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INTRODUCTION |
Significant amounts of the branched
N
(
-glutamyl)lysine isopeptide,
usually a manifestation of the posttranslational
modifications of proteins by
TGase,1 were found in the proteolytic digests of high
molecular weight polymers present in human cataractous lens specimens.
Complementary experiments, in which the intrinsic
Ca2+-activated TGase was allowed to promote the
cross-linking of endogenous proteins, revealed that among the soluble
constituents of the lens, primarily a small subset of crystallins was
targeted by the enzyme (1). Enzyme-specific probes, such as
dansylcadaverine, dansyl-
-aca-QQIV, or similar biotinylated
analogues were synthesized for the identification and analysis of the
substrates of the enzyme. These compounds compete against the
TGase-catalyzed cross-linking of proteins, and by becoming incorporated
in the enzyme-driven reaction into the biological substrates
themselves, they could be employed for locating the Gln (or acceptor)
and the Lys (or donor) functionalities exhibiting the unique
specificities (1-8).
Based on experiments with human red blood cells (9, 10) and with
keratinocytes (11, 12), it seemed reasonable to assume that in addition
to the specific subsets of crystallins, some membrane and cytoskeletal
elements in the lens might also become cross-linked by TGase.
Therefore, we undertook to examine changes occurring in the skeletal
proteins of the lens as the latent TGase becomes activated by
Ca2+ in the tissue. This line of research was greatly
spurred by our recent finding that a TGase antigen became tightly bound
to the IF network in some fibroblasts and keratinocytes (13, 14). The
present report focuses on the intermediate filament protein vimentin as
substrate for the cross-linking enzyme. Notwithstanding the mesenchymal
preference for the expression of the vimentin-encoding gene and the
epithelial origin of the ocular lens (15, 16), vimentin is known to be
actively synthesized in the lens epithelium and in the cortical fiber
cells (17). We have now identified the TGase-reactive Gln and Lys side
chains of vimentin, which can act as acceptor and donor functionalities
of the protein in cross-linking onto itself or to other membrane and
cytoskeletal elements.
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MATERIALS AND METHODS |
Experiments with Lens Homogenates--
Frozen calf lenses
(Pel-Freez, Rogers, AR) were thawed to room temperature and
decapsulated, and the cortex was separated from the nucleus. Cortical
portions from two lenses were homogenized by hand in 3 ml of 50 mM Tris-HCl, pH 7.5, and 100 mM NaCl in a
Potter-Elvehjem tissue grinder. Incubations were carried out for 90 min
and for 21 h at 37 °C in reaction volumes of 200 µl containing about 80 mg of lens protein/ml, 20% glycerol, 50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 2 mM leupeptin (obtained through the United States-Japan
Cooperative Cancer Research Program) and 1-8 mM
CaCl2 or 2 mM EDTA. To some mixtures, 2 mM dansylcadaverine (18, 19) or dansyl-
-aca-QQIV (6, 7)
was also added. Following incubation, the samples were centrifuged
(Eppendorf microcentrifuge, Brinkmann, Westbury, NY; 16,000 × g for 5 min.). Both supernatants and pellets were analyzed
by SDS-PAGE. The supernatants were prepared for electrophoresis by
mixing 5-µl aliquots with 45 µl of solubilization buffer (50 mM Tris-HCl, pH 6.8, 9 M urea, 40 mM dithiothreitol, 2% SDS) at 37 °C for 30 min. The
pellets were washed twice with 1 ml of 50 mM Tris-HCl, pH
7.5, 100 mM NaCl and then solubilized in 65 µl of
solubilization buffer at 37 °C for 30 min and clarified by
centrifugation at 16,000 × g for 5 min.
Electrophoretic Analysis and Western Blotting--
SDS-PAGE was
carried out in 10% acrylamide gel (20), and the proteins (35 µg)
were electroblotted to nitrocellulose (21) and immunostained with a
rabbit antiserum to baby hamster kidney cell vimentin (22) (1:10,000
dilution in 2% milk/phosphate-buffered saline solution) or with
monoclonal IgG against dansyl (23) (1:300,000 dilution in 2%
milk/phosphate-buffered saline solution) overnight at room temperature.
After three washes with 2% milk/phosphate-buffered saline solution,
the blots were treated with alkaline phosphatase-conjugated secondary
antibody (diluted 1:5000 in 2% milk/phosphate-buffered saline
solution) against either rabbit IgG (Promega Corporation, Madison, WI)
or mouse IgG (whole molecule; Sigma) for 2 h at room temperature.
Alkaline phosphatase activity was developed with 5-bromo-4-chloro-indolyl phosphate (Sigma, 300 µg/ml) as substrate and nitro blue tetrazolium (Sigma, 150 µg/ml) as electron acceptor in
100 mM Tris-HCl, pH 9.5, 100 mM NaCl, 5 mM MgCl2.
TGase-mediated Cross-linking of Purified Lens Vimentin and
Inhibition by Dansyl-
-aca-QQIV and Dansylcadaverine--
The
reaction of purified bovine lens vimentin (24, 25) and purified human
recombinant vimentin expressed in Escherichia coli (26) with
a cytosolic TGase (27, 28) was examined in 40-µl mixtures comprising
5 mM Tris-HCl, pH 7.5, 10 mM NaCl, 10 mM dithiothreitol, 10 µg of vimentin, 10 µg/ml TGase,
and 5 mM of either CaCl2 or EDTA. Some mixtures
contained the TGase-specific probes of dansylcadaverine (18, 19) or
dansyl-
-aca-QQIV (6, 7). After 2 h at 37 °C, the reactions
were stopped by addition of 3 µl of 100 mM EDTA.
Electrophoresis was carried out as described above.
Identification of the TGase-reactive Sites in
Vimentin--
Recombinant human vimentin (2 mg) expressed in E. coli (26) was allowed to react with the cytosolic TGase (80 µg/ml) in a 1.2-ml solution of 5 mM Tris-HCl, pH 7.5, 10 mM NaCl, 10 mM dithiothreitol for 2 h at
37 °C with either 2 mM dansylcadaverine or
dansyl-
-aca-QQIV in the presence of 5 mM
CaCl2. At the end of the incubation period, the proteins
were precipitated with 7% trichloroacetic acid and sedimented by
centrifugation (5 min at 14,000 × g), and the pellets
were extracted repeatedly (8 times) either with 1 ml of ethanol:ether
(1:1, v/v) to remove the unreacted dansylcadaverine or with 1 ml of
N,N-dimethylformamide containing 1%
N-methylmorpholine and 5% H2O for removal of
the unreacted dansyl-
-aca-QQIV probe.
The washed pellets were resuspended in 1 ml of 5 mM
Tris-HCl, pH 7.5, 10 mM NaCl and digested with 200 µg of
trypsin treated with
L-(tosylamido-2-phenyl)ethylchloromethylketone (Worthington Biochemicals, Freehold, NY) at 37 °C for 48 h. Digestion was
terminated by addition of 400 µg of phenylmethylsulfonyl
fluoride.
Dansyl-labeled peptides were isolated on a monoclonal anti-dansyl
Affi-Gel 10 column (6, 19) and concentrated by lyophilization. Separation was carried out by HPLC on a Ultrasphere C8 column (Beckman
Instruments) with a gradient of acetonitrile (5-40% for separating
the dansylcadaverine-labeled peptides or 10-60% for the
dansyl-
-aca-QQIV-labeled peptides) in H2O, 0.1%
trifluoroacetic acid over 90 min at a flow rate of 1 ml/min. Effluents
were monitored by absorbance (220 nm) and by fluorescence (excitation,
338 nm; emission, 500 nm). Amino acid sequencing was carried out in an Applied Biosystems Procise sequencer.
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RESULTS AND DISCUSSION |
Experiments with Lens Homogenates--
Examination of proteins by
SDS-PAGE showed that in the control lens specimen, vimentin partitioned
mostly into the insoluble portion of the homogenate (compare bands on
the immunoblots of the 45-60-kDa region (Fig.
1A, lane 4), representing the
supernatant, with those representing the sediment fraction (Fig.
1B, lane 4)), whereas the crystallins (21-32 kDa) (Fig. 1,
A and B, lane 1, stained with Amido Black) were
present mostly in the soluble phase. Except for a faint band, seemingly
corresponding to a trimeric or tetrameric species of vimentin, in Fig.
1B, lane 4, no high molecular weight forms of the
intermediate filament protein were seen in this bovine lens
preparation. However, the mere addition of Ca2+ to the
homogenate generated significant amounts of a series of vimentins with
Mr > 60,000 (Fig. 1, A and
B, lanes 5 and 6, X). The production
of these higher vimentin species seemed to be somewhat enhanced if
leupeptin, a calpain inhibitor that is known to suppress the
proteolytic degradation of vimentin in rabbit lens (29), was included
in the incubation mixture (Fig. 1, A and B,
lane 6). The higher molecular weight vimentin product
separated mainly with the insoluble phase of the homogenate (Fig.
1B, lanes 5 and 6), whereas some of the lower
multiples of vimentin (Mr
200,000) appeared
also in the supernatant (Fig. 1A, lanes 5 and 6).
The endogenous cross-linking of vimentin could also be documented at
lower concentrations of Ca2+ (Fig. 1C) by
incubating the lens homogenate over a longer time period (21 h). These
SDS-PAGE experiments, carried out after reducing the proteins with
dithiothreitol in urea, demonstrated that the cross-linked vimentin
species were produced in the lens tissue by covalent bonds other than
disulfides. The additional findings presented in Fig.
2 provided clear proof for the
involvement of the Ca2+-activated, endogenous TGase in the
observed reaction. As a primary amine, dansylcadaverine is known to
compete against the Lys residues of proteins in the TGase-mediated
cross-linking reaction (18, 19), whereas dansyl-
-aca-QQIV competes
against the enzyme-reactive Gln residues in cross-linking (6, 7). The
presence of either of these compounds (2 mM) during
treatment of the homogenate with Ca2+ reduced the amount of
cross-linked vimentin products, as is best seen in Fig. 2A, lanes
5 and 6. Probing the transblots with a monoclonal
antibody against the dansyl moiety (Fig. 2, lanes 7 and
8) visualized monomeric vimentin at about 55 kDa, as well as
the other TGase-reactive proteins in the lens homogenate.

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Fig. 1.
Ca2+-dependent
cross-linking of vimentin in the lens homogenate. Calf lens
homogenate was incubated for 90 min at 37 °C in the presence of 2 mM leupeptin (lanes 3 and 6) with 2 mM EDTA (lanes 1 and 4) or 8 mM CaCl2 (lanes 2, 3, 5, and
6). The supernatants (A) and solubilized pellets
(B), separated by centrifugation, were analyzed by SDS-PAGE
and then electroblotted to nitrocellulose. Blots were either stained
for protein with Amido Black (lanes 1-3) or developed with
a specific antiserum to vimentin (lanes 4-6). For
experimental details, see "Materials and Methods." The position of
the parent vimentin at 55 kDa (V) and the cross-linked products of vimentin (X) are marked. C
illustrates the production of cross-linked vimentin (partitioned into
the pellet as in B) upon the incubation of lens homogenate
at Ca2+ concentrations of 1 (lanes 1 and
6), 2 (lanes 2 and 7), 4 (lanes 3 and 8) and 8 (lanes 4 and 9)
mM for 21 h at 37 °C. Lanes 5 and 10 are the controls with EDTA (2 mM).
Lanes 1-5 show the Amido Black-stained blots, whereas
lanes 6-10 are immunoblots developed with the antibody to
vimentin.
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Fig. 2.
The inclusion of synthetic substrates for
TGase, i.e. dansylcadaverine or
dansyl- -aminocaproyl-QQIV, in the reaction of the calf lens
homogenate with Ca2+ inhibits the formation of the high
molecular weight vimentin species. Experiments were carried out as
in Fig. 1 in the presence of 2 mM leupeptin, 8 mM CaCl2 with either 2 mM
dansylcadaverine (lanes 2, 5, and 7) or
dansyl- -aminocaproyl-QQIV (lanes 3, 6, and 8)
added. After centrifugation, the supernatants (A) and
pellets (B) were subjected to SDS-PAGE, followed by transfer
to nitrocellulose. Blots were stained for protein with Amido Black
(lanes 1-3) and were immunostained with antibody to
vimentin (lanes 4-6) or to dansyl (lanes 7 and
8). For experimental details, see "Materials and
Methods." The position of vimentin at 55 kDa (V) is
indicated.
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Reactions of the Purified Bovine Lens Vimentin and Recombinant
Human Vimentin with Cytosolic TGase--
Two well-defined preparations
of vimentin were employed to demonstrate that the intermediate filament
component was capable of acting as a bifunctional substrate for TGase,
that its cross-linking could be blocked essentially at the monomeric
stage with the inclusion of either synthetic substrate of TGase
(dansylcadaverine or dansyl-
-aca-QQIV) in the reaction mixture, and
that the dansyl-containing tracers could be employed for labeling
predominantly the monomeric parent vimentin. As illustrated by the
experiments in Fig. 3, all criteria for
TGase catalysis were realized with the purified bovine lens vimentin
(A), as well as with the recombinant human vimentin as substrates (B). Under the experimental conditions employed
(TGase:vimentin ratio of 1:25; pH 7.5; 37 °C), in the absence of
dansylcadaverine, only a trace amount of monomeric vimentin remained at
2 h (Fig. 3, A and B, sets a, lanes 2), and
the large cross-linked products of the enzymatic reaction could barely
enter into the stacking gel during SDS-PAGE. However, with the
inclusion of 2 mM dansylcadaverine (sets b and
c) or dansyl-
-aca-QQIV (sets d and
e) in the incubation mixture, cross-linking of the protein
could be blocked essentially at the monomeric stage. Lower
concentrations of the inhibitors (0.5 and 1 mM) still
allowed for the formation of some vimentin products of intermediate
size. Photographs of the gels taken under UV illumination showed
(sets c for dansylcadaverine and sets e for
dansyl-
-aca-QQIV) that with 2 mM of either blocking
agent in the mixture, the fluorescent label was found almost
exclusively in monomeric vimentin. The incorporation of the
dansyl-
-aca-QQIV tracer, apparently by labeling more than a single
donor site in the parent vimentin molecule, caused a noticeable upward
shift from the original molecular mass value of 55 kDa of the protein in SDS-PAGE (sets d and e).

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Fig. 3.
TGase-catalyzed incorporation of
dansylcadaverine and dansyl- -aminocaproyl-QQIV into vimentin.
Reactions were carried out for 2 h at 37 °C in mixtures
containing either purified bovine lens vimentin (0.25 mg/ml;
A) or recombinant human vimentin (0.25 mg/ml; B),
guinea pig liver TGase (10 µg/ml), 5 mM EDTA (set
a, lane 1) or 5 mM CaCl2 (for all other
lanes). Dansylcadaverine (sets b and c) or
dansyl- -aminocaproyl-QQIV (sets d and e) was included at various concentrations (0.5-2 mM) as shown.
Set a, which did not contain either of these compounds,
served as control. The samples were subjected to SDS-PAGE and
photographed under UV light (sets c and e) prior
to staining the corresponding sets (a, b, and d)
with Coomassie Blue. For experimental details, see "Materials and
Methods."
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Identification of the TGase-reactive Gln and Lys Residues of
Vimentin--
The experiments described in Fig. 3 were used as a guide
for identifying the TGase-reactive acceptor and donor functionalities of vimentin. Details are given under "Materials and Methods," and
Fig. 4 is a summary of the protocol
employed for analyzing the reaction of recombinant vimentin with
cytosolic TGase. A key feature of the procedure was the use of an
anti-dansyl affinity column (6, 19) for separating the fluorescent
peptides that carried the dansyl hapten from all the other fragments in
the tryptic digest of the enzyme-modified vimentin samples. The HPLC profile of the dansylcadaverine-labeled peptides is shown in Fig. 5A, and that of the fragments
obtained from the reaction with dansyl-
-aca-QQIV is presented in
Fig. 5B. Edman degradation (12 cycles) of the two major
fluorescent peaks marked I and II in Fig. 5A, eluting at
approximately 58 and 67 min, respectively, showed that these peptides
were derived from the same segment of the C-terminal tail of the
vimentin molecule, corresponding to the DGXVINETSXHH sequence from
residue 451 through 462 (30). In the sequencing of the peak II
material, no known amino acid (X) was recovered in the 3rd and 10th
Edman cycles where Gln453 and Gln460 would be
expected, and this was taken to indicate that both Gln residues carried
the dansylcadaverine adduct. By contrast, sequencing of the peak I
material comprising the same 451-462 segment of vimentin yielded Gln
in the 3rd and the 10th cycles, although only in amounts of about half
of what would have been expected without derivatization by
dansylcadaverine. The data are consistent with the idea that the peak I
material, actually appearing almost as a double peak on HPLC,
represented a mixture of two peptides of the same sequence, one in
which Gln453 and one in which Gln460 was
decorated with the dansylcadaverine label DGQVINETSXHH and DGXVINETSQHH. Altogether, it was quite remarkable to find that TGase
reacted with high selectivity with only 2 of the 33 Gln residues
present in vimentin. It was equally interesting that the two
TGase-titratable acceptor side chains of this intermediate filament
protein (Gln453 and Gln460) were located close
to each other in the primary sequence in the C-terminal unfolded domain
of the molecule.

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Fig. 4.
Outline of the analytical scheme for
identifying the Gln and Lys residues of human vimentin, which serve as
specific targets for TGase.
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Fig. 5.
Identification of transglutaminase-reactive
residues in vimentin. A, isolation of
dansylcadaverine-labeled peptides from the trypsin digest of
TGase-modified vimentin. The fluorescent peaks marked I and II,
emerging from the C8 HPLC column (see "Materials and Methods") at
58 and 67 min, respectively, were collected, concentrated, and
subjected to amino acid sequencing. B, isolation of
dansyl- -aminocaproyl-QQIV-labeled peptides from the trypsin digest
of TGase-derivatized vimentin. The fluorescent peaks marked I-V were
collected, concentrated, rechromatographed, and submitted for amino
acid sequencing.
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The TGase-mediated titration of Lys side chains with
dansyl-
-aminocaproyl-QQIV provided a more complex picture. Five
fluorescent peaks (marked I-V in Fig. 5B) were collected
following the HPLC separation of the tryptic digest of labeled
vimentin. Each of these was rechromatographed and submitted for
sequencing. High confidence sequences were obtained for the peak II,
III, and IV materials, identifying Lys97 and
Lys104 unambiguously as residues reacting with TGase (Table
I). The peak I material appeared to be a
mixture of several labeled fragments, sequences for which could not be
resolved by Edman degradation. Peak V turned out to be a mixture of two
labeled peptides, present in essentially equal amounts; reliable
reading of the double sequence, however, could be accomplished by
knowing the amino acid sequence of vimentin itself. The two peptides
were derived from protein segments starting with Ser293 and
Glu425, respectively, providing evidence that residues
Lys294 and Lys439 could also act as
TGase-reactive donor side chains. As illustrated in Fig.
6, in the vimentin molecule
Lys294 is located near the interface of the helical 2B
subdomain (121 residues) with the L2 linker segment (8 residues),
whereas Lys439, like the two TGase-reactive acceptor side
chains Gln453 and Gln460, is in the 55 residue-length non-helical C-terminal tail. On the other hand, the
donor residues Lys97 and Lys104, which were
identified with high confidence from sequencing the peak II, III, and
IV peptides (Fig. 5B and Table I), straddle the zone of
transition from the non-helical N-terminal H1 segment (18 residues) to
the helical rod of vimentin, designated as 1A (35 residues).
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Table I
Sequencing the TGase-reactive, dansyl- -aminocaproyl-QQIV-labeled Lys
residues of human vimentin
The superscript denotes residue numbers in the primary sequence of the
protein. Boldface X indicates an Edman cycle without
recovery of a known amino acid and assigned to be an enzyme-derivatized Lys.
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Fig. 6.
Location of TGase-reactive Gln and Lys
residues of vimentin in relation to the architecture of this
intermediate filament protein. A, the amino acid sequence
for human vimentin (27) with the bold letters highlighting the Lys
(97, 104, 294, and 439) and Gln (435 and 460) residues of the protein, which we have shown to
incorporate dansyl- -aca-QQIV or dansylcadaverine in the
TGase-directed reaction. Residues identified in the sequence with
highest confidence (i.e., Lys-97, Lys-104, Gln-435, and
Gln-460) are marked with asterisks. B, organization of
subdomains in vimentin according to Steinert and Parry (53). The number
of amino acid residues assigned to the various segments are indicated
in italics, and the TGase-reactive Gln and Lys residues are
marked by arrows.
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The Gln and Lys residues highlighted in Fig. 6 are potential sites for
various posttranslational possibilities by TGase. These include the
hydrolysis of Gln residues, i.e. a Gln
Glu transition; the
incorporation of low molecular weight primary amines; cross-linking of
vimentin to vimentin or to other proteins by formation of
N
(
-glutamyl)lysine isopeptides or
by a polyamine bridge (spermine, spermidine, or putrescine) between two
reactive Gln residues. It is clear from our data that vimentin could
act as a bifunctional substrate cross-linking onto itself, carrying
TGase-reactive acceptor (Gln) as well as donor (Lys) residues. This may
be contrasted with the substrate qualities of involucrin in human
keratinocytes, the anion transporter band 3 in human red cells or
B
crystallin in bovine lens, each of which is known to act only as a
monofunctional substrate for the enzyme. Involucrin and the cytoplasmic
domain of band 3 contain just a single enzyme-reactive Gln in their
sequences: Gln496, located in the C-terminal region of
involucrin (31), and Gln30, in the N-terminal portion of
band 3 (19); bovine
B crystallin was reported (5, 6) to carry only
an enzyme-reactive Lys, Lys175, at the C-terminal end of
the protein (although there is evidence that in the lens tissue, under
the influence of the intrinsic TGase, the penultimate
Lys174 of
B crystallin also
reacts2).
It is interesting to note that the TGase-reactive sites of the vimentin
molecule (Fig. 6) are present in regions of the protein that are also
known to be susceptible to other posttranslational modifications, such
as phosphorylation and proteolysis (32-37). In general, in terms of
the architecture of intermediate filament proteins, TGase seems to act
with high selectivity at functionally important areas of vimentin. The
tail domain (comprising the TGase-reactive Gln453,
Gln460, and Lys439 residues; see Fig. 6), by
interacting with the
helical rod, is known to stabilize the correct
filamentous assembly of vimentin in vitro as well as
in vivo (38), and was also shown to bind with actin
structures (39). Tail-less vimentin mutants display an unusual tendency
for abnormal type of aggregation (38, 40, 41). A nine-amino acid
sequence
-motif, present in the tail domains of all type III IF
proteins, including vimentin, has been shown to be essential for normal
IF assembly. Remarkably, a single G
V mutation at position 452 (adjacent to the TGase-reactive Gln453 residue) is known to
interfere with normal assembly (42). The area comprising two of the
TGase-reactive Lys donor sites (Lys97 and
Lys104), located at the beginning of the rod domain (Fig.
6), is highly conserved across several types of intermediate filament
proteins. This structural domain is considered to be critical for the
assembly of the keratin units (43); a single point mutation in this
segment can disrupt filament formation in vitro as well as
in vivo (44-50). The Lys residues of keratins involved in
cross-linking to two of the cell envelope proteins, loricrin and
involucrin, are all located in the head domains (cross-linking to
loricrin utilizes Lys73 in keratin 1, Lys70 in
keratin 2e, and Lys9 in keratin 10 (51); cross-linking to
involucrin occurs at Lys73 with keratin 1, Lys69 with keratin 2e, and Lys71 with keratin 5 (52)). It is interesting that this N-terminal domain is missing in the
type III IF vimentin molecule (53).
Intermediate filaments of 10 nm in diameter were long thought to be
static cytoskeletal elements of cells. Recently, however, it has been
shown that their polymerization state in vivo is governed by
an equilibrium between soluble vimentins, V, and the
polymerized form of this protein: nV
Vn (54). TGase activity could conceivably influence the normal distribution and partitioning of vimentin in the
cell. For example, if the enzyme modified mainly the soluble vimentin
species, mV
mV' (e.g. by
deamidation or cross-linking, where m < n),
there might be a disruption of the IF network by depletion of the
soluble vimentin pool available for normal polymerization. If, on the
other hand, TGase acted by preferentially cross-linking (xl)
the assembled subunits of vimentin in situ
((V)n
xl(V)n), the dynamic nature of the
network could become compromised because the covalent fusion of
subunits would preclude the dissociation of the IF ensemble. Either of
these possibilities could have major consequences for cell function and
survival. The change in filament stability may be important in
regulating changes in cell shape that occur during development. In
support of this idea, it has been shown that intermediate filaments
play major roles in cell shape and the mechanical properties of
cytoplasm (55). Close analogy for some of the changes that might occur
in the IF network is the stabilization of fibrin clots by activated
Factor XIII, a member of the TGase family of enzymes. Dissection of the
biochemical steps in the last stages of blood coagulation (56) showed
that the reversible self-assembly of fibrin molecules into the clot, n(fibrin)
(fibrin)n, is followed by the
enzyme-catalyzed cross-linking of the fibrin network:
(fibrin)n
xl(fibrin)n. The
ordered assembly of molecules into the (fibrin)n structure
was shown to accelerate greatly the rate of the enzymatic reaction (57,
58), and the introduction of a few
N
(
-glutamyl)lysine covalent side
chain bridges at strategic locations into the clot results in an
approximately 5-fold increase in clot stiffness (59, 60).
It remains to be seen when and how TGase becomes activated in the lens.
We have recently shown3 that
the cross-linking activity of the purified lens enzyme is inhibited by
GTP. In fact, the sensitivity of the lens TGase to GTP (which probably
reflects on the affinity of the enzyme for binding the nucleotide) is
about an order of magnitude greater than that of the guinea pig liver
enzyme. Thus, like the liver TGase, which was shown to function as a G
protein in signal transduction (61-65), the lens enzyme seems to
represent a gene product potentially with a dual role in the lens cell.
Under the appropriate signal, it may mediate the activation of
phospholipase C. Otherwise, it could function in remodeling reactions,
as described in this report for the modification of vimentin. The
concentration of GTP would affect the cross-linking potential of the
lens enzyme, and a significant drop in the concentration of the
nucleoside triphosphate, as it may occur in cataract formation, would
greatly favor the expression of this activity.
We thank Dr. Ying-Hao Chou for helpful
discussion.