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ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Sept. 1995, p. 1913–1919 Vol. 39, No. 9
0066-4804/95/$04.0010
Copyright q1995, American Society for Microbiology
Transglutaminase-Catalyzed Reaction Is Important for
Molting of Onchocerca volvulus Third-Stage Larvae
SARA LUSTIGMAN,
1
* BETSY BROTMAN,
1
TELLERVO HUIMA,
1
ARLINDO L. CASTELHANO,
2
RAVINDRA N. SINGH,
3
KAPIL MEHTA,
3
AND ALFRED M. PRINCE
1
Department of Virology and Parasitology, Lindsley F. Kimball Research Institute, New York Blood Center, New York,
New York 10021
1
; Syntex Inc., Palo Alto, California 94304
2
; and Department of Clinical Investigations, M. D.
Anderson Cancer Center, The University of Texas, Houston, Texas 77030
3
Received 22 February 1995/Returned for modification 12 June 1995/Accepted 26 June 1995
Highly insoluble proteins, which are probably cross-linked, are common in the cuticle and epicuticle of
filarial parasites and other nematode species. We have investigated the possible involvement of transglutami-
nase (TGase)-catalyzed reactions in the development of Onchocerca volvulus fourth-stage larvae (L4) by testing
the effects of TGase inhibitors on the survival of third-stage larvae (L3) and the molting of L3 to L4 in vitro.
The larvae were cultured in the presence of three specific TGase inhibitors: monodansylcadaverine, cystamine,
and N-benzyloxycarbonyl-D,L-b-(3-bromo-4,5-dihydroisoxazol-5-yl)-alanine benzylamide. None of the inhibi-
tors reduced the viability of either L3 or L4. However, the inhibitors reduced, in a time- and dose-dependent
manner, the number of L3 that molted to L4 in vitro. Molting was completely inhibited in the presence of 100
to 200 mM inhibitors. Ultrastructural examination of L3 that did not molt in the presence of monodansylca-
daverine or cystamine indicated that the new L4 cuticle was synthesized, but there was an incomplete
separation between the L3 cuticle and the L4 epicuticle. The product of the TGase-catalyzed reaction was
localized in molting L3 to cuticle regions where the separation between the old and new cuticles occurs and in
the amphids of L3 by a monoclonal antibody that reacts specifically with the isopeptide «-(g-glutamyl)lysine.
These studies suggest that molting and successful development of L4 also depends on TGase-catalyzed
reactions.
Onchocerciasis, or river blindness, is one of the leading
causes of infectious blindness and severe chronic dermatitis,
afflicting about 18 million people in Africa and Latin America
(43). The parasite is transmitted by bites of Simulium black
flies. Although vector control and the periodic administration
of the drug ivermectin promise a drastic reduction in the bur-
den of skin microfilariae and disease, there is also a need for
alternate strategies for the control of onchocerciasis (19). The
development of a vaccine against components of the infective
stages of the parasite or the identification of key enzymes
essential for their development that could be targeted by che-
motherapeutic agents would provide new means for preventing
infection and disease associated with Onchocerca volvulus in-
fection. Surface and cuticular antigens and the molting process
of larval stages of filarial parasites are considered potential
targets for immunity or chemotherapy (8, 17, 26, 35).
The main structural components of the cuticle of O. volvulus
and other nematodes are collagenous proteins, cross-linked by
disulfide bonds and localized in the basal and inner cortical
layers (6, 36). The external cortical layer and the epicuticle are
highly insoluble and appear to be composed of a protein(s)
cross-linked by nonreducible covalent bonds (6, 10). In some
nematodes these components are referred to as cuticlin (6, 16,
34). In some of the nonreducible and insoluble cuticular pro-
teins more than 25% of the tyrosine incorporated into the
cuticle has been found as dityrosine or isotrityrosine, which
resulted from oxidation of tyrosine residues from adjacent
polypeptides to form a covalent bridge between the phenolic
rings (11, 14, 15, 33). The highly insoluble nature of the epi-
cuticle and cuticle of third-stage larvae (L3) and fourth-stage
larvae (L4) of O. volvulus (28), which is also seen in other
nematodes, suggested the possible presence of a biologically
active transglutaminase (TGase; EC 2.3.2.13).
TGases are a family of enzymes that catalyze the posttrans-
lational modification of proteins by introducing an isopeptide
bond between internal glutamine residues and primary amines,
peptide-bound lysine or polyamine, to form either ε-(g-glu-
tamyl)lysine or ε-(g-glutamyl)polyamine bonds (1, 18). The
covalent isopeptide cross-link is exceptionally stable and resis-
tant to proteolysis and can be broken only after total degrada-
tion of the two peptide chains. Because TGases are widely
distributed enzymes that exist in both intracellular and extra-
cellular forms, TGase-modified proteins are evident in many
mammalian systems, the fibrin network of blood clots, cell
membranes, extracellular matrices, and the cornified features
of the epidermis and its appendages.
The presence of putative TGases and the products of
TGase-catalyzed reactions was reported in other filarial nem-
atodes (29, 31, 41). More recently, an active Brugia malayi
TGase with a molecular mass of 56 kDa was purified (37).
Numerous ε-(g-glutamyl)lysine isopeptide bonds were de-
tected in adult worm extracts of B. malayi (29) and in sheaths
of Litomosoides carinii microfilariae (41). Furthermore, em-
bryos developing in utero contained very high amounts of the
enzyme (29). Inhibition of enzyme activity in vitro by mono-
dansylcadaverine (MDC) and cystamine (CS), two specific in-
hibitors of TGase (13, 25, 40), led to a time- and dose-depen-
dent inhibition of microfilarial production and release by
gravid female B. malayi and Acanthocheilonema viteae (29, 31),
thus indicating that in these parasites, TGase-catalyzed reac-
* Corresponding author. Mailing address: Department of Virology
and Parasitology, Lindsley F. Kimball Research Institute, New York
Blood Center, 310 East 67th St., New York, NY 10021. Phone: (212)
570-3119. Fax: (212) 570-3180. Electronic mail address: lustigm@rock-
vax.rockefeller.edu.
1913
tions play an important role during embryogenesis, the matu-
ration of early embryonic stages to microfilariae, and survival
of the parasites.
In this report we show that TGase-catalyzed reactions also
play an important role in the molting process of O. volvulus L3.
The possible involvement of TGase in the development of L3 to
L4 opens up additional putative targets for drug development.
MATERIALS AND METHODS
In vitro culturing of L3 in the presence of TGase inhibitors. Simulium yahense
black flies were infected with O. volvulus microfilariae, and after 7 to 8 days L3
were harvested as described before (28, 30). L3 were set up for culture in groups
of 10 larvae each in 96-well plates containing 5 310
5
bovine peripheral blood
lymphocytes per ml of culture medium (1:1 NCTC 135 and Iscove’s modified
Dulbecco’s media [IMDM] plus 20% heat-inactivated fetal calf serum, 100 U of
penicillin per ml, 100 mg of streptomycin per ml, and 5 mg of amphotericin B
[Fungizone] per ml). Using these conditions, we routinely achieved 50 to 60%
successful L3 to L4 molting by day 5 in culture. L3 were cultured in vitro for 6
days at 378C in a humidified 5% CO
2
incubator in the presence of increasing
concentrations of TGase inhibitors, and the number of molting larvae was de-
termined on day 6. Molting was manifested by shedding of the thick L3 cuticle
and a marked increase in the motility of the larvae. Larval viability was assessed
visually after the uptake of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tet-
razolium bromide) by the larvae and its reduction into the blue formazan deriv-
ative (5). Briefly, the culture medium at each point was carefully removed from
each well by using finely drawn glass pipettes and was replaced with 150 mlof
phosphate-buffered saline containing 0.1% MTT (Sigma Chemical Co., St. Louis,
Mo.). Metabolically active larvae take up MTT and reduce it to formazan,
subsequently staining themselves blue. Larvae were scored live when they stained
blue uniformly along their entire length and dead when the larvae remained
unstained. Three TGase inhibitors were used: MDC, a competitive pseudosub-
strate that acts as an inhibitor of endogenous protein cross-linking by the enzyme
and CS, an active-site inhibitor, both known to be specific for TGase (13, 25, 40),
and a new synthetic inhibitor of TGase, N-benzyloxycarbonyl-D,L-b-(3-bromo-
4,5-dihydroisoxazol-5-yl)-alanine benzylamide (kindly provided by Allen Krantz,
Syntex Inc., Palo Alto, Calif.), which specifically inactivates mammalian TGases
by binding to the active site of TGase (2, 4). The results shown for each inhibitor
concentration are the average number of larvae that molted or the average
number of larvae that were scored alive in 5 to 10 wells containing a total of 50
to 100 L3. Each experiment was repeated at least twice. The standard error
between experiments never exceeded 10% and did not vary with the concentra-
tion.
Ultrastructure of larvae cultured in the presence of TGase inhibitors. Larvae
that did not molt in the presence of 150 and 200 mM MDC or CS were collected
and fixed overnight at 48C with 3% glutaraldehyde in 0.1 M phosphate buffer (pH
7.3), washed in the same buffer, and processed for electron microscopic exami-
nation as described before (28). For examination of the ultrastructure of larvae
undergoing normal molting, L3 were cultured in vitro for 4 days and larvae from
days 1, 2, 3, and 4 in culture were collected. The larvae were fixed as described
above.
TGase activity in crude extracts of O. volvulus. Worms were homogenized on
ice in prechilled lysing buffer (20 mM Tris-HCl [pH 8.5] containing 150 mM
NaCl, 2 mM dithiothreitol [DTT], 1 mM EDTA, 1 mM phenylmethylsulfonyl
fluoride, 0.1 mM N-tosyl-L-phenylalanine chloromethyl ketone, and 0.1 mM
N-a-p-tosyl-L-lysine chloromethyl ketone). The homogenate was sonicated three
times (the third sonication was in the presence of 0.1% Triton X-100), and
thereafter was spun at 15,000 3gfor 20 min. Extracts were prepared from female
adult worms and from about 1,000 L3 that were cultured for 2 days in vitro.
TGase activity in the crude extracts was determined by using N,N9-dimethylca-
sein (Sigma) as the amine acceptor and 5-(biotinamido)pentylamine (Pierce,
Rockford, Ill.) as the amine donor according to the microtiter plate assay de-
scribed by Slaughter et al. (38). Briefly, a microtiter plate precoated for1hat
378C with 200 ml of dimethylcasein (10 mg/ml) was reacted with 200 ml of crude
extracts (5 mg of adult female worm extract or extract produced from about 100
larvae at day 2 in culture) containing 1 mM 5-(biotinamido)pentylamine, 10 mM
CaCl
2
,and10mMDTTfor2hat378C. For inhibition experiments the reaction
mixture also contained ethylene glycol-bis-(b-amido-ethyl ether)-N,N,N9,N9-tet-
raacetic acid (EGTA), MDC, or CS. The reaction was stopped by washing the
wells with 350 ml of 250 mM EDTA twice. The amount of 5-(biotinamido)pen-
tylamine incorporated into the N,N9-dimethylcasein was determined by detection
with streptavidin-alkaline phosphate and paranitrophenyl phosphate (Sigma).
TGase activity is expressed as the optical density at 405 nm after stopping the
reaction with 50 ml of 2 M sodium bicarbonate.
Localization of the isopeptide «-(g-glutamyl)lysine in larval stages of the
parasite. L3 were cultured in vitro for 4 days, and larvae from days 1, 2, 3, and
4 in culture were collected. The larvae were fixed for 30 min in 0.25% glutaral-
dehyde in 0.1 M phosphate buffer (pH 7.4) containing 1% sucrose and were then
processed for immunoelectron microscopy as described previously (27). Thin
sections of embedded worms were probed with a monospecific immunoglobulin
M (IgM) monoclonal antibody (kindly provided by Gerard A. Quash, Institut
National de la Sante´ et de la Recherche Me´dicale, Oullins, France) which reacts
with the end product of TGase-catalyzed reaction, the isopeptide ε-(g-glutamyl)
lysine (9). The sections were incubated with a second antibody, rabbit anti-mouse
Ig (Accurate Chemical and Scientific Corp., Westbury, N.Y.), before interaction with
10- or 15-nm gold particles coated with protein A (Amersham Life Sciences, Ar-
lington Heights, Ill.). A nonrelated IgM monoclonal antibody was used as a control.
Identification of putative substrate proteins for TGase activity in molting
larvae. Parasite proteins that might serve as substrates for endogenous TGase
during molting were identified by analyzing the incorporation of the pseudosub-
strate MDC into the larval proteins by Western blotting (immunoblotting). A
total of 100 L3 were cultured in the presence of 200 mM MDC or under normal
culture conditions for 2 days and were then collected. Crude extracts of both
molting larvae were prepared by homogenization in sodium dodecyl sulfate
(SDS)-polyacrylamide gel electrophoresis (PAGE) sample buffer (2% SDS in
62.5 mM Tris-HCl [pH 6.8] with 5% 2-b-mercaptoethanol) and centrifugation at
12,000 3g. The crude extracts were separated by SDS-PAGE on a 7.5 to 20%
gradient (22) and were electrophoretically transferred to nitrocellulose (42). The
blot was probed with rabbit anti-MDC antibodies (kindly provided by Laszlo
Lorand, Northwestern University, Chicago, Ill.). Bound antibodies were detected
by
125
I-protein A (0.1 mCi/ml; ICN Biomedicals, Inc., Irvine, Calif.).
RESULTS
Effects of TGase inhibitors on L3 molting. The possible
involvement of TGase-catalyzed reactions in the development
of L4 of O. volvulus was studied in vitro by observing first the
effects of two TGase-specific inhibitors (MDC, a competitive
pseudosubstrate that acts as an inhibitor of endogenous pro-
tein cross-linking by the enzyme, and CS, an active-site inhib-
itor) on the viability of molting L3 and the ability of L3 to molt
to L4. Parasite viability was assessed visually after the uptake
of MTT by the larvae. L3 were cultured in the presence of
increasing concentrations of MDC or CS, and the number of
molting larvae was determined on day 6. Both inhibitors re-
duced, in a dose-dependent manner, the molting ability of L3
in vitro (Fig. 1). In comparison with the typical 50 to 60%
molting under normal culture conditions, 50 to 100% of molt-
ing was inhibited by 50 to 300 mM MDC and CS. Inhibition of
50% was observed with 50 mM CS and 100 mM MDC. CS or
MDC at 150 mM completely inhibited molting. This inhibition
was not due to a lethal effect of the inhibitors on L3. In parallel
experiments larvae were treated with MTT on different days
during culturing in the presence of various concentrations of
the inhibitors, and their viabilities were assessed after 24 h. As
shown in Fig. 1, L3 that were cultured in the highest concen-
tration of CS (300 mM) were still viable. With MDC, only
concentrations greater than 200 mM had any significant effect
on L3 viability. The viability curves in Fig. 1 represent the data
from larvae cultured in the presence of the drugs for 3 days.
We have chosen to present the results for day 3 in culture
because this is the critical time during molting that can indicate
that the larvae are viable and ready for molting. Larvae usually
complete their L3 to L4 molt by day 5 (28). Similar results were
also obtained on successive days; the larvae were motile and
viable during all days in culture (data not shown). Since the
effects of the TGase inhibitors on molting were not due to
lethality, we concluded that the effects were specific to the
molting process. A third TGase inhibitor, N-benzyloxycar-
bonyl-D,L-b-(3-bromo-4,5-dihydroisoxazol-5-yl)-alanine benzyl-
amide, which was developed to specifically inactivate mammalian
TGases by binding to the active site (4), was also tested in our
molting assay. The inhibitor completely inhibited the molting of
O. volvulus L3 to L4 at 100 and 200 mM (95 and 98%, respec-
tively) (data not shown).
To investigate the period during which the molting process is
primarily affected by the inhibitors, two of the inhibitors were
added at different days during the molting process. Culture
medium containing 150 mM MDC or CS was added to the
larvae on day 0 or on days 1, 2, 3, and 4 by replacing the normal
1914 LUSTIGMAN ET AL. ANTIMICROB.AGENTS CHEMOTHER.
culture medium, and the larvae were then allowed to remain in
culture until day 6, when the number of molting larvae was
determined. Both inhibitors induced inhibition of molting but
at different time points during the molting process (Fig. 2). The
presence of the competitive pseudosubstrate MDC during the
first 24 h of the molting process was essential for the complete
inhibition of molting. If the inhibitor was added after 1 or 2
days, 21 and 37% of larvae molted, respectively (63 and 35%
inhibition, respectively), in comparison with 57% molting un-
der normal conditions. When the inhibitor was added after 3 or
4 days, there was no significant effect on molting. By contrast,
the TGase active-site inhibitor CS was completely inhibitory,
even when it was added on day 2 in culture. When the inhibitor
was added on day 3 or 4, only a partial inhibition was observed;
37 and 42% of the larvae molted, respectively, in comparison
with 57% molting in normal cultures. This effect on days 3 and
4 could be due to the inhibitory effect of CS on a subpopulation
of larvae which are slower in their molting process than the
majority of the larvae and which molt only after 5 days in
culture. In conclusion, the effects of MDC and CS on molting
are mostly critical during the first 2 days in culture.
Ultrastructure of larvae that did not molt in the presence of
TGase inhibitors. O. volvulus larvae that did not molt in cul-
tures containing the MDC or CS inhibitors continuously were
collected and processed for electron microscopy. As shown in
Fig. 3d, e, and f, the larvae that did not molt in the presence of
MDC had initiated the molting process but never completed it
as it happens under normal conditions (Fig. 3c). The larvae
had a visible L4 epicuticle and cuticle, in addition to the outer
L3 epicuticle and cuticle, indicating that the new L4 cuticle had
begun to be synthesized (Fig. 3d). In some larvae, irregular
separations between the L4 epicuticle and the L3 cuticle were
observed (Fig. 3e and f), similar to those seen in normal cul-
tures on day 1 (Fig. 3a). However, we could not find any larvae
in which the separation between the cuticles was complete, as
seen in normal cultures on days 2 and 3 (Fig. 3b and c, respec-
tively). The cuticular ultrastructures of the larvae that did not
molt in the presence of CS were similar to the ones cultured in
the presence of MDC (data not shown).
TGase activity in crude extracts of O. volvulus. Extracts of L3
after 2 days in culture and extracts of female adult worms were
tested in a microtiter assay for the presence of endogenous
TGase activity by using N,N9-dimethylcasein as an amine ac-
ceptor and 5-(biotinamido)pentylamine as an amine donor. As
shown in Fig. 4, we detected TGase activity in extracts of both
larvae from 2 days in culture and female adult worms, which
were inhibited by 50 mM EGTA by 81.6 and 97.4%, respec-
tively. TGase activity is Ca
21
dependent and can be inhibited
by EDTA or EGTA (1, 18). Because we did not have enough
material to do more inhibition assays with the larval extracts,
we tested the effects of MDC and CS only on the TGase
activity in female adult worm extracts. In the presence of 20
and 200 mM MDC, TGase activity was inhibited by 88.7 and
86.9%, respectively. In the presence of 20 and 200 mM CS,
TGase activity in the female adult worm extracts was inhibited
by 68.1 and 79%, respectively.
Localization of the isopeptide «-(g-glutamyl)lysine in larval
stages of the parasite. Immunoelectron microscopy staining of
L3 during molting with a monoclonal antibody that is directed
against the isopeptide ε-(g-glutamyl)lysine (9) permitted the
localization of the isopeptide produced by the action of an
endogenous TGase in the intermediate stages of O. volvulus
molting larvae. Examination of thin sections of larvae on days
1 to 3 in culture revealed the newly formed epicuticle and
cuticle of L4 in areas beneath the basal layer of the old L3
FIG. 1. Molting and viability of O. volvulus L3 in the presence of TGase
inhibitors. A total of 50 to 100 L3 were cultured in the presence of increasing
concentrations of MDC (a) or CS (b), and the molting rate was determined on
day 6. A total of 50 to 60% L3 molted by day 5 under normal control culture
conditions. Larval viability after 3 days in culture in the presence of the drugs was
assessed visually after the uptake of MTT by the larvae and its reduction into the
blue formazan derivative. Each experiment was repeated at least twice, and the
standard error between experiments never exceeded 10%.
FIG. 2. Molting of O. volvulus L3 in the presence of TGase inhibitors that
were added at different days during molting. The inhibitors MDC or CS were
added at different days during the molting process of 50 L3 and were kept in
culture until day 6, when the number of molting larvae was determined. A total
of 57% of the larvae molted under normal culture conditions without inhibitors.
VOL. 39, 1995 TRANSGLUTAMINASE AND MOLTING OF ONCHOCERCA LARVAE 1915
cuticle (Fig. 5a, arrowhead) and the separation between the
two cuticles below the old basal layer of L3 (Fig. 5b, arrow-
head), as seen during normal molting (Fig. 3a and b). The
monoclonal antibody recognized the isopeptide mostly in areas
around the region where the separation between the cuticles
takes place, in some areas of the cuticle of the newly formed
L4, and in the lower part of the old cuticle, where the separa-
tion occurs (Fig. 5a and b). Interestingly, the density of staining
is higher in the larvae after 1 day in culture, just before the
separation between the cuticles starts, than in larvae 2 to 3 days
FIG. 3. Ultrastructures of larvae during normal molting and larvae that did not molt in the presence of the MDC inhibitor. L3 during normal molting were collected
on days 1, 2, and 3, and L3 that did not molt in the presence of 150 and 200 mM MDC were collected on day 6. Thin sections of larvae during the normal molting process
for larvae on day 1 (a), day 2 (b), and day 3 (c) and thin sections of three different worms that did not molt in the presence of MDC (d, e, and f) are presented (bars,
0.25 mm). Separations between the L4 epicuticle and the L3 cuticle are marked by arrows and arrowheads, respectively.
FIG. 4. TGase activity in O. volvulus worm extracts. Extracts equivalent to 100 L3 2 days in culture (L3 2D) or 5 mg of female adult worm extracts (OVA) were
tested in a microtiter plate assay as described in Materials and Methods. For specific inhibition, the reaction mixture also contained 50 mM EGTA, 20 or 200 mM MDC,
and 20 or 200 mM CS. The amount of 5-(biotinamido)pentylamine that was incorporated into the N,N9-dimethylcasein by the parasite endogenous TGase was
determined by staining with streptavidin-alkaline phosphate and paranitrophenyl phosphate. TGase activity is expressed as the optical density (OD) at 405 nm after
stopping the reaction with 50 ml of 2 M sodium bicarbonate.
1916 LUSTIGMAN ET AL. ANTIMICROB.AGENTS CHEMOTHER.
in culture, when the separation between the cuticles is almost
complete. A control IgM monoclonal antibody did not cross-
react with any proteins in the larvae (data not shown). When
longitudinal cross sections in the head region of L3 were
probed with the isopeptide-specific monoclonal antibody, we
found that the antibodies also bound to proteins in the am-
phids of the larvae (Fig. 5c), near the amphidial opening and
the amphidial cilia.
Identification of endogenous substrates for larval TGase.
Preliminary studies that were directed toward the identifica-
tion of the substrate proteins to be cross-linked by the TGase-
catalyzed reaction in larvae took advantage of the incorpora-
tion of the exogenous MDC into larval proteins during the
molting process. Larvae were cultured with 200 mM MDC for
2 days and were then collected, and the proteins were extracted
by homogenization as described in Materials and Methods.
Proteins bound to MDC were identified by Western blot anal-
ysis with rabbit anti-MDC antibodies. As shown in Fig. 6, two
major protein bands were detected: a single band of 80 kDa
and a band of 68 to 74 kDa (lane 1). Protein extracts of larvae
that were cultured without MDC did not cross-react with these
antibodies (lane 2).
DISCUSSION
In the present study we have shown that TGase appears to
be an important enzyme for the molting process of L3 to L4
and, consequently, for the successful development of O. volvu-
lus L4. Our conclusion is based on results from four different
independent experiments. First, we have shown the inhibitory
effects of three specific TGase inhibitors on the molting of L3
to L4: MDC, a known high-affinity pseudosubstrate for TGase;
CS, which binds to the active site of TGase (13, 25, 40); and a
new synthetic inhibitor of TGase, N-benzyloxycarbonyl-D,L-b-(3-
bromo-4,5-dihydroisoxazol-5-yl)-alanine benzylamide, which was
developed by Syntex Inc. for potential use in chemotherapy and
which as CS specifically inactivates TGases by binding to the
active site of the enzyme (2, 4). These structurally unrelated
inhibitors, which interact with two different steps of the TGase-
catalyzed reaction, are all inhibitory and exert the same effect on
L3 during molting. Because these inhibitors were not lethal to the
larvae, it was suggested that the inhibitory effect was specific to
FIG. 5. Ultrastructural localization by immunoelectron microscopy of the
isopeptide ε-(g-glutamyl)lysine product of the TGase-catalyzed reaction in O.
volvulus. Thin sections of O. volvulus L3 during molting in vitro after 1 and 2 days
in culture (a, b) and a longitudinal section of the head region of O. volvulus L3
(c) were first incubated with a monoclonal antibody directed against the isopep-
tide ε-(g-glutamyl)lysine. The sections were then reacted with rabbit anti-mouse
Ig and protein A coupled to 10- or 15-nm gold particles for indirect antigen
localization (bars, 0.25 mm). Note the area of the L4 epicuticle and cuticle (a;
arrowhead), where the separation between cuticles takes place, and the partial
separation between the basal layer of the L3 cuticle and the L4 epicuticle (b;
arrowhead). The amphidial opening (arrowhead) and amphidial cilia (ac) are
marked in panel c.
FIG. 6. Identification of putative parasite substrate proteins for endogenous
larval TGase. Protein extracts of molting L3 in the presence of 200 mM MDC
(lane 1, 100 larvae per lane) or during normal culture conditions (lane 2, 100
larvae per lane) were subjected to electrophoresis and were transferred to ni-
trocellulose. The Western blot was probed with rabbit anti-MDC antibodies and
125
I-protein A. Molecular mass markers are indicated on the left.
VOL. 39, 1995 TRANSGLUTAMINASE AND MOLTING OF ONCHOCERCA LARVAE 1917
the molting process and, therefore, indirectly indicated that a
specific TGase that catalyzes cross-linking is present and active in
the larvae during molting. Both inhibitors, MDC, which competes
with the endogenous larval proteins that serve as a substrate(s)
for TGase-catalyzed cross-linking of proteins, and CS, the active-
site inhibitor, had to be present during the first 24 to 48 h, the
beginning of the molting process, to exert a complete inhibitory
effect (Fig. 2). After the third day in culture, when complete
separation of the cuticle usually occurs (Fig. 3c), a complete
inhibitory effect was not observed. These findings implied that the
effect of the inhibitors was specific to some of the first changes
that occur in the cuticle during the molting process: development
of the new L4 epicuticle and cuticle and the beginning of the
separation between the cuticles (Fig. 3a and 5a). This conclusion
is supported by the ultrastructural studies (Fig. 3d to f). Larvae
that did not molt in the presence of the inhibitors were found to
be at the different stages of development that precede complete
separation between the cuticles. Some larvae had visible L4 epi-
cuticles and cuticles, and in some we observed irregular separa-
tions between the L4 epicuticle and the L3 cuticle. However, we
never observed within unmolted larvae a complete separation
between the cuticles (Fig. 3b and c). This indicated that when we
generated inhibition of TGase-catalyzed reactions we indirectly
prevented the complete separation between the cuticles and con-
sequently ecdysis, shedding of the old cuticle. Second, the product
of a TGase-catalyzed reaction, the isopeptide ε-(g-glutamyl)-
lysine, was found to be mostly localized in the lower part of the L3
cuticle and the L4 epicuticle and cuticle at the start of the molting
process around the sites where the separation between the old
and new cuticles occurs (Fig. 5a and b). This underscores the
presence of products produced by the action of TGase activity
and indirectly indicates that an active TGase is also present in the
cuticles during the molting process. In preliminary immunogold
labeling experiments we were able to localize a putative parasite
TGase with a cross-reacting monoclonal antibody raised against a
guinea pig liver TGase. Interestingly, this monoclonal antibody
reacted in molting larvae with a protein present in the same areas
where the monoclonal antibody against the isopeptide ε-(g-glu-
tamyl)lysine bound (data not shown). Third, the activity of an
endogenous parasite TGase, which was inhibited by EGTA, was
detected in extracts of molting larvae on day 2 in culture and in
extracts of adult female worms. TGase activity in adult worm
extracts was also inhibited by 200 mM MDC and CS (86.9 and
79%, respectively). The incomplete inhibition is probably due to
competition with endogenous substrates or other proteins present
in the worm extracts. In studies with the B. malayi TGase (37), it
was demonstrated that 50% of the purified enzyme activity was
inhibited by 500 mM CS. We have not been able to characterize
the enzyme biochemically because of the difficulty in obtaining
sufficient material for a proper analysis. Lastly, we have shown as
well that during molting MDC was cross-linked to endogenous
larval proteins, which were detected with anti-MDC antibodies
(Fig. 6), thus implying the presence of an endogenous TGase in
molting larvae which catalyzed the cross-linking of MDC to par-
asite proteins. On the basis of the results presented above, we
conclude that an endogenous larval TGase is active during the
molting process and that inhibition of the TGase-catalyzed reac-
tions apparently prevents complete separation of the cuticles and,
consequently, ecdysis.
Two observations were unexpected; first, the development of
the new epicuticle and cuticle of L4 apparently was not inhib-
ited, as might have been anticipated if a TGase would have
been responsible for cross-linking epicuticular and cuticular
proteins during cuticle formation in the developing L4; second,
the ε-(g-glutamyl)lysine products of the TGase-catalyzed reac-
tion were also localized in the amphids of the larvae. Experi-
mentally, our observations clearly indicated that the specific
stage during the molting process when the cuticles are sepa-
rated was somehow affected and consequently caused incom-
plete molting. The question is how TGase regulates or controls
the molting process if its inhibition prevents complete molting.
It appears most likely that a complete and normal develop-
ment of the L4 epicuticle and cuticle, which might not be
evident only by ultrastructural analysis, is necessary before
complete separation between the cuticles can occur. It is pos-
sible that TGase in the larval stages of O. volvulus is respon-
sible for cross-linking proteins that are an important part in the
assembly of the L4 epicuticle and cuticle and that must evolve
during the molting process and precede complete separation of
the cuticles. Therefore, failure of those proteins to be cross-
linked in the presence of inhibitors might have prevented the
normal development of the L4 epicuticle and cuticle and, con-
sequently, the separation between the L3 and L4 cuticles to
complete the molt. In addition, it could be that proteins in the
amphids are responsible for regulating the molting process,
and when their complete posttranslational modification is in-
hibited, the molting process is incomplete. It was suggested
that the amphids, which are nerve cells with a secretory activ-
ity, may, under neurosecretory control, allow products to get to
the surface of molting larvae. At present, the role of the am-
phids during the molting process of nematodes is not clear.
The only study that found some direct relationship between
amphids and molting was described by Delves et al. (7) in
Dirofilaria immitis. They found that in molting larvae a fuch-
sinophilic substance in the amphids was associated with the
pre-ecdyzed stages of L3; once the ecdysis started the staining
disappeared. The significance of TGase activity and the prod-
ucts of the TGase-catalyzed reaction in the amphids and their
relationship to molting need more study.
The challenge will be to identify the specific components in
the epicuticle, cuticle, or amphids that provide the substrate(s)
for this reaction. Interestingly, one of the known substrates for
TGase in mammalian systems was shown to be collagen (3, 20,
21). Collagens have also been shown to be one of the major
protein groups that are significant for the development of
larval cuticles (6, 10, 34, 36). The amphids consist of invagi-
nated channels that are covered with a cuticular layer which is
structurally similar to the external parts of the cortical layer of
the cuticle of O. volvulus L3 (39). Our preliminary results have
identified an 80-kDa protein and proteins of 68 to 74 kDa that
are potential substrates for an endogenous TGase in molting
larvae (Fig. 6). Analysis of these proteins would indicate if they
are collagen-like proteins. Interestingly, collagen-like proteins
in nematodes have molecular masses of 30 to 120 kDa on
reducing gels (12, 36).
It is likely that TGase-catalyzed reactions are important to the
molting process of not only O. volvulus but other nematodes as
well. This is supported by the studies with the synthetic TGase
inhibitor N-benzyloxycarbonyl-D,L-b-(3-bromo-4,5-dihydroisox-
azol-5-yl)-alanine benzylamide, which was tested against helminth
infections and which was found to be inhibitory (4). This inhibitor
was able to reduce in vitro (100 mM drug for 7 days) the ability of
L4 of Nippostronglus brasiliensis to molt to adult stages and also
affected the viability and mobility of N. brasiliensis. In addition,
the inhibitor reduced the number of parasites that developed in a
mixed infection with Nematospiroides dubius and Hymenolepsis
nana in mice. The same inhibitor at 100 mM completely inhibited
the molting of O. volvulus L3 in our study. Recently, an active
TGase from an adult B. malayi worm was biochemically purified
and characterized and its partial amino acid sequence was deter-
mined (37). In previous studies (29, 31), it was reported that
TGase inhibitors were lethal to B. malayi L3 and, in B. malayi and
1918 LUSTIGMAN ET AL. ANTIMICROB.AGENTS CHEMOTHER.
A. viteae, also affected other stages of development, microfilaria
production, and microfilaria release by gravid female worms. In
addition, an indirect support for the importance of TGase-cata-
lyzed reactions for the molting of Onchocerca larvae could be
derived from the report by Lok et al. (24), showing that synthetic
retinoids can inhibit the L3 to L4 molt of O. lienalis in vitro. It has
been shown in mammalian systems that the levels of keratinocyte
TGase are specifically suppressed by retinoic acid (23, 32). It is
possible that the presence of synthetic retinoids in the study of
Lok et al. (24) also reduced the levels of TGase in the molting
larvae and thus caused, indirectly, arrested molting.
In conclusion, TGase-catalyzed posttranslational modifica-
tion of proteins seems to be important to the successful devel-
opment of O. volvulus L4, because inhibition of TGase activity
prevents the complete separation between L3 and L4 cuticles,
and therefore ecdysis. The identification of TGase as an es-
sential enzyme for the molting process of O. volvulus L3 and
the development of L4 opens up important new avenues for
drug development. Other important areas of study include the
molting process in nematodes, the role of amphids during
development, and the regulation of epicuticle and cuticle for-
mation during molting.
ACKNOWLEDGMENTS
We are grateful to Birger E. Blomback for drawing our attention to
the possible role of TGases in giving rise to the insolubility of proteins
derived from the O. volvulus epicuticle and cuticle. We are grateful to
Laszlo Lorand for providing the rabbit anti-MDC antibodies, to Ger-
ard A. Quash for providing the monoclonal antibody to the isopeptide
ε-(g-glutamyl)lysine, and to Allen Krantz and Syntex Inc. for providing
us with the TGase inhibitor N-benzyloxycarbonyl-D,L-b-(3-bromo-4,5-
dihydroisoxazol-5-yl)-alanine benzylamide.
This study was supported in part by grants from The Edna Mc-
Connell Clark Foundation and the Onchocerciasis Programme in West
Africa-UNDP/World Bank/WHO Special Programme for Research
and Training in Tropical Diseases Macrofil Chemotherapy Project.
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VOL. 39, 1995 TRANSGLUTAMINASE AND MOLTING OF ONCHOCERCA LARVAE 1919
