BIOLOGY OF REPRODUCTION 77, 648–657 (2007)
Published online before print 11 July 2007.
A Role for Tissue Transglutaminase in Stabilization of Membrane-Cytoskeletal
Particles Shed from the Human Placenta
Nicola J. Robinson, Philip N. Baker, Carolyn J.P. Jones, and John D. Aplin1
Maternal and Fetal Health Research Centre, Division of Human Development, University of Manchester, St. Mary’s
Hospital, Manchester M13 0JH, United Kingdom
Tissue transglutaminase (TGM2; also known as TG2 or tTG)
localizes to the syncytial microvillous membrane (MVM) of the
human placenta, the primary interface between maternal and
fetal tissue. To identify TGM2 substrates in the MVM, membrane
vesicles were prepared and labeled with biotinylated acyl donor
or acceptor probes. Biotinylated species were selected on an
avidin affinity matrix and identified by mass spectrometry of
tryptic peptides. The most abundant were cytoskeletal (actin,
tubulin, and cytokeratin) and membrane-associated (annexins,
integrins, and placental alkaline phosphatase) proteins. During
pregnancy, apoptotic particulate material, the end product of
the trophoblast life cycle, is shed from the MVM into maternal
circulation. Shed material was isolated from primary trophoblast
cultures in which syncytial-like masses develop by fusion. A
substantial fraction of actin in the particles was in the form of
covalent polymeric aggregates, in contrast to cellular actin,
which dissociated completely into monomer in SDS-PAGE.
When cells were cultured in the presence of transglutaminase
inhibitors, actin in the shed particles remained exclusively in
monomeric form, and a reduction in trophoblast intercellular
fusion and differentiation was observed. These findings suggest
that transglutaminase-mediated cross-linking stabilizes the
particulate material shed from the placenta.
actin, cytoskeleton, immunology, placenta, plasma membrane,
Tissue transglutaminase (TGM2; also referred to as
transglutaminase type 2, TG2, or tTG) belongs to a family of
cross-linking enzymes responsible for catalyzing Ca2þ-depen-
dent acyl-transfer reactions, resulting in the formation of an
isopeptide bond . In addition to its transamidating capacity,
TGM2 possesses GTPase activity, with the ability to hydrolyze
both GTP and ATP, and intrinsic kinase activity . TGM2 is a
cytosolic protein that has also been observed in the nucleus and
can be externalized to the cell surface or extracellular matrix [3,
4]. Despite ubiquitous expression, its functions remain poorly
understood. The observation that TGM2 activity correlates with
cellular regression in rat livers after induction of hyperplasia
first suggested its involvement in apoptosis . TGM2-
mediated cross-linking is thought to be important in stabilizing
apoptotic cells by formation of intracellular cross-linked
protein polymers, preventing leakage of intracellular compo-
nents into the surrounding tissues. This protein scaffold may
stabilize dying cells, ensuring their clearance by phagocytosis
and thereby preventing inflammatory reactions .
TGM2 has been identified as the major autoantigen in celiac
disease , which several authors suggest is associated with
adverse pregnancy outcome, including recurrent miscarriage,
low birth weight babies, stillbirths, and intrauterine growth
restriction in untreated women [8–12]. The reasons for the poor
outcomes are unknown. TGM2 is widely expressed in human
placenta, with strong activity at the syncytial microvillous
membrane [13, 14], which is directly exposed to maternal
blood and autoantibodies. During pregnancy, particulate
material continuously sheds from this surface into maternal
circulation [15, 16], where it is thought to interact with, and
skew, responses in maternal immune cells. Identification of the
principal target proteins of TGM2 at this interface may help
elucidate its normal function as well as the pathogenic
mechanism of altered pregnancy outcome in celiac disease.
We have used a proteomic strategy to identify TGM2 target
proteins in the placental microvillous membrane (MVM). To
test the hypothesis that material shed from terminally
differentiated syncytiotrophoblasts may be cross-linked by
TGM2, shed microparticulate material was isolated from
primary cytotrophoblast cells in the presence and absence of
TGM2 inhibitors. Trophoblast cell fusion and differentiation
was also assessed following TGM2 inhibition. The results
suggest a role for TGM2 in the organization and turnover of the
MVM and its associated cytoskeleton.
MATERIALS AND METHODS
Local Ethical Committee Approval, with patient consent, enabled the
collection of placental tissue. Term placenta was obtained at cesarean section or
vaginal delivery following uncomplicated pregnancy.
The preparation of MVMs was based on an established method  and
used either method 1 or method 3. Greater purification was obtained with
vesicles made from method 1, as demonstrated by the higher enrichment of
alkaline phosphatase activity. The protein recovery (milligram per gram of
placenta) was 5-fold higher by method 3 (stir-prep). The final vesicle
preparation was spun down from 150 mM NaCl, 4 mM KCl, 2 mM MgCl2,
and 2 mM Tris-HCl, pH 7.4, and stored at ?808C.
Membrane-Associated TGM2 Activity Assay with Acyl-
Two biotinylated probes were used for labeling TGM2 substrate proteins in
MVM vesicles: biotin-cadaverine (BTC), which represents the acyl-acceptor
probe, and a biotinylated glutamine-containing hexapeptide (biotinyl-
TVQQEL) , which represents the acyl-donor probe in the cross-linking
reaction catalyzed by TGM2. Aliquots of frozen MVM isolated from term
1Correspondence: John Aplin, Division of Human Development,
University of Manchester, St. Mary’s Hospital, Research Floor Room
74, Hathersage Rd., Manchester M13 0JH, U.K.
FAX: 44 0 161 276 6134; e-mail: email@example.com
Received: 30 March 2007.
First decision: 30 April 2007.
Accepted: 1 July 2007.
? 2007 by the Society for the Study of Reproduction, Inc.
ISSN: 0006-3363. http://www.biolreprod.org
human placental syncytiotrophoblasts were spun down in buffer at 230003g,
15 min, 48C. One hundred micrograms of MVM protein was resuspended in
TGM2 activity buffer containing 5 mM CaCl2, 100 mM Tris-HCl (pH 8.3),
0.15 M NaCl, 5 mM acyl-donor or -acceptor probe, and 10 mM dithiothreitol
(DTT) (added just before the incubation) in a total volume of 0.2 ml. This was
incubated at 378C for 18 h, and then SDS-PAGE/Western blotting was carried
out. N,N0-dimethylcasein was labeled with guinea pig TGM2 as a positive
control for the assay.
Identification of Biotinylated Peptides
The biotinylated TGM2 target polypeptides in MVM were separated from
the unbiotinylated peptides by avidin affinity chromatography. MVM protein
was biotinylated as described and spun down at 130003g for 5 min to recover
the membrane fraction; the pellet was solubilized in 1.0% SDS/Tris-HCl (100
mM, pH 8.3), with approximately a 1:5 ratio of MVM:SDS. This was boiled for
3 min and diluted 10-fold to 0.1% SDS. Dialysis against 0.1% SDS was carried
out to remove free biotin, and the solution was mixed overnight with prewashed
avidin beads (Softlink soft release avidin resin; Promega) that had been
prepared by 33 washes in Tris-HCl (pH 8.3), with a final wash and a 30-min
incubation at room temperature in 0.1% Tris-HCl. Following the overnight
incubation, the beads were washed in Tris-HCl (pH 8.3), and the supernatant
containing the unbiotinylated peptides was retained. The biotinylated peptides
were released from the beads by boiling in 50 ll of SDS sample buffer. The
bound and unbound fractions were loaded onto SDS-PAGE gels and visualized
by silver staining.
SDS-PAGE/Silver Staining/Protein Blotting
Protein concentration was determined with the BCA Protein Assay Kit
(Pierce) according to the manufacturer’s instructions. Aliquots of the MVM
vesicles obtained were run on 7.5% SDS-PAGE, and filter transfers were
analyzed with avidin peroxidase to detect biotinylated proteins. Silver staining
of SDS-PAGE gels was performed by briefly washing gels in dH2O and then
fixing them in 50% (v/v) methanol and 5% (v/v) acetic acid for 20 min; this
was followed by fixation in 50% (v/v) methanol for 10 min. Gels were
sensitized in 0.02% (w/v) sodium thiosulfate for 1 min and then washed twice
for 1 min each in dH20. Gels were then submerged in 0.1% (w/v) silver nitrate
for 20 min at 48C. After two 1-min washes in dH2O, gel staining was developed
in 0.04% (v/v) formalin and 2% (w/v) sodium carbonate until bands were
visible (5–10 min). The development was stopped with 5% acetic acid, and the
gels could be stored in 1% acetic acid at 48C.
Western blotting, antibody probing, and enhanced chemiluminescence
detections were carried out according to the procedure previously described
. Monoclonal mouse anti-b-actin antibody was used at a final concentration
of 0.5 lg/ml, and polyclonal goat anti-mouse-HRP (DAKO) was used at 1 lg/ml.
This was carried out in the Biomolecular Analysis Core Facility in the
Faculty of Life Sciences at the University of Manchester. Gel bands from the
fraction bound to the avidin beads were excised with a disposable glass pipette,
reduced with 10 mM DTT in 25 mM NH4HCO3at 568C for 1 h, and then
alkylated with 55 mM iodoacetamide in 25 mM NH4HCO3for 45 min at room
temperature in the dark. After washing in acetonitrile, proteins were digested
overnight with ;5 ll of trypsin at 12.5 ng/ll (sequencing grade; Promega) in
;50 ll of 25 mM NH4HCO3. The resultant peptides were extracted from the
gel with 25 mM NH4HCO3, with further extraction with 5% formic acid in 50%
acetonitrile. Samples were dried to approximately 20 ll, of which 6 ll was
analyzed by liquid chromatography-tandem mass spectrometry (LC-MSMS)
with a Q-TOF Micromass spectrometer. Data acquired were searched against
both SWISSPROT and TrEMBL by ProteinLynxGlobalServer software.
Positive identification was defined as three or more distinct peptides from
the same polypeptide sequence by tandem MS. Two or one peptide was classed
as a tentative identification.
Biotinylated MVM protein was spun down to recover the membrane pellet
and solubilized in 1% (v/v) Brij35 (Surfact-Amps 35; Pierce) in Tris-buffered
saline containing 1% (v/v) commercial protease inhibitors (containing pepstatin
A, bestatin, E-64, leupepin, and aprotinin) and mixed at 48C for ?4 h. The
extract was centrifuged at 13000 3 g at 48C for 15 min to separate Brij35-
soluble and -insoluble proteins. The pellet was taken up into 40 ll of SDS
sample buffer and stored at ?208C. The supernatant, containing detergent-
soluble proteins, was retained at this stage for use in SDS-PAGE/Western
blotting. For immunoprecipitation experiments, rabbit polyclonal anti-human
annexin A5 antibody (final concentration, 4 lg/ml; Abcam) was added to the
supernatant and mixed for 1 h at room temperature. Protein A beads were
swollen in buffer A (0.02 M NaH2PO4and 0.15 M NaCl, pH 8.0) for 30 min
and then washed in buffer A (23), with a final wash in Brij35/buffer A. The
MVM plus antibody solution was then added to the Protein A beads and mixed
overnight at room temperature. The beads were spun down, and the first
supernatant or ‘‘flow-through’’ was retained. The beads were washed in Brij35/
buffer A (33) with a final wash in buffer A without Brij35. The antibody plus
antigen complex was released from the beads by boiling in SDS sample buffer.
Mouse monoclonal anti-human annexin A5 antibody (final concentration, 0.4
lg/ml; Abcam) was used for Western blotting.
Isolation of Primary Trophoblast Cells from Term Placenta
The method of isolation employed was based on methods used by Kliman
et al.  with later modifications . Mononucleate cytotrophoblast cells
were cultured in Dulbecco modified Eagle medium/F12/10% fetal calf serum
aggregate and fused for 2–4 days in culture . The culture medium contained
1.05 mM calcium. Cytotrophoblasts from term placenta were plated onto glass
coverslips precoated with 10% (v/v) collagen/0.1 M acetic acid at a density of 1
3 106per coverslip. Cells were fixed with ice-cold methanol at 66 h. Anti-
cytokeratin 7 antibody was used as a positive control for trophoblast cells, and
anti-vimentin antibody was used to detect fibroblast or macrophage
Antibody staining and imaging were carried out as previously described
. Trophoblast cells were incubated with primary antibodies for 1 h at room
temperature. Mouse monoclonal anti-TGM2 antibody (CUB7402; Neomarkers)
was used at a final concentration of 1 lg/ml, mouse monoclonal anti-annexin
A5 (Abcam) was used at a final concentration of 4 lg/ml, and goat polyclonal
anti-annexin A2 (Abcam) was used at a final concentration of 5 lg/ml. Cells
were incubated with fluorescein-conjugated secondary antibodies for 1 h at
room temperature, either polyclonal goat anti-mouse fluorescein isothiocyanate
(FITC; DAKO) or polyclonal rabbit anti-goat FITC, both at a final
concentration of 40 lg/ml. Nuclei were counterstained with propidium iodide
In Situ TGM2 Activity Assay
An assay was developed for measuring in situ tTG cross-linking activity in
the trophoblast cell cultures with a biotinylated probe. By this method, a
biotinylated primary amine such as BTC acts as the acyl-acceptor in the
transamidating reaction catalyzed by tTG and becomes incorporated into
endogenous protein substrates of tTG . Cells were preincubated with 1 mM
BTC for 1 h at 378C prior to fixation, and fluorescently labeled streptavidin
(streptavidin-FITC) was used to detect the biotinylated product.
Exposure of Trophoblast Cultures to TGM2 Inhibitors
The competitive and irreversible TGM2 inhibitor cystamine [23, 24], which
is thought to undergo disulfide exchange with the active-site cysteinyl residue,
and the primary amine competitor monodansylcadaverine (MDC) , which
has been widely used to inhibit TGM2 activity in vivo, were introduced into the
cytotrophoblast culture system. Following the determination of the optimum
nontoxic inhibitor doses, cystamine concentrations of 1.5 mM and MDC of 150
lM were used in the cultures. As TGM2 is known to be important for cell-
matrix adhesion [26, 27], the inhibitors were added to the cultures after 18 h to
allow sufficient time for stable cell attachment and the formation of
Assessment of Trophoblast Differentiation
Trophoblast differentiation was assessed by measuring the extent of
multinucleated trophoblast formation at the end of the culture period . Cells
were fixed and permeabilized with methanol as described. Cell-cell borders and
nuclei were detected by staining cultures simultaneously with antibody to E-
cadherin, which detects intertrophoblastic contact surfaces, and with PI. Total
numbers of nuclei were counted from three random fields per culture flask. At
least 50 nuclei were counted per field. Nuclei present in a single cell (cells were
identified by E-cadherin staining at cell-cell borders) were also counted. Nuclei
present as two or more in a single cell were expressed as a percentage of the
total nuclei and were defined as multinucleated.
TGM2 AND SHED PLACENTAL MICROPARTICLES
Levels of immunoreactive hCG in trophoblast culture media were
determined with a solid-phase enzyme-linked sandwich immunosorbent assay
(DRG Diagnostics). Medium was collected from 18–66 h of culture. The
concentration of p-nitrophenol product was assessed by measuring the
absorbance at 405 nm. All measurements were carried out in duplicate.
Isolation of Syncytiotrophoblast Microparticles from
To isolate shed syncytiotrophoblast microparticles, supernatants from 18 to
66 h in culture from three or more 25-cm3flasks of trophoblast cells were
collected and subjected to a three-step centrifugation procedure at 48C. The
supernatant was spun at 1000 3 g for 10 min, at 10000 3 g for 10 min to
remove cellular debris, and finally at 70000 3 g for 90 min to pellet the
microparticulate material . The final pellet was collected and resuspended
into 20 ll of SDS sample buffer and stored at ?208C until use.
Electron Microscopy Sample Preparation
For electron microscopy preparation, specimens were resuspended in
human serum and embedded as previously described .
All experiments were performed three times, and each experiment used
cells obtained from different placentas. Cells were not pooled. Within an
experiment, means were obtained from duplicate determinations. Data were
compared by the nonparametric Kruskal-Wallis test for repeated-measures
analysis of variance with the Dunn post hoc test by GraphPad Prism software,
version 4 (GraphPad Software). Significance was taken as P , 0.05.
Proteomic Analysis of Target Proteins for TGM2-Mediated
Cross-Linking in the Placental MVM
TGM2 transamidating activity was detected in isolated
MVM vesicles with two different biotinylated affinity probes
in the presence of 5 mM calcium. The biotinylated primary
amine BTC acted as an acyl-acceptor (Lys-donor) , and the
hexapeptide biotinyl-TVQQEL  acted as an acyl-donor
(Glu-donor) probe. Both were incorporated into numerous
endogenous protein substrates of TGM2 in the MVM, as
shown by separating MVM reaction mixtures on SDS-PAGE
acyl-donor and acceptor TGM2 protein
substrates in the MVM. TGM2-mediated
labeling of MVM vesicles with (A) the acyl-
acceptor probe BTC or (B) the acyl-donor
probe biotinyl-TVQQEL. MVM protein (M)
from term placenta was reacted with probe
in the presence of Ca2þ. Aliquots were
fractionated on avidin-conjugated beads. S
shows nonretained material, while E1 and
E2 are proteins eluted from the beads.
Replicate gels were stained with silver (top),
or blots were prepared and probed with
avidin peroxidase to detect TGM2 substrate
proteins (bottom). Bands in a wide range of
Mrvalues are present in the unfractionated
MVM and in eluates from the beads, but no
bands are present in the supernatant frac-
tions, indicating efficient selection of bio-
tinylated proteins. Note that the two probes
yield different protein profiles. *, þ, # ¼
bands in common from eluted fraction
between silver-stained gel and immunoblot.
Positions of molecular weight standards
(kiloDaltons) are indicated. C) TGM2 pro-
tein localization in 66-h trophoblast cul-
tures. Primary term trophoblast cells were
fixed in methanol after 66 h of culture and
(left) stained with mouse monoclonal anti-
TGM2 antibody and an FITC-conjugated
secondary antibody (green). TGM2 is ob-
served at lateral cell boundaries, especially
in residual mononucleate cells, in associa-
tion with stress fibers, and more weakly in
punctate deposits. D) TGM2 transamidating
activity in 66-h trophoblast cultures. TGM2
cross-linking activity was visualized with a
BTC incorporation assay with streptavidin-
FITC to detect biotinylated products (right,
green). Labeling is partially seen in punctate
deposits, with activity not obviously asso-
ciated with lateral cell boundaries or major
cytoskeletal elements. Nuclei were coun-
terstained with propidium iodide (PI) (red).
Bar ¼ 20 lm.
Identification and fractionation of
ROBINSON ET AL.
and probing blots with avidin peroxidase (Fig. 1, A and B).
Substrates ranged from ;15 to 230 kDa, with high-molecular-
mass cross-linked products sometimes observed. No bands
were present when nonbiotinylated MVM was run on SDS-
PAGE and probed with avidin peroxidase (data not shown).
Biotinylated target proteins in the MVM were isolated with
an avidin-affinity column. The presence of 0.1% SDS in the
washing buffer ensured that only directly biotinylated targets
would be recovered. Multiple proteins were present in fractions
eluted from the beads. No biotinylated products remained in
the supernatants when either the acyl-acceptor or acyl-donor
probe was used, indicating that efficient separation had
occurred in both cases. Fractions eluted from beads were run
on SDS-PAGE. Several regions of each gel were excised,
alkylated, and digested with trypsin. Samples were then
directly analyzed by LC-MSMS. Supplemental Tables 1 and
2 (available online at www.biolreprod.org) show the range of
acyl-donor and acyl-acceptor TGM2 substrate proteins identi-
fied, respectively, in descending order of apparent abundance.
This proteomic strategy led to the identification of .30
TGM2 protein substrates in the MVM. Table 1 is a functional
classification of the TGM2 protein substrates identified. Some
target proteins were identified with both probes, indicating that
they are able to function as both acyl-acceptor and -donor in
the transamidation reaction and therefore form homo-oligo-
mers. The two main target types are cytoskeletal and
membrane-associated proteins, suggesting a role for TGM2
in the organization and turnover of trophoblast plasma
membranes and associated cytoskeleton.
TGM2 Distribution and Activity in Cultured
Having established the presence of TGM2  and
identified target proteins in an in vitro assay with MVM
isolated from placental tissue, it was important both to
characterize the enzyme activity and confirm selected target
proteins in living cells. Primary cytotrophoblasts isolated from
term placenta were used for studying in vitro TGM2 expression
and transamidation activity. Mononucleate cells aggregate and
fuse over 2–4 days in culture . TGM2 localization in the
66-h cultures was widespread and strong. It was prominent at
the lateral borders of the cells, both at cell contacts and at
noncontact surfaces, in the cytoplasm and in association with
stress fibers and peripheral actin bundles (Fig. 1C).
To detect acyl-donor (Gln-donor) TGM2 substrates, the
acyl-acceptor probe BTC was added to the cultures and
visualized with FITC-labeled streptavidin. TGM20s trans-
amidating activity appeared much more variable between
different cells, perhaps consistent with differentiation depen-
dence. It was most prominently seen in a punctate cytoplasmic
compartment (Fig. 1D). Generally, the activity of TGM2 was
far more restricted than the enzyme’s distribution. Biotinylation
TABLE 1. Functional classification of identified TGM2 substrates.
Functional groupProteins Acyl donor/acceptor
Cytoskeletal network Actin; beta, gamma 1 and alpha 2
Myosin heavy chain 9
Myosin light chain 6
Heat shock protein 27-kDa protein 1
Alkaline phosphatase, placental
Chloride intracellular channel protein 4
Clathrin heavy chain
Solute carrier family 2 (facilitated glucose
ATPase, Naþ/Kþtransporting, alpha 1 polypeptide
Protein disulphide isomerase A3
Heat shock protein HSP90-alpha
Heat shock protein HSP90-beta
Fibrinogen alpha chain
Fibrinogen beta chain
Fibrinogen gamma chain
S100 calcium-binding protein P
S100 calcium-binding protein A8
S100 calcium-binding protein A11
Guanine nucleotide-binding protein (G protein)
beta polypeptide 1
Chorionic somatomammotropin hormone
Ras-related protein Rab-1A
Pyruvate kinase, isoforms M1/M2
Monoamine oxidase type A
Donor and acceptor
Donor and acceptor
Donor and acceptor
Donor and acceptor
Donor and acceptor
Donor and acceptor
Donor and acceptor
Donor and acceptor
Donor and acceptor
Donor and acceptor
TGM2 AND SHED PLACENTAL MICROPARTICLES
MVM and cultured trophoblast. A) MVM
vesicles were reacted with BTC, detergent
solubilized, and fractionated on avidin
beads. The top panel shows annexin A5
immunoreactivity (33 kDa) detected with a
mouse monoclonal anti-human annexin A5
antibody in the unfractionated MVM (M),
the pellet (P) and the detergent solubilizate
(S), the flow-through (FT) after solubilization
of the MVM vesicles with nonionic deter-
gent, and the immunoprecipitate (IP) with a
rabbit polyclonal anti-human annexin A5
antibody. The lower panel shows an im-
munoblot of the MVM (M), flow-through
(FT), and immunoprecipitate (IP) probed
with avidin peroxidase, indicating that
immunoprecipitated annexin A5 is biotiny-
lated and also that a significant fraction of
biotinylated A5 is not retained on the beads.
B) Immunolocalization of TGM2 substrates
annexin A2 and A5 (green) in term tropho-
blast at 66 h in culture. Punctate annexin A2
staining is observed (arrowheads) mostly
around the edges of the syncytium (arrows).
Annexin A5 appears mostly associated with
mononucleate unfused cells (arrows), with
less staining observed in the large syncytia
(arrowheads). C) Serial Z-sections of an-
nexin A2 localization within multinucleated
66-h trophoblast cells, from the basal (top
left) to the apical (bottom right) surface.
Focal annexin A2 immunoreactivity is
detected at the apical surface (arrows),
above the plane of the nuclei. Nuclei were
counterstained with PI (red). Bar ¼ 25 lm
(B) and 20 lm (C).
Annexins are TGM2 substrates in
ROBINSON ET AL.
in living cells could be inhibited by the function-blocking anti-
TGM2 monoclonal antibody CUB7402 (data not shown).
Annexins are one of the most abundant target protein
families observed in the MVM (see Supplemental Tables 1 and
2 available online at www.biolreprod.org). Evidence of direct
annexin biotinylation in the MVM was obtained with annexin
A5 as target. Immunoprecipitation of annexin A5 was carried
out from biotinylated MVM, and biotinylation was verified by
the binding of avidin to the annexin A5 immunoreactive band
(Fig. 2A). A fraction of biotinylated annexin A5 was not
retained by the avidin beads and appeared in the flow-through,
suggesting that the binding capacity of the beads was limiting.
Immunolocalization of selected annexins was carried out with
cultured cytotrophoblasts from term placenta. Punctate annexin
A2 staining was observed in trophoblast cultures fixed at 66 h
(Fig. 2B), with the prominent localization seen around the
edges of the syncytium. Annexin A5 appeared mainly
associated with mononucleate, unfused cells, with less seen
in the syncytia (Fig. 2B). Serial Z-sections were taken, showing
some focal expression of annexins at the apical surface above
the plane of the nuclei, possibly associated with membrane-
bound fractions breaking away from the multinucleated
syncytium. Annexin A2 immunoreactivity was observed,
associated with blebs at the apical surface (Fig. 2C).
Effect of Inhibiting TGM20s Transamidating Activity on
The main functional classifications of TGM2 substrates in
the placental MVM were cytoskeletal and membrane-associ-
cadaverine incorporation assay with streptavidin-FITC to detect biotinylated product (green). TGM2 cross-linking activity apparent in the control culture
(upper left) is lost from the cultures treated with either TGM2 inhibitor (upper middle and right). Minimal cross-linking activity is observed in the
cystamine (125 mM) treated culture (arrows), with no evidence of TGM2 cross-linking in the MDC (150 lM) treated culture. Bar ¼ 25 lm. B)
Cytotrophoblast cells were cultured in the presence or absence of TGM2 inhibitors, and cultures were stained with E-cadherin protein to detect sites of
cell-cell contract (green). In untreated trophoblast cells at 66 h, little E-cadherin staining is apparent (lower left), suggesting fusion to form multinucleated
syncytia has occurred. More E-cadherin staining is observed between trophoblast cells cultured with either cystamine or MDC (lower middle and right),
indicating less fusion with a higher proportion of mononucleate cells remaining. Nuclei were counterstained with propidium iodide (PI) (red) in each case.
Bar ¼ 20 lm. C) By E-cadherin immunoperoxidase staining, nuclei were counted, and those present as single mononucleate cells with a complete E-
cadherin cell border staining were scored in the presence or absence of either TGM2 inhibitor. The extent of multinucleated cell (i.e., syncytiotrophoblast)
formation was calculated as described in Materials and Methods. Use of either inhibitor resulted in a significant reduction in syncytium formation.
Asterisks indicate values significantly different from untreated controls (*P , 0.05, ***P , 0.001, Kruskal-Wallis ANOVA followed by the Dunn post hoc
test). D) Culture media were collected from trophoblast cells cultured in the presence or absence of TGM2 inhibitors, and the secretion of bioactive hCG
was analyzed as described in Materials and Methods. A significant reduction (***P , 0.001, the Dunn post hoc test) in hCG secretion was seen in cultures
treated with either cystamine or MDC. After removal of culture supernatants, the protein content of the adherent cells was measured (E). No significant
differences in the protein content were determined after treatment with either TGM2 inhibitor.
The effect of TGM2 on trophoblast turnover. A) TGM2 cross-linking activity in primary trophoblast cells fixed at 66 h, measured with the biotin-
TGM2 AND SHED PLACENTAL MICROPARTICLES
ated proteins, suggesting a role for TGM2 in the organization
and turnover of trophoblast plasma membranes and associated
cytoskeleton. To explore a functional role for TGM2 in the life
cycle of trophoblasts, TGM2 inhibitors were introduced into
primary cytotrophoblast monolayer cultures. Following the
addition of either the TGM2 competitive substrate inhibitor
MDC  or the TGM2 active site inhibitor cystamine [23,
24], almost complete inhibition of TGM2 cross-linking activity
was observed with the BTC incorporation assay. There was no
evidence for transamidating activity in the MDC-treated
cultures, and minimal levels of TGM2 cross-linking activity
were detected in the cultures containing cystamine (Fig. 3A).
The normal process of trophoblast cell turnover in the
placental villus consists of the proliferation and differentiation
of underlying mononuclear cytotrophoblast cells, followed by
intercellular fusion of differentiated cytotrophoblasts to form
the continuous multinucleate syncytiotrophoblast layer, which
continues to differentiate; finally, there is an apoptotic release
of old material into the maternal circulation .
Differentiation was assessed in the cultures by measuring
the extent of multinucleated trophoblast at 66 h, using a nuclear
stain together with anti-E-cadherin to reveal intercellular
borders. During cytotrophoblast aggregation, E-cadherin as-
sembles at cell-cell contact surfaces, and its expression is
down-regulated and finally lost as the mononucleate cells
undergo cellular differentiation and fusion . Figure 3B
shows the effect of either TGM2 inhibitor on the E-cadherin-
nuclear-staining pattern. When cells were fixed after 66 h with
no inhibitor present, some E-cadherin staining was apparent,
but many of the cells were multinucleated, with three or more
nuclei in a single cell. In contrast, when trophoblasts were
cultured in the presence of either cystamine or MDC, increased
E-cadherin expression was observed, with a higher proportion
of mononucleate cells. Mononucleate cells with continuous E-
cadherin-positive cell borders were scored in the presence or
absence of TGM2 inhibitors (Fig. 3C). Nuclei present as two or
more in a single cell were expressed as a percentage of the total
nuclei. In the control cultures, more than 85% of the cells were
multinucleated, with a significant reduction when either
cystamine (64.5%) or MDC (56.5%) was used (Kruskal-Wallis
test, P , 0.0001, and Dunn post hoc test, P , 0.05 control
versus cystamine, P , 0.001 control versus MDC).
Syncytiotrophoblast are the major source of the glycoprotein
hCG, a widely used marker of trophoblast differentiation. It is
thought that hCGa is initially produced and is followed by the
production of hCGb as trophoblast differentiation progresses.
Therefore, hCGb is the limiting factor for the production of
hCG and is measured in the hCG assay as an indicator of the
presence of total hCG. Levels of hCG were measured in the
medium of cells cultured with and without the addition of
TGM2 inhibitors (the untreated and treated cells were passaged
and maintained in culture at the same time). The inhibitors
were added to the trophoblasts before differentiation had
occurred, as described in Materials and Methods. Culture
supernatants were removed after 66 h for the assay of
immunoreactive hCG. There were significant reductions in
hCG concentration between control cultures and those treated
with either cystamine or MDC (Fig. 3D). The hCG
concentration with cystamine (52.2 6 4.5 mIU/ml) was
reduced by 60.3% (control hCG, 131.56 11.3 mIU/ml), and
competitive substrate inhibitor MDC reduced hCG production
by 61.8% (50.3 6 4.0 mIU/ml). Use of either inhibitor caused
a significant difference compared with the control (Kruskal-
Wallis test, P , 0.0001, and Dunn post hoc test, P , 0.001, in
both cases). The cellular protein content of the cultures was not
altered by treatment with either inhibitor (Fig. 3E), suggesting
the effects seen of TGM2 inhibition on trophoblast differen-
tiation were not just due to decreased cell viability.
TGM2-Mediated Cross-Linking in Shed Placental
TGM2 is thought to stabilize apoptotic cells by preventing
the leakage of intracellular components by means of cross-
linking. TGM2 cross-linking of proteins in the MVM could
therefore occur before the shedding of apoptotic particulate or
vesicular material into the maternal circulation. Material shed
from primary trophoblast monolayer cultures between 18 and
TEMs of material shed by trophoblast cultures and isolated by high-speed
centrifugation. A) Low-power image of sheets of mononuclear cyto- (C)
and syncytial (S) trophoblasts showing numerous microvilli (arrowheads)
on the apical surface as well as processes basally. B) At high power,
microvilli can be seen to contain longitudinal actin filaments (arrows),
while microtubules and cytokeratin filaments are also observed. C) Actin
filaments are also apparent in transverse section (arrows) of microvilli and
larger cytoplasmic particles. Bar¼5 lm (A), 0.25 lm (B), and 0.2 lm (C).
D) TGM2 cross-linking activity in trophoblast cell homogenates and
syncytiotrophoblasts shed material in the presence of TGM2 inhibitors.
Western blot analysis of the 66-h trophoblast cell homogenates (C) and
material shed (SM) from trophoblast cultures, probed with (i) avidin
peroxidase or (ii) anti-b-actin antibody and in the presence of TGM2
inhibitors cystamine (Cys) or monodansylcadaverine (MDC). Note that
actin immunoreactivity is present in a monomer band and in a high Mr
fraction, with loss of the high Mrband from the shed material treated with
either TGM2 inhibitor. Molecular weight markers are indicated.
Microvilli with actin cytoskeleton in shed trophoblast material.
ROBINSON ET AL.
66 h was isolated by differential centrifugation . Trans-
mission electron micrographs (TEMs) of the shed material
demonstrated the presence of various different sizes and types
of material ranging from large, apical, syncytial fragments with
intact microvilli to microvesicles (Fig. 4, A–C). Actin filaments
were seen running longitudinally in microvilli, and cytokeratin
filament bundles and microtubules, the protein constituents of
which had been identified at TGM2 substrates (Table 1), were
TGM2 protein was readily detectable by Western blotting in
the shed material (data not shown). To ascertain whether this
fraction of the enzyme was active, the acyl-acceptor probe BTC
was added to the cells in the final few hours of culture (from
;63 to 66 h), a time point reflecting the final stages of
trophoblast differentiation in the cell cultures, when most
nuclei have become incorporated into syncytium, from which
shedding of apical material occurs. Shed material was isolated
from the culture medium, and blots of the high-speed pellet
were probed with avidin peroxidase (Fig. 4D). Numerous
bands were present in both fractions, indicating that TGM2
cross-linking activity is present in shed material as well as in
living cells. Both the cell homogenate and shed material were
found to contain biotinylated b-actin, consistent with its
identification as a TGM2 substrate in situ (Table 1). Strikingly,
a high-molecular-mass immunoreactive band appeared in shed
material but not in live cells and contained actin immunore-
activity, suggesting cross-linking into large multimeric aggre-
gates. The actin monomer at ;42 kDa was of a lower intensity
in the shed material than in the cell homogenate, suggesting
much of the actin in the shed trophoblast microparticles was
The TGM2 inhibitors MDC and cystamine were added to the
trophoblast cultures to examine the role of TGM2 in the
shedding process. Syncytiotrophoblast microparticulate frag-
ments were collected from the medium from 18 to 66 h in culture
in the presence or absence of an inhibitor. The presence of tTG
inhibitors did not affect the quantity of shed material isolated,
with an average yield of 123 6 10.8 lg of protein per 303106
cells without inhibitor, 119 6 12.3 lg of protein per 30 3 106
cells in the presence of cystamine, and 121 6 8.0 lg of protein
per 303106cells in the presence of MDC (n¼5 preparations).
Western blots of the shed material from inhibitor-treated cells
showed that b-actin was exclusively in the monomeric 42-kDa
form (Fig. 4D), with disappearance of the high-molecular-mass
band apparent in the untreated shed material.
The syncytiotrophoblast MVM acts as an interface between
the placenta and maternal blood in the intervillous space and
shows strong TGM2 expression and reactivity [14, 32]. We
aimed to identify substrate proteins in the MVM in order to
advance understanding of TGM2’s function at this key tissue
interface. Assays were carried out in the presence of calcium
ions, which are required for the transamidating activity of
TGM2. Of .30 proteins cataloged, some were known TGM2
substrates, whereas others had not been previously identified.
Several proteins acted both as acyl-donor and -acceptor
substrates in the transamidating reaction, strongly suggesting
these targets are cross-linked by TGM2 into homo-oligomers.
Substrates fall into several functional groups. Cytoskeletal
proteins such as actin, myosin heavy and light chains, tubulin,
and intermediate filament polypeptides of the cytokeratin
family are highly represented. TGM2-mediated polymerization
of cytoskeletal proteins has implications for the enzyme in
apoptosis. Changes to cytoskeletal organization have been
proposed to determine the morphologic events of programmed
cell death . TGM2 is often highly expressed during
programmed cell death, and its induction has been associated
with apoptosis in several cell systems .
Despite the appearance of TGM2 in various cells and tissues
undergoing apoptosis, it is unclear whether the increase in
TGM2 activity causes cell death in various pathological
processes or whether the two occurrences are related non-
causally. Recent reports have supported the idea that TGM2’s
GTP-bound form plays a role in promoting cell survival, with
the calcium-dependent transamidating activity being involved
in apoptosis . Modification of cytoskeletal proteins such as
actin or myosin by TGM2 could lead to the formation of a
highly cross-linked protein scaffold that stabilizes apoptotic
cells, preventing the release of cellular components into
surrounding tissues and thus circumventing the inflammatory
response . Various cytoskeletal proteins have previously
been identified as TGM2 substrates; for example, actin was
identified as a TGM2 substrate in human leukemia cells
undergoing apoptosis , and myosin and spectrin were
recently identified as TGM2 substrates in an intestinal
epithelial cell line .
MVM proteins, including transferrin receptor, placental
alkaline phosphatase (ALPP, also known as PLAP-1), and
integrins alpha V and alpha IIb, were also identified as TGM2
substrates. ALPP is attached to the outer leaflet via an inositol
link, and its presence in the substrate fraction suggests that
TGM2 is active at the outer face of the membrane as well as the
inner face, as evidenced by the presence in the target pool of
cytoskeletal components and several members of the annexin
family of Ca2þ-dependent phospholipid-binding proteins.
Annexins have various functions, including anticoagulation,
cytoskeletal interactions, anti-inflammatory activity, and signal
transduction. The anticoagulant properties of annexin A5,
which binds anionic phospholipids and shields coagulant sites,
have been proposed to be crucial for maintaining placental
integrity . Annexin A5 may also play a role in trophoblast
differentiation, as anti-annexin A5 antibody was demonstrated
to block trophoblast fusion in culture, and antisense oligonu-
cleotides to annexin A5 reduced fusion by about 50% [37, 38].
There is evidence that annexin A5 may be present at both the
outer and inner surface of the MVM [36, 39, 40].
Syncytiotrophoblast membrane microparticles isolated from
normal placenta have previously been shown to inhibit
endothelial cell proliferation in vitro . Eight proteins were
identified from purified syncytiotrophoblast MVM and found
to assemble a self-aggregating complex, which may be
responsible for the antiproliferative activity . Seven of
these eight proteins have been identified in the current study as
TGM2 substrates in MVM (transferrin and its receptor,
integrins aV and a5, ALPP, a-actinin, and monoamine oxidase
type A), suggesting that TGM2 transamidation is involved in
the formation of this aggregating cluster of proteins that
becomes shed from the syncytial MVM.
In vitro, TGM2 is seen both in syncytio- and cytotropho-
blasts in association with cytoskeletal structures, including
peripheral actin bundles and stress fibers. Colocalization of
TGM2 with stress fibers has previously been demonstrated in
human umbilical vein endothelial cells , where immuno-
precipitation studies suggested that association with stress
fibers was due to TGM2’s cross-linking of myosin. The use of
a biotinylated substrate allowed transamidating activity to be
localized and compared with the protein distribution in live
cells. TGM2 localized predominantly to lateral cell borders,
whereas cross-linking activity was restricted to a small number
of cells and was not obviously associated with lateral cell
TGM2 AND SHED PLACENTAL MICROPARTICLES
boundaries. This suggests that subcellular regulation of TGM2
transamidating activity occurs within the MVM, perhaps by
means of intracellular Ca2þconcentration . Because there
are significant amounts of intracellular TGM2 in trophoblasts
that are not associated with the MVM, TGM2 may carry out
distinct functions in different cellular compartments in this
The involvement of TGM2-mediated cross-linking in
trophoblast differentiation was examined by the use of
TGM2 inhibitors during differentiation in vitro and monitoring
of the formation of multinucleated syncytiotrophoblasts (which
occurs by fusion) and hCG secretion. Fusion was reduced by
treatment with either TGM2 inhibitor and was accompanied by
no loss in cell viability. Cystamine or MDC was added to the
cytotrophoblast cultures prior to the onset of differentiation and
significantly inhibited hCG secretion. Undifferentiated cyto-
trophoblast cultures produce low levels of hCG, and a
reduction in secretion in the presence of inhibitor is consistent
with the inhibition of syncytiotrophoblast formation. Although
care must be applied in extrapolation of these in vitro data to
the in vivo situation, these findings suggest that TGM2
enzymatic activity is important for normal trophoblast turnover
in the placental villus.
In vivo, after cytotrophoblast differentiation and fusion to
form syncytiotrophoblast, terminal differentiation occurs with-
in the syncytium. This eventually leads to the packaging of
apoptotic material into syncytial knots and smaller vesicular
structures, which are shed into maternal circulation .
TGM2 cross-linking of cytoskeletal proteins has implications
in this normal process of cell turnover in the placental villus.
TGM2 cross-linking of actin in the MVM could occur at the
end of the trophoblast life cycle, before shedding. Previous
analytic and functional studies have utilized either particles
isolated from maternal venous blood  or microvillus-
derived membrane fractions derived by various methods
(perfusion, explant, and mechanical dissection) [16, 41] from
villous tissue. In the former case, the particles represent a
fraction that has survived in circulation, while in the latter case,
the preparation is from the intact cell membrane and not
derived by a natural process of shedding. We have now
established that microparticulate fragments are shed by primary
trophoblasts in culture, suggesting the suitability of this model
for such studies. These microparticles were found to contain
abundant TGM2 protein but had less TGM2 cross-linking
activity than equivalent loadings of cell protein (data not
shown). The biotinylated acyl-probe was added to the
trophoblast cells from 63 to 66 h in culture; thus, the presence
of biotinylated bands in the shed particles shows TGM2
activity occurring in the final few hours of culture. This
suggests that the predominant TGM2 transamidating activity
occurs before material is shed from the syncytiotrophoblast.
TGM2 inhibitors did not prevent shedding in vitro, as indicated
by the presence of b-actin in approximately equivalent amounts
in both the inhibitor-treated and control shed fragments. In the
presence of either inhibitor, actin monomer was present in the
shed material, in comparison to the nontreated shed material,
where high-molecular-mass multimeric actin was present. This
is consistent with less actin becoming cross-linked into larger
complexes in the shed material from inhibitor-treated cultures.
TGM2’s cross-linking activity may be important in the
preparation of apically associated membrane material for
shedding, suggesting that it has as a vital role to play in
placental homeostasis and function. TGM2 cross-linking of
material destined to be shed from the placenta may enhance
phagocytosis by the maternal reticuloendothelial system and
contribute to reducing the maternal immune reaction to the
high quantities of fetal antigen shed during pregnancy.
To summarize, substrates for TGM2 have been identified at
the primary maternal-fetal interface, the most abundant types
including cytoskeletal and membrane-associated proteins.
Syncytial microparticles shed from primary trophoblast
cultures contain abundant TGM2 protein and cross-linking
activity. A substantial proportion of actin in the shed particles
is in the form of polymeric aggregates, in contrast to cellular
actin, which dissociates completely into monomers in SDS-
PAGE. Primary trophoblast cells cultured in the presence of
transglutaminase inhibitors showed decreased trophoblast
differentiation, and actin in the shed particulate material
remained exclusively in monomeric form. These results
suggest a role for TGM2-mediated cross-linking in stabilizing
particulate material shed from the placenta and, more generally,
in the organization and turnover of trophoblast plasma
membrane and associated cytoskeleton.
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