Clinical Science (1985) 68, 513-519
573
Human jejunal transglutaminase: demonstration of activity,
enzyme kinetics and substrate specificity with special relation
to gliadin and coeliac disease
S. E . B R U C E , I . B J A R N A S O N
AND
T. J. PETERS
Division of Clinical Cell Biology, MRC Clinical Research Centre, Harrow, Middlesex, U.K.
(Received 5 March116 August 1984; accepted 30 November 1984)
Summary
1. By use of a radiometric assay transglutaminase activity was demonstrated for the first time in
human jejunal mucosa. The activity is similar to
that in other tissues, with a pH optimum of 9.0, an
absolute requirement for Ca2+ and an apparent
K , for putrescine of 0.15 mmol/l.
2. Assay of jejunal transglutaminase activity
with a variety of dietary proteins as acceptors
showed high activity with gliadin, comparable with
that of the standard substrate, dimethylcasein.
Deamidation of the gliadin markedly reduced its
acceptor activity. Collagen, ovalbumin, elastin
and zein exhibited very low acceptor activities.
3. Increased transglutaminase activity was
demonstrated in jejunal biopsies from four
patients with untreated coeliac disease compared
with 14 control subjects and eight patients with
inflammatory bowel disease. Eight patients with
coeliac disease in remission, with normal levels of
brush border a-glucosidase, showed elevated
transglutaminase activities compared with those of
controls.
4. It is postulated that intestinal transglutaminase activity may be important in gliadin binding
to tissues and thus in the pathogenesis of coeliac
disease.
Key words: coeliac disease, gliadin, transglutaminase.
Introduction
Transglutaminase (R-glutaminyl-peptide :amine yglutamyl transferase: EC 2.3.2.13) has been
Correspondence: Professor T. J. Peters, Division
of Clinical Cell Biology, MRC Clinical Research
Centre, Watford Road, Harrow, Middlesex HA1
3UJ, U.K.
demonstrated in a variety of tissues including liver
[ l ] , skin [2, 31, erythrocytes [4, 51, seminal fluid
[6], kidney [7] and brain [8]. The activity first
characterized in detail was plasma fibrin stabilizing
factor (factor XIII) [9]. The enzyme catalyses
the formation of isopeptide bonds between ycarboxyl groups of glutamine residues in one
polypeptide with €-amino groups of lysine residues
in another. Thus the activity converts recently
formed fibrin monomers into highly cross-linked
oligomers [9]. Similarly, epidermal cell transglutaminase stabilizes keratin molecules by forming isopeptide bonds [lo].
The role of transglutaminase in other tissues is
less well defined. It has been implicated in cell
division and proliferation, cell-cell interactions
and endocytosis (review: [ 111). Increased activities
have been reported in rapidly proliferating tissues
[12], with a relative reduction in frankly neoplastic cells [13]. However, the role of transglutaminase in cell turnover remains to be
determined.
Although the causal role of gliadin and its peptide fragments in the aetiology of the coeliac
lesion has been well documented, the pathogenic
mechanism remains to be determined: both
biochemical and immunological [ 141 mechanisms
have been proposed.
The initial effect in gliadin toxicity is presumably binding of gliadin to the mucosa with
either a direct effect on enterocytes via, perhaps,
endocytosis of the gliadin to lysosomes [15, 161
or an immunological reaction to gliadin fragments
[ 171 due to an abnormally permeable intestinal
mucosa [18, 191. Both of these hypotheses implicate a selective binding of gliadin to cell surfaces.
The possible mechanisms of gliadin binding have
been little considered although it has been
suggested that gliadin can act as a lectin [20,21].
5 74
S. E. Bruce e t al.
Consideration of the substrate requirements for
transglutaminase suggested that gliadin would be
a highly preferred acceptor substrate. The protein
contains approximately 40% of glutaminyl
residues, of which over 90% are in the form of
amidc residues [22], an absolute substrate requirement by transglutaminases [l 11. It has been long
established that deamidated gliadin is non-toxic
to patients with coeliac disease [23-251 and thus a
study of intestinal transglutaminase was indicated.
. A preliminary report of this work has been
published [26].
Methods
Materials
[ 1,4-'4C]Putrescine (2-10 Ci/nimol) and PCS
scintillant were purchased from Amersham International, putrescine dihydrochloride, dithiothreitol, zein, elastin, ovalbumin and collagen from
Sigma (London) Chemical Co. (Poole, Dorset,
U.K.) and N,N-dimethylcasein and gliadin from
BDH Chemicals (Chadwell Heath, Essex, U.K.).
All other reagents were of AnalaR grade. Gliadin
(100 mg) was deamidated by heating at 100°C for
45 min in 50 ml of 1 mol/l HC1 [25]. After
cooling, the mixture was exhaustively dialysed
for 48 h against several changes of distilled water
at 4OC and the protein collected by freeze-drying.
Experimen tal
Tissue samples. Jejunal biopsy samples were
collected just distal to the ligament of Treitz, with
a Crosby capsule under fluoroscopic control, from
patients with coeliac disease in relapse, i.e. before
treatment by gluten withdrawal, and from patients
with coeliac disease in remission who had been
treated for various periods, between 5 and 20
years, by gluten exclusion. Control tissue was
from patients of similar age and sex distribution
who were initially suspected of having small bowel
disease but in whom subsequent review revealed
no significant pathology. Most of these patients
suffered from the irritable bowel syndrome.
Patients with inflammatory bowel disease comprised four with ulcerative colitis in partial or
complete repression and four with Crohn's diseasc
affecting the teminal ileum, as judged by radiological criteria.
The tissue was divided into two portions. One
was immediately snap-frozen and stored in liquid
nitrogen for up to 6 months before assay; the
other was processed for routine histological
assessment, morphometric measurements [27] and
for intra-epithelial lymphocyte counts [28]. These
studies were approved by the Harrow Health
Authority Ethical Committee.
Biochemical analyses. All samples were coded
and enzyme assays were performed without
knowledge of the diagnostic categories. Immediately before assay they were thawed in a volume of
sucrose (0.25 mol/l) containing disodium EDTA
(1 mmol/l), pH 7.2, to give a final homogenate
concentration of 100 mg wet weight/ml and disrupted on ice in a small Dual1 homogenizer.
Portions of the homogenate were assayed for
brush border Zn2+-resistanta-glucosidase [29] and
protein [30] with bovine serum albumin as
standard. Transglutaminase was assayed by a
modification [31] of the method of Lorand
et al. [32]. Details are given of the routine assay
procedure adopted. Reaction mixture (45 p1)
contained, on addition of tissue sample, final
concentrations of 0.25 mmol of putrescine/l
(approx. 135 000 c.p.m.), 50 mmol of dithiothreitol/l, 10 mmol of CaCI2/1 and 4% (w/v)
dimethylcasein in Tris-HC1 buffer (50 mmol/l),
pH 9.0, containing 0.1% (w/v) Triton X-100.
Tissue homogenates (30 p1) were added and the
mixture was incubated, with shaking, at 37OC
foi 20 min. A portion (20 p l ) was spotted onto
Whatman 3 MM filter paper (2 cm x 1 cm) (Reeve
Angel, Maidstone, U.K.) and immediately plunged
into ice-cold 10% (w/v) trichloroacetic acid ( I 0 ml
per filter paper). After 15 min, the papers were
washed twice in 5% (w/v) trichloroacetic acid for
a further 15 min, followed by brief washing in
ethanol/acetone (1 : 1, w/v) and in zcetone. After
drying, the filter papers were counted in 3 ml
of PCS sctintillant. Blank incubations, standards
and controls were similarly processed.
Results
Demonstration of enzyme activity
Fig. 1 shows the pH-activity profile for jejunal
transglutaminase activity. There is a broad peak
with maximal activity at pH 9.0. Fig. 2(a) shows
the activity-time and Fig. 2(b) the activityconcentration studies. Activity is reasonably linear
for up to 50 min incubation and at least 350 pg
of homogenate protein. Linear kinetics were
obtained for up to 10% incorporation of
putrescine into acceptor protein. Activity was
shown to have an absolute requirement for Ca2+
with optimal activity at 10 mmol/l of incubation
medium. The activity showed saturation kinetics
with respect to putrescine concentration (Fig. 3)
with an apparent K , of 0.12 mmol/l and a V,,,.
of 0.17 nmol min-' mg-' of homogenate protein.
Acceptor substrates
The standard assay mixture contained 2.5 mg
of dimethylcasein/ml of incubation medium.
Coeliac disease and jejunal transglutaminase
100 90 80 706050 C 40 5 30 2 20-
24
.4
slv
6.5
I
I
I
I
I
I
7
7.5
8
8.5
9
9.5
575
1
/
q /
12
PH
FIG. 1. pH optimum for human jejunal transglutaminase. Normal mucosal homogenate (70 pg
of protein) was incubated with piitrescine and
dimethylcasein in standard assay mixture over a
range of Tris buffers at different pH values.
Results show means of duplicate analyses.
’
-100
0
100
200
300
400
S (mmol/l)
FIG. 3. Apparent K, determination for putrescine
for human jejunal transglutaminase. Mucosal
homogenate was incubated with various concentrations of putrescine by the standard assay
procedure. Apparent Km determined by direct
linear plot computation [44] is 0.12 mmol/l.
V , Activity (units)/ml of incubation medium.
Incubation time, 30 min.
75
50
0
10
20
30
.40
50
100
200
300
400
500
- (b)
1.6 1.4
-
1.2 -
1.0 -
..-+-
x
Y
0.8
s
0.6 -
-
OA -
02 0
0
0
Homogenate (pg of protein)
FIG. 2. Activity kinetics of human jejunal transglutaminase. Linear kinetics with respect to ( a )
incubation time (70 pg of normal mucosal
homogenate protein) and ( b ) homogenate protein
concentration. Activities were assayed in duplicate by the standard method.
Fig. 4 shows the activity of transglutaminase with
gliadin as acceptor substrate. Optimal activity was
obtained at a concentration greater than 2.5
mg/ml in the incubation meS.ium, although gliadin
shows limited solubility at this pH.
Table 1 shows the relative acceptor activity of
various proteins. Gliadin shows similar acceptor
activity t o dimethylcasein, the most active substrate used in transglutaminase assays. Deamidation of the gliadin led to a striking decrease in
acceptor activity similar to that found with
collagen. Ovalbumin, elastin and zein show
negligible activity.
Morphometric analyses of jejunal biopsies
Table 2 shows morphometric analysis of
jejunal biopsies from patients and controls.
Patients with coeliac disease in relapse show a
normal villous height with an increase in crypt
depth; patients in remission and patients with
inflammatory bowel disease show normal values.
Patients with untreated coeliac disease show a
striking reduction in villous/crypt height ratios and
S. E. Bruce et al.
516
an increase in intra-epithelial lymphocyte counts.
Patients in remission show a higher villous/crypt
height ratio, which, however, is still significantly
lower than in control subjects. Similarly the intraepithelial lymphocyte counts were higher in the
patients jn remission than in the control subjects
but are within the laboratory normal range.
Patients with inflammatory bowel disease show
normal values for morphometric studies. These
biopsies, however, show increased intra-epithelial
lymphocyte counts compared with controls but
within the kboratory normal range.
h
'5
-
0.08 1
se 0.07
'2
'S w
2
5 0.06
s 5E 0.05
5 2 0.04
z - 0.03
5 5 0.02
b '2
c
7
0.01
v
0
6
0
5
10
15
20
Gliadin (mg/ml of incubation medium)
FIG. 4 Transglutaminase acceptor substrate
activity of gliadin. Mucosal homogenate was
assayed for transglutaminase activity with various
concentrations of gliadin in the incubation
medium. Incubation time, 30 min.
TABLE
1. Dietary proteins as acceptor substrates
for intestinal transglu ta rn inase
Each substrate was incubated in triplicate in
standard assay medium for 30 min and proteinbound radioactivity determined as described in the
Methods section. Results are expressed as m-unit/
mg of homogenate protein where 1 m-unit
corresponds to the incorporation of 1 nmol of
putrescine into acceptor protein.
Substrate
(2.5 mg/ml of medium)
Dim ethylcasein
Gliadin
Deamidated gliadin
Collagen
Ovalbumin
Elastin
Zein
Activity
(m-unit/mg of protein)
0.188
0.170
0.022
0.021
0.001
0.008
0.002
*
Enzymic analyses o f jejunal biopsies
Table 3 shows the transglutaminase activity in
jejunal biopsies. Patients with coeliac disease, both
in relapse and remission, have increased transglutaminase activities. Although patients in
remission show lower activities than patients in
relapse, the differences are not statistically significant. Detailed comparison of enzyme activities
with morphometric measurements showed no
significant relationship. The activity of ZnZ+resistant a-glucosidase, a highly specific brush
border marker enzyme, is also shown in Table 3.
Biopsies from patients with coeliac disease in
relapse show reduced activities. Patients in remission and those with inflammatory bowel
disease show normal values.
Discussion
The data in this paper demonstrate, for the first
time, transglutaminase activity in human jejunal
mucosa. The properties and the activity are
similar to those reported for other tissues, most
notably liver, for which much information is
available. The pH optimum of 9.0 is higher than
that reported for guinea pig liver (8.0-8.5) but the
TABLE2. Morphometric analyses of jejunal biopsies
Results show means SD. Statistical analysis by one-way analysis of variance compares control subjects
with the patients groups: N.S., P > 0.05. Laboratory normal ranges are given in parentheses.
Control subjects
(11 = 14)
Villous height (win)
Crypt depth ( p n )
Villous/crypt height ratio
Intra-epithelial lymphocytes
(cells/100 enterocytes)
485 t 102
(323-553)
116f.17
(68-160)
4.18 t0.79
(2.87-6.1 1)
19.9 t 7 . 8
(<40)
Coeliac disease
Coeliac disease
Inflammatory bowel
in relapse (n = 4) in remission (n = 8)
disease ( n = 8)
404 c68
N.S.
356 r67
(P< 0.001)
1.15 cO.08
(P < 0.001)
69.8 c6.3
(P< 0.001)
478 f. 117
N.S.
151 261
N.S.
3.29 f.0.68
(P< 0.01)
37.4 c 13.1
(P < 0.01)
508 t 79
N.S.
118r16
N.S.
4.41 t0.78
N.S.
28.9 c8.7
(P< 0.05)
577
Coeliac disease and jejunal transglutaminase
TABLE3. Enzyme activities in jejunal biopsies
Activities are expressed as means k sE. Statistical analysis by one-way analysis of variance and by Student's
t-test: N.S., P > 0.05.
Activity (m-units/mg of protein)
Control subjects
(n = 14)
Transglutaminase
Zn2+-resistantaglucosidase
0.191 t0.154
1.59 t0.57
activity shows a similar absolute requirement for
Ca2+, with an optimum at 10-15 mmol/l. The
apparent K , for putrescine is also similar to that
reported for guinea pig liver [l]. Linear kinetics
are obtained with respect to time and tissue
homogenatc concentrations. Comparison of
activities with different substrates indicates that
gliadin is as effective as dimethylcasein, the
standard acceptor substrate. Other proteins were
much less effective. Deamidation of the gliadin
led to a marked reduction in activity, emphasizing the importance of amidated glutamic acid
residue in this reaction.
Compared with activities reported in other
tissues, the mucosal transglutaminase activity is,
in normal intestinal tissue, approximately onethird that in rat liver [31]; activities in coeliac
disease are of the same order. However, more
detailed studies, including molecular weight
determination, electrophoretic studies and
immunochemical investigations are necessary
before intestinal transglutaminase can be considered identical with that in hepatic tissue. The
plasma [9] and epidermal [2, 31 activitics have
been clearly distinguished from hepatic activity.
The importance of these observations to the
pathogenesis of coeliac disease also requires
further investigation. However, the demonstration
of significant transglutaminase activity in human
jejunum and particularly the increased activity in
coeliac disease, both in relapse and remission,
could indicate a role for this enzyme. An important question is the subcellular and cellular
localization of transglutaminase activity in the
jejunal mucosa of both normal and coeliac tissue.
This has not so far been investigated in man but
recent animal studies [33] suggest that mature
enterocytes contain less than 1% of the total
jejunal activity. Mucosa stripped of enterocytes
contains 20% of the activity, but the contribution
of the crypt cells remains to be determined.
Studies in regenerating rat liver have claimed
Coeliac disease
in relapse (n = 4)
0.616 k0.261
(P< 0.06 1)
1.059 tr0.21
(P< 0.01)
Coeliac disease in Inflammatory bowel
remission (n = 8)
disease (n = 8)
0.441 t0.275
(P< 0.01)
1.49 t0.53
N.S.
0.170 t0.053
N.S.
1.29 t0.34
N.S.
increased transglutaminase levels as a physiological
response to cell proliferatiori [121, but a recent
study in partial hepatectomized rats does not confirm this finding [31]. It is, however, possible that
the increased activity in coeliac mucosa reflects
the proliferative response of mucosal cells and it
is of interest that ornithine decarboxylase, the
enzyme responsible for putrescine formation, does
show a marked increase in rapidly proliferating
intestinal epithelial cells [34].
Other possible locations of the transglutaminase
activity include chronic inflammatory cells of the
lamina propria and these may be responsible for
the increased levels in coeliac mucosa. Although
peripheral blood lymphocytes contain at most
trace amounts of transglutaminase activity, after
stimulation there is a rapid increase in their
enzyme activity [35]. Similarly a recent report
notes high levels of transglutaminase activity in
activated macrophages [36]. A submucosal
localization of transglutaminase would not itself
exclude its role in the pathogenesis of coeliac
disease since the coeliac mucosae is abnormally
permeable to macromolecules despite treatment
and histological normality [18, 191. Clearly
further studies with histochemical cytochemical
and immunological techniques are necessary to
determine the cellular location of transglutaminase
activity in the small intestine.
The demonstration of significant activity with
gliadin as substrate could implicate transglutaminase in the cellular binding of gliadin. Selective
binding of gliadin to coeliac mucosa has been
claimed [21, 221 but recently refuted [37]. The
mechanism is uncertain but claims of a significant
carbohydrate content in gliadin [38] have lead to
suggestions it may act as a lectin [22] or a substrate for cell surface glycosyltransferases [39].
Isopeptide bonds are resistant to normal proteolytic enzymes [40-421 and specific isopeptidases
have only recently been identified in mammalian
systems [43]. It would clearly be of interest to
578
S. E. Bruce et al.
identify such peptide bonds in the mucosa after
administration of gliadin in vivo or in vitro.
Acknowledgments
We are grateful to Ms S. E. Ember for secretarial
assistance and The Wellcorne Trust for financial
support (I.B.).
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