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* Institut für Zellbiologie, Universität Bonn, Ulrich-Haberland-Str. 61a, D-53121 Bonn, Germany;
Washington University, School of Medicine, Department of Cell Biology, St. Louis, Missouri, 63110 USA; and
Northwestern University, Medical School, Department of Cell and Molecular Biology, Chicago, Illinois 60611, USA
1Correspondence: Institut für Zellbiologie, Universität Bonn, Ulrich-Haberland-Str. 61a, D-53121 Bonn, Germany. E-mail: herzog{at}uni-bonn.de
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(
-glutamyl)lysine
cross-links and that only traces of these can be found in the soluble
form of TG. Because such isopeptide bridges are generated usually by
the action of a transglutaminase, it is reasonable to propose that the
covalent polymerization of TG in the globules is under the control of
this enzyme. Soluble TG was shown to be a substrate for
transglutaminase in vitro; moreover, the presence of
transglutaminase was demonstrated by immunofluorescence and by
immunoblotting in freshly isolated bovine thyroid globules. With
immunoelectron microscopy, transglutaminase was detected in the
cytoplasm of thyrocytes, but not in compartments of the secretory
pathway. Only one messenger RNA for transglutaminase was found by
Northern blotting. Sequencing of the cloned gene failed to reveal a
secretory signal, which supports the notion that the thyroid
transglutaminase is the cytosolic type. Apparently, the enzyme reaches
the lumen of the follicle by an as yet unknown pathway to catalyze the
covalent cross-linking of thyroid globules in this extracellular
compartment.—Saber-Lichtenberg, Y., Brix, K.,
Schmitz, A., Heuser, J. E., Wilson,
J. H., Lorand, L., and Herzog, V. Covalent cross-linking of
secreted bovine thyroglobulin by transglutaminase |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 2)
.
Thyroglobulin (TG) is the macromolecular precursor of thyroid hormones
and the most abundant protein in the thyroid gland, where it
constitutes up to 75% of the total protein content (3)
.
Synthesis and secretion of thyroid hormones and TG are controlled by
TSH, insulin, and insulin-like growth factor-1. These processes include
iodination of TG and the coupling of iodotyrosine residues by thyroid
peroxidase (1
, 2)
, the extracellular storage and
endocytosis of TG and its proteolytic degradation resulting in the
release of thyroid hormones (4
, 5)
. In TG, only 20–40
residues per molecule become iodinated, but only 4–16 participate in
the coupling reaction (6
, 7)
. As iodine incorporation into
TG and coupling of tyrosines represent the only means for the organism
to store iodine and thyroid hormones, storage of large quantities of TG
in the follicle lumen is necessary for maintaining constant levels of
thyroid hormones (8)
. Storage of TG is made possible by
compactation (i.e., the tight packaging of TG molecules). It is assumed
that this condensation process of TG in the follicle lumen serves to
increase the storage capacity of the thyroid.
TG from the follicle lumen can be collected by micropuncture, and
protein concentrations of 100–400 mg/ml have been reported (3
, 9
, 10)
. Higher lumenal concentrations have been assumed to exist
but have not been determined because of the viscosity of lumenal TG
(3)
. We have shown that TG from human (11)
or
bovine (12)
thyroid glands can be isolated in at least two
distinct forms of aggregation: 1) soluble TG, which occurs
as TG subunits, in monomeric, dimeric, or trimeric forms or as small
oligomers, and 2) solid TG in multimerized state in which TG
forms large colloidal globules. These globules may fill the entire
follicle lumen and can be isolated in an intact state. We have
developed techniques for the isolation of these globules and found
lumenal TG concentrations ranging up to 750 mg/ml. Since then,
colloidal globules have been discovered in other species, including pig
(13)
and rat (unpublished results). The nature of the
protein cross-linking differs widely, however, among various species.
In human insoluble TG, the cross-linking is mainly accomplished by
intermolecular disulfide bonds (11)
. In contrast, in
bovine globules, the intermolecular disulfide bonds represent only
~22% of cross-links, whereas the vast majority (~75%) of the
globular protein was found to be covalently cross-linked by
nondisulfide bonds (12)
. Obviously, distinct
species-specific mechanisms have evolved to achieve multimerization of
TG. The formation of thyroid globules might, therefore, be of
functional relevance in the biology of the thyroid gland.
As yet, the precise nature of intermolecular cross-linking in bovine TG has been unknown. In this paper we present evidence that part of the cross-linking process of bovine TG is mediated by the action of a transglutaminase. Given that covalently cross-linked TG has to undergo partial proteolysis before endocytosis, our findings imply that thyroid hormone liberation from multimerized TG is not restricted to lysosomes, but may also occur in the follicle lumen.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Isolation and purification of bovine thyroid globules
Bovine thyroid glands were freed of fat and connective tissue
and minced with razor blades into 1–2 mm pieces. The tissue fragments
were collected in H2O and homogenized by the use
of a Polytron homogenizer (15 s, position 4.5, Kinematika, Kriens,
Luzern, Switzerland). The homogenate was diluted with
H2O and filtered (150 µm Thermapor nylon gauze,
Reichelt Chemietechnik, Heidelberg, Germany), and the filtrate was
centrifuged (60 s, 100 g). The composition of the pellet was
usually found to be enriched in colorless translucent globules
contaminated with remnants of thyroid tissue. The colloidal globules
were collected by the use of capillaries (tip diameter, 0.5 mm)
connected to the vacuum device of a micromanipulator (ECET 5170,
Eppendorf-Netheler-Hinz, Hamburg, Germany). This technique resulted in
the preparation of small, but highly purified, amounts of
well-preserved thyroid globules.
Isolation of soluble thyroglobulin
For isolation of soluble TG, thyroid tissue was minced as
described above. The tissue fragments were collected in PBS containing
protease inhibitors (1 mM N
-p-tosyl-L-arginine methyl ester, 1
µg/ml antipain, 1 µg/ml pepstatin A, 4 µg/ml aprotinin, and 0.5
mM phenylmethylsulfonylfluoride), and treated in a Polytron homogenizer
(30 s, position 4). Homogenates were filtered through nylon gauze (150
µm), centrifuged (30 min, 22,000 g, 4°C, L7–65
ultracentrifuge, Beckman Instruments, Palo Alto, Calif.), and the
supernatant was subjected to precipitation with ammonium sulfate (35
and 45% saturation). The TG fraction, which precipitated at 45%
saturation, was collected by centrifugation (10 min, 10,000
g, 4°C), resuspended, repeatedly washed with 45%
saturated ammonium sulfate, finally cleared by centrifugation, and
dialyzed against H2O or buffer containing 0.02%
NaN3. Soluble TG was stored at -80°C in
aliquots.
In vitro iodination of soluble TG
Iodination of TG with [125I]NaI was
performed in PBS for 30 min at room temperature using iodobeads
(Pierce, Rockford, Ill.) (14)
. Free
[125I]NaI was removed by
desalting (Econo Pac 10 DG, Bio-Rad, Hercules, Calif.), yielding a
specific radioactivity of 4.7 µCi/mg protein.
[125I]TG was used for in vitro
cross-linking reactions with transglutaminase (see below).
Determination of protein content
Protein concentrations of individual thyroid globules were
determined by estimating the volumes of the globules first in a
hemocytometer (i.e., the flattened area of the globule multiplied by
the 90 µm height of the chamber) (11)
; the globules were
then hydrolyzed in 6N HCl (110°C, 24 h) and
N2 for measuring free amino groups by a
colorimetric assay (16)
. Soluble bovine TG, processed in
parallel, was used as a standard; its protein content was determined by
the Bradford procedure (15)
. Typically, protein
concentrations were measured for 10 globules and averaged. The total
colloid globule protein was related to the globule volume.
The ratio between soluble and insoluble TG was estimated by
homogenizing the bovine tissue and fractionating it by Percoll gradient
centrifugation (12)
. Protein determination was carried out
by the colorimetric ninhydrin method, referenced above
(16)
.
Cell culture
Follicle fragments were prepared from bovine thyroid tissue as
described previously (17)
. The fragments were seeded, and
monolayers of thyrocytes were cultured at 37°C and 5%
CO2 in Eagle’s Minimum Essential Medium
supplemented with 10% FCS, 100 U/ml penicillin, 0.1 mg/ml
streptomycin, and 0.2 µg/ml amphotericine B.
Immunolabelling of thyroid globules
Purified bovine thyroid globules were fixed with 8%
paraformaldehyde in 200 mM Hepes (pH 7.4) for 4 h at room
temperature, washed by repeated centrifugation, and resuspended in 10%
gelatin dissolved in PBS at 40°C. After cooling to 4°C, the gelatin
embedded globules were postfixed in 8% paraformaldehyde/200 mM Hepes
for 8 h at 4°C, infiltrated overnight with 2.3 M sucrose as a
cryoprotectant, and frozen in liquid propane. Cryosections of 500 nm to
1 µm were prepared with a cryotome (Reichert-Jung, Wien, Austria) and
mounted on microscope slides. Cryosections were immunolabelled with
rabbit anti-human erythrocyte transglutaminase (provided by Dr. G.
Aumüller, Marburg, Germany) and TRITC-coupled goat anti-rabbit
antisera (Dianova, Hamburg, Germany). Immunolabelled sections were
viewed with a fluorescence microscope (Axiophot, Zeiss, Oberkochen,
Germany).
Immunolabelling of thyrocytes for electron microscopy
Cells were fixed as described above, infiltrated with sucrose,
and frozen in liquid propane. Cryosections were immunolabelled with
rabbit anti-human erythrocyte transglutaminase and goat anti-rabbit IgG
coupled to Au6 and to Au12
(Dianova). Sections were stained with 0.4% uranyl acetate in 0.2%
methylcellulose (10 min) and examined with an electron microscope
(CM120, Philips, Kassel, Germany). Photographic Scientia EM film was
from Agfa-Gevaert (Leverkusen, Germany).
Sodium dodceyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE) and immunoblotting
Samples of soluble TG or colloidal globules dissolved by
mechanical dissociation in 0.5% sodium dodceyl sulfate (SDS) and 50 mM
dithiothreitol (DTT) were run on 5–18% polyacrylamide gradient gels
according to the method of Laemmli (18)
in a horizontal
gel electrophoretic apparatus (Pharmacia LKB Biotechnology, Uppsala,
Sweden). Staining of gels was performed by the silver technique
(19)
. Rainbow marker kits were used as mass markers
(Amersham Buchler, Braunschweig, Germany). After gel electrophoresis,
proteins were transferred to nitrocellulose (Schleicher and Schuell,
Dassel, Germany) according to the method of Towbin et al.
(20)
. Blot membranes were blocked and probed with rabbit
anti-human erythrocyte transglutaminase and goat anti-rabbit antisera
coupled to horseradish peroxidase (Dianova). Visualization of the
antigens was performed by enhanced chemiluminescence (ECL, Amersham
Buchler).
Isolation and analysis of
N(-glutamyl)lysine cross-links
Purified bovine thyroid globules as well as the soluble TG were
digested with trypsin [50 µg of trypsin (sequencing grade,
Boehringer Mannheim, Mannheim, Germany) per mg TG in 0.1 M Tris-HCl, pH
8.5 for 20 h at 37°C]. Trypsin, without the TG substrates,
served as control. After addition of
NH4HCO3 (to 0.1 M) and a
crystal of thymol, the samples were further processed as described by
Cariello et al. (21)
. Serial digestion by proteases
necessary for the release of
N(-glutamyl)lysine was accomplished by
consecutive treatment (12 h, 34°C) with 2% of subtilisin, Pronase,
and carboxypeptidase Y, and 1% of leucine aminopeptidase and
prolidase. Each treatment was repeated twice before the application of
the next enzyme [1:50 (w/w) of enzyme:substrate] in the series. After
digestion, the samples were dried and taken up in 2 mM HCl. Amino acid
analysis was performed with precolumn derivatization by
o-phtalaldehyde, and N(-glutamyl)lysine
content was measured as described by Cariello et al. (21)
and by Murthy et al. (22)
by high-performance liquid
chromatography (HPLC) with a Waters model 600 E solvent delivery system
and Waters maxima 825 workstation. Verification of the peak emerging at
~30 min as N(-glutamyl)lysine was
obtained by 1) augmentation with inclusion of 22 pmol of the
authentic isopeptide, and also 2) by eliminating it through
treatment of the sample with -glutamine cyclotransferase
(23)
.
Transmission electron microscopy and freeze etching of
thyroid globules, soluble TG, or transglutaminase cross-linked TG
Soluble or in vitro cross-linked TG (see below) was
spread onto carbon- and Formvar-coated grids. After washing the grids
in H2O, negative staining was for 25 s with
1% uranyl acetate. After air-drying, grids were viewed with the
electron microscope (CM120) in the low-dose mode.
For freeze-etch electron microscopy, bovine thyroid globules were
quick-frozen by abrupt application of a pure copper block cooled to
liquid helium temperature, then freeze-fractured and deep-etched in a
Balzers 400 device according to standard procedures (24)
.
After freeze-drying for 15 min at -18°C, the specimens were rotary
replicated with 2 nm of Pt-C by applying to an electron beam gun
mounted 24° above the horizontal and were backed with 6 mm of pure
carbon. Replicas were then separated by flotation on hydrofluoric acid,
washed by flotation on several changes of water, and picked up on 75
mesh Formvar-coated electron microscope grids. The grids were viewed at
100 kV in a JEOL 100 CX electron microscope.
In vitro cross-linking of TG by transglutaminase
Radioiodinated TG was cleared by centrifugation (10 min, 14,000
g). Incubation of 5 mg/ml radioiodinated TG with 0.1 mg/ml
transglutaminase from guinea pig liver (activity of 1 U/mg protein) was
for time periods of up to 60 min at room temperature in 50 mM Tris-Cl
(pH 8.0) supplemented with 10 mM CaCl2. Controls
were incubated without transglutaminase. After the indicated time
periods, aliquots of the incubation mixtures were removed and stopped
by the addition of 100 mM ethylenediaminetetraacetate and boiling in
sample buffer containing SDS and DTT. Analysis was on horizontal
SDS-gels and by autoradiography of the dried gels. To quantify the
amounts of cross-linked TG, films were scanned using a transmitted
light scanner device (Hewlett-Packard, Palo Alto, Calif.), and protein
bands of the stacking gel corresponding to cross-linked TG were
quantified by densitometry using standard computer software.
Sequencing of bovine thyroid transglutaminase
Messenger RNA (mRNA) was isolated from bovine thyrocytes using
an mRNA isolation kit according to the manufacturer’s protocol (Perkin
Elmer, Applied Biosystems, Langen, Germany). mRNA dissolved in elution
buffer was used for oligo dT primed first strand cDNA synthesis by
Superscript II RNase H- reverse transcriptase (Gibco BRL,
Life Technologies, Karlsruhe, Germany). Transglutaminase specific
primers (MWG Biotech, Ebersberg, Germany) with the sequence 5'-CCT TGG
AAT TTT GGG CAG TTT TAA GA-3' for the sense primer and 5'-CCG GAT CCA
GTC CAC CAC GTC A-3' for the antisense primer were used in polymerase
chain reactions (PCR) with Superscript polymerase (Gibco BRL). Usually,
30–35 cycles with 30 s at 95°C, 30 s at 58°C, and 1 min
at 72°C were performed. PCR products were analyzed on 1.0–1.5%
agarose gels and sequenced (Sequiserve, Vaterstetten, Germany).
For rapid amplification of transglutaminase cDNA 5'-ends (5'-RACE)
(25
26
27)
an anchor of the sequence 5'-P-CAC GAA TTC ACT
ATC GAT TCT GGA ACC TTC AGA GG-NH3-3' was ligated
to the 5'-end of the oligo dT-primed first strand by incubation with 10
U of T4-RNA-ligase (Boehringer Mannheim) for 16 h at 22°C. PCR
amplification was with the ligation product, the sense anchor primer
5'-CCT CTG AAG GTT CCA GAA TCG ATA G-3', and the transglutaminase
specific antisense primer 5'-TCT TCA AAC TGC CCA AAA TTC CAA GG-3'
(GSP) by using the Expand Long Template PCR system (Boehringer
Mannheim) and according to the manufacturer’s protocol for system 1. A
total of 30 cycles with 45 s at 94°C, 45 s at 60°C, and 2
min at 72°C were performed. The PCR product was sequenced
(Sequiserve).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(-glutamyl)lysine cross-links are found in TG
globules
. The
average protein concentration in the globules was ~550 mg/ml
(500–625 mg/ml). Percoll gradient centrifugation (12)
yielded an estimate of 10–16% for the insoluble form of TG; however,
because of the difficulty of extracting all insoluble TG from the
tissue, the figure is probably an underestimate. Nevertheless, the data
suggest that the proportion of the insoluble TG in the bovine gland is
higher than in the porcine (13)
and lower than in the
human gland (11)
. Because the main portion of bovine
globules cannot be dissolved by treatment with chaotropic agents (e.g.,
urea) even in the presence of reducing thiols, intermolecular disulfide
bonds do not seem to be involved in the polymerization of bovine TG. No
hydroxylysine or dityrosine bridges could be detected by chromatography
after enzymatic digestion of the globules (not shown). Therefore, we
examined the possibility that the polymerization of TG might be brought
about by the action of a transglutaminase. First, we sought to gather
evidence to demonstrate the presence of
N(-glutamyl)lysine cross-bridges, essential
footprints of transglutaminase activity, in the intact TG globules. As
seen from the data in Fig. 1
, substantial amounts of the isopeptide could be isolated from the total
proteolytic digest of TG globules (marked by arrow in panel
a). This peak, emerging from the HPLC column at ~30 min,
was absent in the mock digest that contained all the proteases but not
the TG (not shown). Its identity as
N(-glutamyl)lysine was confirmed by two
independent means of verification. Addition of the authentic isopeptide
to the sample (panel b) caused a selective increase of the
30 min peak, whereas treatment with the isopeptide hydrolyzing enzyme
-glutamylamine cyclotransferase made the peak disappear (panel
c). From the data in Fig. 1a
, the presence of
~40.7 pmol of cross-links in the 1.98 µg digest of TG globules was
calculated; this, given the 660 x 103
molecular mass of monomeric TG (28)
, corresponds to
generating ~14 mol of N(-glutamyl)lysine
cross-links per mole of TG in forming insoluble globules. By contrast,
only trace amounts of the cross-link (~0.5 mol per mol of TG) were
found in the soluble form of TG (data not shown). Electron microscopic
differences between the two forms of TG are highlighted in Fig. 2
.
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Transglutaminase in TG globules
To test for the presence of transglutaminase in isolated and
highly purified lumenal content, TG globules were cryosectioned
(Fig. 3a
) and transglutaminase was visualized by immunofluorescence
microscopy using an antiserum raised against human erythrocyte
transglutaminase (Fig. 3b
). Immunofluorescence was found
over the whole TG globule with stronger labeling of the globular
surface, which had been exposed to the apical surface of thyrocytes
before isolation of the globules. To ensure the specificity of the
antiserum, TG globules were extracted by boiling in SDS and DTT,
separated by SDS gel electrophoresis, and examined by immunoblot
analysis using purified transglutaminase from guinea pig liver as a
standard (Fig. 3c
). In solubilized TG globules, the
antiserum detected a single band with the same molecular mass as
purified transglutaminase (Fig. 3c
).
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TG is a substrate for transglutaminase-catalyzed cross-linking
in vitro
To demonstrate that TG could serve as a transglutaminase
substrate, soluble TG was incubated in vitro with
transglutaminase and [3H]-putrescine as a
second substrate. The reaction products were analyzed by SDS-PAGE and
autoradiography. The results showed that
[3H]-putrescine was incorporated into TG (data
not shown). Hence, TG has exposed -carboxyamide groups that can
serve as acceptor sites for the transglutaminase-catalyzed
incorporation of putrescine.
Because TG is a substrate for transglutaminase in vitro, the
transglutaminase-catalyzed in vitro formation of TG polymers
was examined. Soluble TG and purified transglutaminase were incubated
for up to 1 h in the presence of calcium, and the reaction
products were prepared for negative staining and electron microscopy.
In control samples lacking transglutaminase, only dimeric or
occasionally tetrameric TG was detected (Fig. 4a
). In contrast, after incubation of TG with
transglutaminase, aggregates containing 20–40 TG molecules were
observed (Fig. 4b
). For biochemical analysis, the same
in vitro cross-linking reaction was performed using
[125I]-TG. The reaction products were analyzed
after the indicated periods of time by reducing SDS gel electrophoresis
and autoradiography (Fig. 4c
). In the presence of
transglutaminase, a time-dependent increase in high molecular weight
[125I]-TG that did not penetrate the stacking
gel was observed (Fig. 4c
, +). In the absence of
transglutaminase, only a small amount of aggregated TG was observed,
the amount of which, however, did not increase during the incubation
period (Fig. 4c
, -). Densitometric analysis of the
autoradiographs revealed a 5.4-fold increase in the polymerized TG
during 1 h of incubation with transglutaminase (Fig. 4d
).
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Thyrocytes do not contain a secretory form of transglutaminase
Although some transglutaminases, such as the one secreted by the
anterior lobe of the prostate in rodents (29
, 30)
,
function extracellularly, the mechanism of transglutaminase release is
still unknown. Because transglutaminase was detected in the
extracellularly located TG globules, we tested whether transglutaminase
was present in organelles of the secretory route of primary bovine
thyrocytes. Immunofluorescence microscopy showed diffuse staining,
indicating that the major part of transglutaminase was localized to the
cytosol (not shown). Because the weak labeling of transglutaminase
within the organelles of the secretory pathway might have been
disguised by the prominent cytosolic signal, the subcellular
localization of transglutaminase was studied by immunoelectron
microscopy (Fig. 5
). Transglutaminase was found almost exclusively in the cytosol. Only
few gold particles were found within or at the cisternae of the
endoplasmic reticulum or the Golgi apparatus.
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To corroborate the nonsecretory nature of thyroid transglutaminase, the
complete cDNA of transglutaminase, including 72 bases of the 5'
untranslated region, was cloned from primary bovine thyrocytes
(Fig. 6
). Sequencing of the PCR product revealed a 98% homology between the
bovine thyrocyte transglutaminase (bTT) (Fig. 6)
and the bovine
endothelial transglutaminase (bET). Most important, the 5' untranslated
regions of the cDNA of the thyrocyte and endothelial cell proteins were
almost identical. No signal peptide that would indicate a secretory
form of transglutaminase was detected. In agreement with this finding,
Northern blot hybridization with a transglutaminase-specific probe
revealed a single mRNA species even after prolonged exposures, also
indicating the presence of only one form of transglutaminase (Fig. 6b
). Thus, as judged by data on its subcellular localization
and on its gene structure, the bovine thyroid transglutaminase does not
seem to be targeted to enter the secretory pathway.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, in which it is
known to comprise the major portion of soluble tissue protein. In
previous studies it was shown, however, that in addition to the soluble
form of TG, an almost insoluble multimerized form can be isolated from
bovine thyroid glands. Bovine multimeric TG occurs in the form of large
globules that resist commonly used protein dissociation procedures
(12)
. In this study, we identify the nature of the
cross-links underlying the multimerization of bovine TG as
N(-glutamyl)lysine isodipeptides. This type
of the covalent cross-linking of bovine TG implies a need of some
extracellular solubilization mechanism (a proteolytic step) that would
have to precede the endocytosis of TG molecules.
Transglutaminase-mediated multimerization of bovine TG
Covalent nondisulfide cross-links are known to occur in a variety
of proteins. In collagen and elastin, intermolecular cross-links are
formed between modified lysine side chains. During this process,
certain lysine and hydroxylysine residues are deaminated by the action
of the extracellularly located lysyl oxidase, resulting in the
formation of highly reactive aldehyde groups that spontaneously form
covalent bonds with each other or with other lysine or hydroxylysine
residues (32)
. Other possibilities of covalent
intermolecular cross-linking might be related to the highly iodinated
state of cross-linked bovine TG. Whereas an average of ~12 iodine
atoms per 12-S TG subunit was measured in soluble TG, the insoluble
form proved to carry ~55 iodine atoms (12)
.
Intermolecular dityrosine bridges have been described in other systems
(e.g., in sea urchin oocytes) where ovoperoxidase is known to harden
the fertilization membrane because of the formation of phenolic
dityrosine cross-links (33
, 34)
. In porcine TG, this
mechanism of multimerization appears to be established
(13)
, whereas in bovine TG, we neither detected
hydroxylysine- nor phenolic dityrosine-mediated cross-linking
(unpublished results).
Our observations show that
N(-glutamyl)lysine-bonds contribute to the
formation of insoluble TG polymers. Quantitatively, the presence of
~14 mol of the isopeptide per mol TG was demonstrated in the globules
whereas only ~0.08 mol of these per mol TG was found in soluble TG.
The presence of N(-glutamyl)lysine in TG
globules was demonstrated by the identification of the isopeptide using
HPLC separation as well as its specific cleavage by -glutamylamine
cyclotransferase. Finding ~175 times as many of these bonds in
insoluble TG than in soluble TG is a clear indication for the
participation of transglutaminase in the process of bovine TG
cross-linking. This was confirmed by in vitro experiments
that showed that TG had the ability to catalyze the formation of
insoluble homopolymeric assemblies.
Transglutaminases have been shown to operate in the cross-linking of
fibrin gamma chains (35
, 36)
, in the biogenesis of sea
urchin egg fertilization membranes (33
, 34
, 37)
, or in the
covalent intermolecular cross-linking of glycoproteins in the plant
cell wall (38)
. In addition, transglutaminases play a
major biological role in the morphogenesis of the cornified envelope
during terminal differentiation of keratinocytes (39)
and
of the vaginal plug of rodents (29)
and other
extracellular matrix constituents (40)
. Hence,
transglutaminases are ubiquitously distributed cytosolic enzymes that
are, however, also found in the extracellular space. No signal sequence
for any of these enzymes has been detected that would direct the enzyme
into the secretory pathway. Possibly, transglutaminase is released from
the cytosol by an apocrine mechanism. This process has been shown to
occur during the hormone-regulated release of transglutaminase in the
rat dorsal prostate and in the coagulating gland (41)
. Our
observations indicate that a signal sequence is also lacking in the
primary translation product of thyroid transglutaminase. Its export
pathway into the follicle lumen is unknown but it may also involve an
apocrine mechanism, given that the apocrine formation of cytoplasmic
blebs on the apical surface of thyrocytes has been described
(42)
.
Possible biological role of TG multimerization
Storage of high concentrations of single TG molecules without
cross-linking would result in increased osmolality. That TG
cross-linking indeed renders the lumenal content of thyroid follicles
osmotically inert has been shown in human thyroid globules in which TG
is cross-linked by intermolecular disulfide bridges and in which an
influx of water can be evoked by the application of reducing conditions
(11)
. In contrast to human globules, bovine thyroid
globules are not dissociable by reducing agents, but it is conceivable
that the condensation process of bovine TG also serves to increase the
storage capacity in the follicle lumen. Thyroid globules represent
probably a storage form of the thyroid hormones or iodine, or both. The
actual hormone content of bovine globules is yet to be determined, but
it is known that insoluble TG contains a 4- to 5-fold higher amount
(~55 iodine atoms per 12-S TG) of iodine than the soluble TG (~12
iodine atoms per 12-S TG) (12)
. It has been suggested that
at first newly exported TG molecules are internalized and hydrolyzed,
whereas previously secreted TG is stored in the follicle lumen and
mobilized only when the organism requires large quantities of thyroid
hormones. This selectivity has been summarized in the ‘last come,
first served-concept’ (43)
. The extracellular
compactation and storage of TG described here is in support of this
hypothesis and might be the structural basis for the sorting of newly
exported soluble TG from the covalently cross-linked storage form of
TG.
Proteolysis and release of thyroid hormones have long been considered
to be restricted to lysosomes (3)
and to be separated in
space and time, therefore, from the extracellular storage of TG in the
follicle lumen. However, the covalent intermolecular cross-linking of
TG requires mechanisms of globule solubilization to facilitate
endocytosis. Hence, it has been postulated that extracellular
proteolysis of TG might occur (44)
. Recent work from our
laboratory has provided evidence that limited extracellular proteolysis
of TG indeed occurs (5)
and that a variety of plasma
membrane-associated cathepsins might be involved in this process
(5
, 45)
.
In summary, our results show that the multimerization of bovine TG is at least in part a transglutaminase-mediated process enabling storage of TG in an osmotically inert form. Covalent cross-linking of TG might explain the sorting of a ready-to-use soluble form from a storage form of TG. The mode of transglutaminase release from thyrocytes, however, is still unknown.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-(L--glutamyl)–L-lysine and related compounds. Proc. Natl. Acad. Sci. USA 77,4564-4568-glutamyl--lysine dipeptide from clotted secretion protein of guinea pig seminal vesicle. Proc. Natl. Acad. Sci. USA 69,2322-2225This article has been cited by other articles:
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