The Intermediate Filament Protein, Vimentin, in the Lens Is a Target for Cross-linking by Transglutaminase*

Sophie ClémentDagger , Pauline T. VelascoDagger , S. N. Prasanna MurthyDagger , James H. WilsonDagger dagger , Thomas J. Lukas§, Robert D. GoldmanDagger , and Laszlo LorandDagger

From the Departments of Dagger  Cell and Molecular Biology and § Molecular Pharmacology and Biological Chemistry, Northwestern University Medical School, Chicago, Illinois 60611

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results & Discussion
References

Mere addition of Ca2+ to a lens cortical homogenate (bovine) generates a series of products composed of a variety of high molecular weight vimentin species. The Ca2+-induced cross-linking of this cytoskeletal element seems to be mediated by the intrinsic transglutaminase of lens, because the reaction could be blocked at the monomeric state of vimentin by the inclusion of small synthetic substrates of the enzyme dansylcadaverine or dansyl-epsilon -aminocaproyl-Gln-Gln-Ile-Val. These compounds are known to compete against the Gln or Lys functionalities of proteins that would participate in forming the Nepsilon (gamma -glutamyl)lysine protein-to-protein cross-links. The cytosolic transglutaminase-catalyzed reactions could be reproduced with purified bovine lens vimentin and also with recombinant human vimentin preparations. Employing the latter system, we have titrated the transglutaminase-reactive sites of vimentin and, by sequencing the dansyl-tracer-labeled segments of the protein, we have shown that residues Gln453 and Gln460 served as acceptor functionalities and Lys97, Lys104, Lys294, and Lys439 as electron donor functionalities in vimentin. The transglutaminase-dependent reaction of this intermediate filament protein might influence the shape and plasticity of the fiber cells, and the enzyme-catalyzed cross-linking of vimentin, in conjunction with other lens constituents, may contribute to the process of cataract formation.

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results & Discussion
References

Significant amounts of the branched Nepsilon (gamma -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-epsilon -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.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results & Discussion
References

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-epsilon -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-epsilon -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-epsilon -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-epsilon -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-epsilon -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-epsilon -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.

    RESULTS AND DISCUSSION
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Abstract
Introduction
Materials & Methods
Results & Discussion
References

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-epsilon -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-epsilon -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-epsilon -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.

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-epsilon -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-epsilon -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-epsilon -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-epsilon -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-epsilon -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-epsilon -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."

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-epsilon -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-epsilon -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.

The TGase-mediated titration of Lys side chains with dansyl-epsilon -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-varepsilon -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-epsilon -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.

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 Glnright-arrowGlu transition; the incorporation of low molecular weight primary amines; cross-linking of vimentin to vimentin or to other proteins by formation of Nepsilon (gamma -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 alpha 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 alpha 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 alpha 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 alpha  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 beta -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 Gright-arrowV 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 left-right-arrows  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 right-arrow 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 right-arrow 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) left-right-arrows  (fibrin)n, is followed by the enzyme-catalyzed cross-linking of the fibrin network: (fibrin)n right-arrow 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 Nepsilon (gamma -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.

    ACKNOWLEDGEMENT

We thank Dr. Ying-Hao Chou for helpful discussion.

    FOOTNOTES

* This research was supported by Grants EY-03942 and NIGMS-36806 and 30861 from the National Institutes of Health. Some aspects of these investigations were presented at the meeting of the Association for Research in Vision and Ophthalmology (Lorand, L., Velasco, P. T., Murthy, S. N. P., Clement, S., Quinlan, R., and Goldman, R. D. (1996) Invest. Ophthalmol. Visual Sci. 37, S600, Abstr. 2767-B612).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

dagger This work is dedicated to the memory of our colleague, James H. Wilson, who died on June 20, 1997.

To whom correspondence should be addressed: Dept. of Cell and Molecular Biology, Northwestern University Medical School, 303 East Chicago Ave., Chicago, IL 60611-3008. Tel.: 312-503-0591; Fax: 312-503-0590

1 The abbreviations used are: TGase, transglutaminase; dansylcadaverine, N-(5-aminopentyl)-5-dimethylaminonaphthalene-1-sulfonamide; dansyl, 5-dimethylaminonaphthalene-1-sulfonyl; dansyl-epsilon -aca-QQIV, dansyl-epsilon -aminocaproyl-Gln-Gln-Ile-Val; HPLC, high performance liquid chromatography; PAGE, polyacrylamide gel electrophoresis; IF, intermediate filament.

2 S. N. P. Murthy and L. Lorand, unpublished data.

3 S. N. P. Murthy, P. T. Velasco, and L. Lorand, unpublished data.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results & Discussion
References

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