-
ity was 2 residues downstream suggests that either (i) furin and
the C. parvum protease activity cleave gp40/15 at slightly dif-
ferent sites or (ii) both cleave at the same site, but the gp15
product of cleavage by the C. parvum protease activity is pro-
cessed further by other proteases present in the lysate. The
latter possibility is consistent with a previous report of a C.
parvum aminopeptidase (24), the presence of aminopeptidase
genes in the C. parvum genome (1), and the finding by Winter
et al. (31) of four forms of native S16 (same as gp15) with
different N termini (two of which are shown in Table 1). These
investigators also suggested that an aminopeptidase activity of
C. parvum might be responsible for the observed “ragged end”
of the S16 (gp15) cleavage product. Additional processing fol-
lowing furin cleavage has also been previously reported for
other substrates (18).
The C. parvum-derived protease activity is similar to the
enzymatic activity of furin in many respects. In addition to
similarities in their substrate specificity, cleavage of rgp40/15
by both enzyme activities was inhibited by the highly specific
furin inhibitor Dec-RVKR-cmk. Like furin, the C. parvum
protease activity was active across a broad pH range, with an
optimum at 7.0, and is Ca2?dependent. The inhibitor profile
of cleavage of the furin-specific substrate Boc-RVRR-AMC by
the C. parvum protease activity also demonstrated similarities
with furin. Aprotinin, a serine protease inhibitor that has no
activity against subtilisins, had no effect on the C. parvum
protease activity, whereas the other serine protease inhibitors,
PMSF, TLCK, and AEBSF, were inhibitory, albeit at relatively
high concentrations. Although inhibition by PMSF and related
sulfonyl fluorides are considered diagnostic for serine pro-
teases, subtilisin-like proconvertases such as furin and Kex2
are also only inhibited by these compounds at high concentra-
tions (21). Although the results of our study support the pres-
ence of furin-like protease activity in C. parvum lysates, we
have no evidence at present to suggest that this activity is due
to a single enzyme.
Previous studies indicated that gp40 and gp15 are important
in mediating infection of host cells, raising the possibility that
cleavage of gp40/15 is also essential for this process. This is
supported by our finding that C. parvum infection of HCT-8
cells was inhibited by Dec-RVKR-cmk. Since Dec-RVKR-cmk
is lipid soluble, it can penetrate cells and could thus inhibit
proteolysis by parasite-derived enzymes. This inhibitor has also
been shown to prevent processing of viral proteins by furin (7).
Previous studies have suggested a role for serine proteases in
excystation of C. parvum oocysts (6, 14). In our study, the
specific furin inhibitor Dec-RVKR-cmk did not affect excysta-
190 WANYIRI ET AL.INFECT. IMMUN.
tion, suggesting that the inhibition of infection was not due to
an effect on excystation. Inhibition of infection by Dec-RVKR-
cmk also did not appear to be due to an effect on host cell furin
activity, implicating the parasite rather than the host cell pro-
tease activity in mediating infection. However, it remains to be
determined whether the inhibition of infection was due to an
effect on processing of gp40/15 or on that of other parasite
Although furin or other proprotein convertases have not
been reported in apicomplexans, these parasites express sub-
tilisin-like serine proteases, including PfSUB-1, PfSUB-2, and
PfSUB-3 in Plasmodium falciparum (32), TgSUB1 (19) and
TgSUB2 (20) in Toxoplasma gondii and NcSUB1 in Neospora
caninum (17). At least one gene encoding a subtilisin-like
serine protease has been identified in each of the Cryptospo-
ridium genomes (1, 34), designated CpSUB1 for C. parvum and
ChSUB1 for C. hominis. The catalytic domains of the Crypto-
sporidium sp. subtilases have significant homology with those
of other apicomplexan subtilisins as well as with bacterial sub-
tilisins and human furin (Fig. 6). However, like bacterial and
other apicomplexan subtilisins, the C. parvum enzymes lack the
characteristic P domain of proprotein convertases, such as
furin and Kex2 (26). Although there is no direct evidence that
these enzymes are responsible for the furin-like protease ac-
tivity in C. parvum lysate, CpSUB1 and ChSUB1 are candidate
proteases that might cleave C. parvum or C. hominis gp40/15.
Current efforts are directed at cloning and expressing enzymat-
ically active recombinant forms of these enzymes and deter-
mining if they are capable of processing gp40/15.
Previous reports have raised the possibility that subtilisin-
like proteases involved in processing of proteins implicated in
pathogenesis of viral, bacterial, and parasitic infections may be
potential therapeutic targets (13, 29). It is possible that, when
identified and characterized, the protease(s) responsible for
processing gp40/15 may also serve as a potential target for
intervention. However, since furin and other subtilisin-like
proprotein convertases are ubiquitous in host cells, it remains
to be determined if specific inhibitors can be designed that will
FIG. 6. The catalytic domain of CpSUB1 is homologous to that of other apicomplexan and bacterial subtilisins and to human furin. Sequences
of the catalytic domains of C. parvum subtilisin CpSUB1 (AAEE01000002), Bdellovibrio bacteriovorus subtilase BbSUB (BX842649), Toxoplasma
gondii subtilisins TgSUB1 (AY043483) and TgSUB2 (AF420596), Neospora caninum subtilisin NcSUB1 (AAF04257), Plasmodium falciparum
subtilisins PfSUB1 (AJ002233) and PfSUB2 (AJ132422), and human furin (X17094) were aligned using the Clustal W algorithm of the Vector NTI
v8.0 program (Invitrogen). GenBank accession numbers of the sequences are in parentheses. The catalytic triad residues for furin, aspartic acid,
histidine, and serine and the putative catalytic triad residues aspartic acid, histidine, and serine for the other subtilisins are indicated by single
asterisks; the oxyanion hole residue asparagine for furin and the putative oxyanion hole residue asparagine for the other subtilisins are indicated
by double asterisks. Identical residues are in gray with a black background, and similar residues are in black with a gray background. The catalytic
domain of CpSUB1 is 55% identical to that of B. bacteriovorous subtilase, 45% to that of TgSUB1, 45% to that of TgSUB2, 44% to that of NcSUB1,
38% to that of PfSUB1, 37% to that of PfSUB2, and 19% to that of human furin.
VOL. 75, 2007 PROCESSING OF CRYPTOSPORIDIUM GLYCOPROTEIN gp40/15191
block infection by pathogens but have minimal toxicity for host Download full-text
In summary, we have shown that gp40/15 is proteolytically
processed into gp40 and gp15 by human furin as well as a by a
C. parvum serine protease activity that is very similar to furin
in its substrate specificity, enzyme kinetics, protease inhibitor
profile, calcium dependence, and pH optimum. Further, a spe-
cific furin inhibitor abrogates C. parvum infection of host cells.
Additional studies are required to definitively identify the pro-
tease or proteases responsible for processing of gp40/15 and to
determine their role in mediating C. parvum infection.
This work was supported by NIH grants RO1 AI05786 (to H.D.W.),
RO1 DE015844 (to A.G.P.), RO1 AI46985 (to K.K.), K01 DK062816
(to R.O.), and P30 DK34928-18 and the GRASP Digestive Diseases
Center at Tufts-New England Medical Center. J.W. is supported by
NIH training grant T32 AI007329. G.A. is supported by NIH training
grant T32 AI07389.
We thank Sitara Rao and Gagandeep Kang for C. hominis subtype
1e DNA; the Intestinal Microbiology Core of the GRASP Center for
reagents, preparation of media, plasmids, and recombinant proteins;
and the Tufts University Core facility for nucleotide and amino acid
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Editor: W. A. Petri, Jr.
192WANYIRI ET AL.INFECT. IMMUN.