![Laminin-Sepharose chromatography of E. histolytica extracts and conditioned media. Detergent extracts of trophozoites were incubated in the presence of laminin-Sepharose, fibronectin-Sepharose, or collagen-Sepharose and analyzed by gel electrophoresis (for details, see Materials and Methods). (A) Fluorogram of gel from [ 35 S]methionine-labeled trophozoites. Total lysate is shown in lane 1. Fractions eluted with 4 M urea after binding to collagenSepharose (lane 2), fibronectin-Sepharose (lane 3), and laminin-Sepharose (lane 4) are also shown. (B) [ 35 S]methionine-labeled conditioned media incubated with laminin-Sepharose. (C) Coomassie blue-stained unlabeled cysteine protease eluted from laminin-Sepharose. Molecular weight standards (in thousands) are shown in the margins.](https://www.researchgate.net/profile/Ellen-Li/publication/242692392/figure/fig1/AS:669465192722432@1536624348175/Laminin-Sepharose-chromatography-of-E-histolytica-extracts-and-conditioned-media_Q320.jpg)
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INFECTION AND IMMUNITY, Oct. 1995, p. 4150–4153 Vol. 63, No. 10
0019-9567/95/$04.0010
Copyright q1995, American Society for Microbiology
Interaction of Laminin with Entamoeba histolytica Cysteine
Proteinases and Its Effect on Amebic Pathogenesis
ELLEN LI,
1,2
* WEN-GANG YANG,
1
TONGHAI ZHANG,
1
AND SAMUEL L. STANLEY, JR.
1,3
Departments of Medicine,
1
Biochemistry and Molecular Biophysics,
2
and Molecular Microbiology,
3
Washington University School of Medicine, Saint Louis, Missouri 63110
Received 13 April 1995/Returned for modification 22 May 1995/Accepted 31 July 1995
The Entamoeba histolytica 27-kDa cysteine proteinases exhibit striking binding specificities for immobilized
laminin over other components of the extracellular matrix, such as collagen and fibronectin. Inactivation of the
proteinase with the active-site inhibitor L-trans-epoxysuccinyl-leucylamido(4-guanidino)butane abolishes lami-
nin binding by the enzyme, and conversely, laminin inhibits cleavage of a fluorogenic dipeptide substrate of the
amebic cysteine proteinase, suggesting that the substrate binding pocket of the enzyme is involved in the
binding of laminin. The addition of laminin but not fibronectin or collagen to E. histolytica trophozoites
significantly reduces amebic liver abscess formation in severe combined immunodeficient mice, further sup-
porting the hypothesis that E. histolytica cysteine proteinases play an important role in amebic pathogenesis.
The specific interaction of amebic proteinases with laminin may be exploited in designing new inhibitors of
these enzymes.
The protozoan parasite Entamoeba histolytica is the caus-
ative agent of human amebiasis. Invasive disease is character-
ized by ulcerations of the intestinal wall and in some cases by
invasion through the wall and dissemination to the liver, re-
sulting in the clinical syndromes of amebic dysentery and liver
abscess, respectively (8). Ultrastructural studies of experimen-
tal E. histolytica infection revealed the degeneration of epithe-
lial cells adjacent to invading trophozoites and the penetration
of trophozoites into the lamina propria through the basement
membrane (16). The interaction of amebic factors with com-
ponents of the extracellular matrix potentially plays an impor-
tant role in the penetration of trophozoites through the intes-
tinal mucosa. In order to identify E. histolytica proteins that
interact with components of the extracellular matrix, amebic
lysates were fractionated over laminin-Sepharose, fibronectin-
Sepharose, and collagen-Sepharose. We report here that E.
histolytica 27-kDa cysteine proteinases exhibit striking binding
specificities for immobilized laminin over immobilized fi-
bronectin or collagen. Furthermore, the coinjection of laminin
but not fibronectin or collagen with E. histolytica trophozoites
greatly reduces liver abscess formation in severe combined
immunodeficient (SCID) mice.
MATERIALS AND METHODS
Materials. Laminin isolated from Engelbreth-Holm-Swarm sarcoma was pur-
chased from Boehringer Mannheim Biochemicals (Indianapolis, Ind.) and was
generously provided by Hynda Kleinman. Bovine fibronectin was purchased from
Calbiochem (San Diego, Calif.). Calf skin type I collagen coupled to Sepharose
(1 mg of beads per ml) was generously provided by Samuel Santoro.
Cells. E. histolytica HM1-IMSS trophozoites were obtained from the American
Type Culture Collection (Rockville, Md.). The amebas were grown in BI-S-33 as
previously described (3).
Ameba radiolabeling. Cultures (72 h) of amebic trophozoites were metaboli-
cally labeled with
35
Strans label (85% methionine, 15% cysteine, 50 mCi/ml;
ICN) in methionine-free minimal essential medium-alpha, supplemented with
0.1% bovine serum albumin (BSA)–5.7 mM cysteine for4hat358C. The cells
were harvested by chilling and low-speed centrifugation, washed with phosphate-
buffered saline (PBS), and solubilized. The solubilization buffer consisted of 1%
Nonidet P-40, 0.15 M NaCl, 1 mM CaCl
2
, 1 mM MgCl
2
, 5 mM phenylmethyl-
sulfonyl fluoride, and 5 mML-trans-epoxysuccinyl-leucylamido(4-guanidino)bu-
tane (E-64) in 10 mM Tris-Cl buffer, pH 8.0. The cell extracts were clarified after
centrifugation at 10,000 3gfor 5 min.
Affinity chromatography. Engelbreth-Holm-Swarm laminin or fibronectin was
conjugated to Sepharose CN 4B (Pharmacia LKB Biotechnology Inc.) at a ratio
of 1 mg of protein to 1 ml of Sepharose according to the manufacturer’s instruc-
tions. Under these conditions, 90% coupling was achieved, as monitored by the
A
280
of the protein solution after coupling. Aliquots of detergent extracts of
amebic trophozoites (0.5 ml, containing 1 310
6
to 1.5 310
6
cells) were incu-
bated with an equivalent volume of laminin-Sepharose overnight at 48C with
gentle agitation. The beads were washed with 10 volumes of buffer A (0.1%
Nonidet P-40, 150 mM NaCl, 10 mM Tris-HCl, pH 8.0), 10 volumes of buffer B
(0.1% Nonidet P-40, 500 mM NaCl, 10 mM Tris-HCl, pH 8.0), and 5 volumes of
buffer C (50 mM Tris-Cl, pH 6.8) as described by Woo et al. (21). Proteins bound
to laminin-Sepharose were eluted with Laemmli sample buffer or with 4 M urea.
In some experiments, 0.1 M N-acetyllactosamine or heparin (Sigma Chemical
Co., St. Louis, Mo.) (5 mg/ml) was added to the laminin-Sepharose incubations.
In order to inactivate the cysteine proteinase, trophozoite lysates were incubated
with 500 mM E-64 (1) at 258C for 20 min.
Protein purification and microsequencing analysis. Proteins bound to lami-
nin-Sepharose were resolved by sodium dodecyl sulfate–10% polyacrylamide gel
electrophoresis, electroblotted onto polyvinyldene difluoride, stained with Coo-
massie blue, excised, and subjected to microsequencing as described previously
(9).
Solid-phase binding assay of
125
I-labeled affinity-purified E. histolytica cys-
teine assay. Laminin affinity-purified E. histolytica cysteine proteinase (10 mg)
was iodinated by incubation of 1 mCi of Na
125
I in the presence of two Iodobeads
(Pierce) according to the manufacturer’s recommendations. The labeled cysteine
proteinase was further purified by gel filtration with a Superose-12 column in
PBS–2 mM dithiothreitol. Laminin, fibronectin, collagen, or BSA (8 mg) in PBS
was applied to 96-well polystyrene radioimmunoassay wells overnight at room
temperature. The plates were washed and blocked for 1 h with 0.5% BSA. The
wells were incubated at 48C with labeled E. histolytica cysteine proteinase (20 ng,
1,000 cpm/ng) and were aspirated, and the plates were washed three times with
0.5% BSA. The wells were excised and counted in a gamma counter. Each
experiment was carried out in quadruplicate.
Assays for proteolytic activity. Protease activity was assessed by gelatin sub-
strate gel electrophoresis as described previously (12). Protease activity was also
assayed by monitoring cleavage of a fluorogenic substrate, Boc-arginine-argi-
nine-4-amino-7-methylcoumarin (ZRR-AMC), as described previously (12). A
1-ml aliquot of laminin affinity-purified cysteine proteinase in 4 M urea was
added directly to the reaction mixture.
Effect of laminin on amebic liver abscess formation in SCID mice. SCID mice
were inoculated with 10
6
E. histolytica trophozoites in either 100 ml of BI-S-33
medium, 100 ml of BI-S-33 plus 20 mg of laminin, 100 ml of BI-S-33 plus 20 mg
of fibronectin, or 100 ml of BI-S-33 plus 20 mg of collagen as described previously
(14). After 48 h, SCID mice were sacrificed, their livers were removed and
inspected for the presence of amebic liver abscesses, abscesses and livers were
weighed, and the percentage of liver abscessed was calculated (14).
* Corresponding author. Mailing address: Washington University,
School of Medicine, Campus Box 8051, 660 S. Euclid Ave., St. Louis,
MO 63110. Phone: (314) 362-1070. Fax: (314) 362-9230.
4150
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RESULTS AND DISCUSSION
Detergent extracts of [
35
S]methionine-radiolabeled E. histo-
lytica trophozoites were incubated in the presence of laminin-
Sepharose. The beads were washed with 0.5 M NaCl, and the
bound proteins were analyzed by sodium dodecyl sulfate-poly-
acrylamide gel electrophoresis. As shown in Fig. 1A, two
closely spaced protein bands estimated to be approximately 27
kDa in molecular mass were observed. These were the only two
bands detected which withstood a high-concentration salt wash
of the column. Under these conditions, binding to neither
fibronectin-Sepharose nor collagen-Sepharose (Fig. 1A) was
observed. Binding was not inhibited by the addition of 0.1 M
N-acetyllactosamine or heparin (5 mg/ml), indicating that
binding was not lectin mediated or heparin dependent, as re-
ported for several other nonintegrin laminin-binding proteins
(5, 10, 15, 22). As shown in Fig. 1B, the E. histolytica laminin-
binding protein can also be isolated from conditioned culture
media. Coomassie blue staining of the affinity-purified protein
fraction, shown in Fig. 1C, confirmed that the 27-kDa laminin-
binding proteins were the predominant species retained on
laminin-Sepharose after the high-concentration salt wash.
To further characterize the 27-kDa laminin-binding pro-
teins, the affinity-purified proteins were subjected to microse-
quence analysis. The two protein bands could not be easily
separated on the polyvinylidene difluoride membrane. The
N-terminal sequence obtained from affinity-purified protein,
shown in Fig. 2, consequently revealed heterogeneity in the
sample. Comparison of the sequence with the deduced amino
acid sequence of two genes encoding homologous E. histolytica
27-kDa neutral cysteine proteinases, recently cloned by Tan-
nich and coworkers (17, 18), suggests that the laminin-Sepha-
rose affinity-purified fraction includes both cysteine protein-
ases.
The levels of binding of
125
I-labeled affinity-purified E. his-
tolytica cysteine proteinase to laminin, fibronectin, collagen,
and BSA adsorbed to the wells of a polystyrene radioimmu-
noassay plate were measured (Table 1). The addition of 50 mg
of soluble laminin to each well inhibited binding of the labeled
cysteine proteinase by 95% 62% (mean 6standard deviation;
n52). Thus, the E. histolytica cysteine proteinases appear to
exhibit a relative binding specificity for immobilized laminin
compared with those for other proteins which also serve as
substrates for these enzymes (6, 7, 13). The 27-kDa E. histo-
lytica cysteine proteinases belong to the papain superfamily
(17, 18).
125
I-labeled papain, a cysteine proteinase homologous
to the amebic cysteine proteases, does not exhibit increased
binding to immobilized laminin compared with those of fi-
bronectin, collagen, and BSA in this assay (Table 1).
The proteinase activity of the affinity-purified protein was
assessed by gelatin substrate gel electrophoresis. As shown in
Fig. 3, several clear bands, ranging from 30 to 69 kDa in
molecular mass, were observed, indicating that the affinity-
purified protein had proteinase activity. When the [
35
S]methi-
onine-labeled affinity-purified material was subjected to elec-
trophoresis through a gelatin substrate gel, several higher-
molecular-mass bands were observed, corresponding to the
bands of clearing (Fig. 3). That the cysteine protease has a larger
apparent molecular mass when subjected to electrophoresis
through a gelatin substrate gel under nonreducing conditions has
been reported by a number of investigators (6, 7).
E-64 is an inhibitor of cysteine proteinases which forms a
covalent link between the sulfur of the active-site cysteine and
the C-2 atom of the inhibitor (1). Although the inclusion of the
active-site protease inhibitor E-64 at concentrations as high as
0.5 mM in lysis buffer at 48C fails to inhibit binding of the
proteases to laminin (data not shown), preincubation of the
lysates at room temperature with 0.5 mM E-64 for 20 min
abolishes binding to laminin (Fig. 4). The temperature depen-
dence for the inhibitory effect of E-64 may reflect the relatively
low affinity of E-64 relative to laminin for the substrate pocket.
In the papain–E-64 complex, the inhibitor interacts with the S
subsites on the enzyme and causes a slight widening of the
active-site cleft (19), so that E-64 could also have an allosteric
effect on laminin binding.
Hydrolysis of the fluorogenic peptide substrate ZRR-AMC
by 0.5 U of laminin affinity-purified E. histolytica cysteine pro-
teinase is inhibited 93% 64% (mean 6standard deviation;
n54) by the addition of 0.1 mg of laminin per ml. In contrast,
FIG. 1. Laminin-Sepharose chromatography of E. histolytica extracts and
conditioned media. Detergent extracts of trophozoites were incubated in the
presence of laminin-Sepharose, fibronectin-Sepharose, or collagen-Sepharose
and analyzed by gel electrophoresis (for details, see Materials and Methods). (A)
Fluorogram of gel from [
35
S]methionine-labeled trophozoites. Total lysate is
shown in lane 1. Fractions eluted with 4 M urea after binding to collagen-
Sepharose (lane 2), fibronectin-Sepharose (lane 3), and laminin-Sepharose (lane
4) are also shown. (B) [
35
S]methionine-labeled conditioned media incubated
with laminin-Sepharose. (C) Coomassie blue-stained unlabeled cysteine protease
eluted from laminin-Sepharose. Molecular weight standards (in thousands) are
shown in the margins.
FIG. 2. Comparison of the N-terminal sequence of the 27-kDa laminin-binding
proteins with the amino acid sequences of two E. histolytica cysteine proteases.
At positions 3, 4, 7, and 10, two amino acid residues were detected. The se-
quences of Eh-CPp1 and EH-CPp2 are derived from the work of Tannich et al.
(18).
TABLE 1. Binding of
125
I-labeled affinity-purified E. histolytica
cysteine proteinase and papain
a
Binding to: cpm (mean 6SD) of bound:
Cysteine proteinase Papain
Laminin 1,900 6200 100 620
Fibronectin 70 610 150 620
Collagen 70 620 60 620
BSA 40 620 50 610
a
Each experiment was carried out in quadruplicate wells as described in
Materials and Methods. The values shown represent the means 6standard
deviations of two separate experiments.
VOL. 63, 1995 E. HISTOLYTICA CYSTEINE PROTEINASE BINDING TO LAMININ 4151
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the addition of 0.1 mg of collagen per ml has no significant
effect on hydrolysis of the fluorogenic peptide substrate by the
laminin affinity-purified E. histolytica cysteine proteinase.
These results also suggest that the substrate binding pocket of
the enzyme is involved in the binding of laminin.
E. histolytica cysteine proteinases appear to play important
roles in the pathogenesis of invasive amebiasis. The degrees of
inflammation and necrosis produced by different strains of E.
histolytica correlate with proteinase activity in animal models
(4, 11, 12). Inhibition of amebic protease activity markedly
reduces the inflammatory lesions resulting from highly virulent
E. histolytica (11). We have previously reported immunohisto-
chemical studies of tissue sections of a SCID mouse amebic
liver abscess which indicate that significant quantities of extra-
cellular E. histolytica cysteine proteinase are present within the
amebic liver abscess and have shown that treatment of E.
histolytica trophozoites with the cysteine proteinase inhibitor
E-64 blocked or greatly reduced liver abscess formation in
SCID mice (14). We have shown that E. histolytica cysteine
proteinases bind to laminin tightly and that laminin inhibits the
cleavage of other substrates. In order to examine the effect of
laminin on amebic pathogenesis in vivo, we examined whether
the coinjection of trophozoites with laminin would alter ame-
bic liver abscess formation (Table 2). SCID mice were injected
with 10
6
trophozoites in 100 ml of BI-S-33 (serum-free) me-
dium alone or medium containing 20 mg of either laminin,
fibronectin, or collagen. The addition of laminin, fibronectin,
or collagen did not affect the viability (90 to 95% by trypan
blue exclusion), nor did it visibly affect the motility of the
trophozoites in the inoculum prepared as described after in-
cubation at 378C for up to 4 h. All 12 control animals had
amebic liver abscesses, with a mean of 24% 616% (mean 6
standard deviation) of the total liver abscessed. The mean
abscess size for the 19 mice receiving trophozoites treated with
laminin was 4% 67%, with 9 animals having no detectable
abscesses. Thus, the amebic liver abscesses were significantly
smaller in mice inoculated with trophozoites that had been
coinjected with laminin than those in the control animals (P,
0.001). In contrast, the amebic liver abscesses in mice inocu-
lated with trophozoites that had been coinjected with fibronec-
tin or with collagen were not significantly smaller than those in
the control animals (Table 2).
The effect of laminin compared with the effects of fibronec-
tin and collagen on amebic liver abscess formation in vivo thus
appears to correlate with the tight binding of E. histolytica
cysteine proteinases to immobilized laminin compared with
their binding to immobilized fibronectin and collagen observed
in vitro. The results are consistent with the hypothesis that the
E. histolytica cysteine proteinases play important roles in the
development of amebic liver abscess in the SCID mouse model
and with the inhibitory effects of laminin on amebic cysteine
proteinase activity. However laminin is a large (;900-kDa)
multidomain complex that exhibits multiple biological activi-
ties, including cell adhesion and cell migration (2), and one
cannot exclude the possibility that the in vivo effects of laminin
on amebic liver abscess formation are due to interactions of
laminin with the trophozoite other than inhibition of proteo-
lytic activity. It is interesting that in an animal model of a
FIG. 3. Gelatin substrate gel electrophoresis of proteinase activity of the
27-kDa laminin-binding proteins. Lane 1, Coomassie blue-stained gelatin sub-
strate gel of
35
S-labeled 27-kDa affinity-purified laminin-binding protein. Lane 2,
fluorogram of the gelatin substrate gel of
35
S-labeled 27-kDa affinity-purified
laminin-binding protein. Molecular weight standards (in thousands) are noted in
the margin.
FIG. 4. Inhibition of laminin binding by inactivation of the cysteine protein-
ase with E-64. Shown are fluorograms of laminin-Sepharose affinity-purified
fractions from detergent lysates of [
35
S]methionine-labeled trophozoites incu-
bated in the presence (1) or absence (blank) of 0.5 mM E-64 prior to incubation
with laminin-Sepharose. Molecular weight standards (in thousands) are indi-
cated in the margin.
TABLE 2. Inhibition of amebic liver abscess formation in
SCID mice by administration of laminin with
E. histolytica trophozoites
Inhibitor n
a
% liver abscessed
(mean 6SD) P
b
Control medium 12 24 616
Laminin 19 4 67,0.001
Fibronectin 10 15 66 0.124
Collagen 10 17 69 0.25
a
n, number of SCID mice in each group. The results represent the totals from
three separate experiments for control and laminin groups and two experiments
which included the fibronectin and collagen groups.
b
Pvalues are derived from the two-tailed ttest for comparisons between each
of the listed inhibitors and the control group for percentages of liver abscessed.
The mean abscess size for SCID mice to which laminin was administered as an
inhibitor was also significantly smaller compared with that when fibronectin (P,
0.001) and collagen (P,0.001) were used as inhibitors.
4152 LI ET AL. INFECT.IMMUN.
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fungal pathogen, Paracoccidioides brasiliensis, which binds to
laminin via a surface glycoprotein receptor, the coinjection of the
organisms with 20 mg of laminin enhances pathogenicity (20).
In summary, the E. histolytica cysteine proteinases exhibit
striking binding specificities for immobilized laminin over
other components of the extracellular matrix. Treatment of
amebic trophozoites with laminin at the time of their inocula-
tion into SCID mice results in significantly decreased liver
abscess size, which may be secondary to the inhibition of the
amebic cysteine proteinases. The structural basis for the inter-
action of the amebic cysteine proteinases with laminin may be
potentially exploited in designing specific inhibitors of these
enzymes.
ACKNOWLEDGMENTS
We thank R. Mecham and E. Brown for many helpful discussions.
We thank K. Myung for excellent technical assistance.
This work was supported by the National Institutes of Health grants
RO1 AI30084 and PO1 AI37977 and a grant from the Monsanto Co.
E.L. is the recipient of NIH Research Career Development Award
DK-02072. S.L.S. is the recipient of NIH Research Career Develop-
ment Award AI01231.
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