Loading...
Please wait, while we are loading the content...
Similar Documents
Lens transglutaminase and cataract formation [ e-( y-glutamyl ) lysine crosslinks / aging lens / p-crystallins ]
| Content Provider | Semantic Scholar |
|---|---|
| Author | Lorand, Laszlo Siefring, Gerald |
| Abstract | A protein polymer characteristically present in human cataract was shown to contain significant amounts of Yglutamyl-e-lysine isopeptides. It is proposed that these crosslinks are produced by the action of transglutaminase (R-glutaminylpeptide:amine-glutamyl-yltransferase, EC 2.3.2.13), which is all the more plausible because lens contains the enzyme and endogenous protein substrates for it. The enzyme is similar to that obtained from liver and is Ca2+ dependent. Highest apparent activity is found in lens cortex. When cortex homogenate from the rabbit was incubated in the presence of Ca'+ with either ['4C]putrescine or with dansylcadaverine, a selective incorporation of the radioactive or fluorescent amine into the heavier subunits (Mr 26,000 and 30,000) of 13-crystallins could be demonstrated. Possible modes of regulating the crosslinking activity of this enzyme in lens are discussed. Loss of transparency of the aging lens, culminating in senile cataract, is thought to be related to posttranslational modifications affecting the cell proteins themselves. Deamidation, proteolytic degradation, changes in properties of aggregation, and polymerization of constituent proteins by disulfide bonds have been observed (1, 2). However, for the purpose of the present paper, the most pertinent finding is that cataract tissue contains polymeric structures that cannot be dissociated into smaller components by solubilization in sodium dodecylsulfate and urea after reduction of disulfides (3, 4). Thus, the possibility exists that these unusual polymers may have been assembled under the catalytic influence of a Ca2+-dependent transglutaminase (R-glutaminyl-peptide:amine-y-glutamyl-yltransferase, EC 2.3.2.13) (i.e., an endo-y-glutamine:E-lysine transferase; ref. 5) and are held together by y-glutamyl-e-lysine side-chain bridges. In addition to promoting this crosslinking reaction, transglutaminases also catalyze the specific incorporation of amines into proteins and, in fact, these enzymes in tissues can be assayed readily by amine incorporation (6). Recent work on human erythrocytes from this laboratory (7-9) and on cultured keratinocytes by Rice and Green (10, 11) suggests a general role for transglutaminases in some remodeling reactions during the process of cell aging. Usually an increase in Ca2" concentration seems to trigger the activity of these otherwise latent enzymes, although the possibility of regulation by other factors also has to be considered. Accumulation of Ca2" in the cytoplasm might be brought about by the failing of an outward-directed Ca2" pump in the plasma membrane (as in the erythrocytes) or by the disintegration of mitochondria (as in the keratinocytes), but, whatever the cause, the ensuing activation of transglutaminase seems to be the common denominator for determining further events. The structural consequences of the polymerization reaction vary from cell type to cell type. In the case of erythrocytes, y-glutamyl-E-lysine crossbridging involves proteins in the membrane and in the infrastructure. In keratinocytes, a circularly arranged cornifying envelope forms just underneath the plasma membrane. However, the net result in both cases is the irreversible rigidification of the cell. We sought to explore the possibility that polymer formation by covalent bonds other than disulfide in cataract might be due to transglutaminase action. Thus far we have been able to show that the polymer isolated from human cataract, indeed, contains significant amounts of y-glutamyl-E-lysine crosslinks. Furthermore, there is a Ca2"-dependent transglutaminase in lens that acts on protein substrates in this tissue in a rather selective manner. An account of this work was presented in New Orleans at the meeting of the American Society of Biological Chemists (12). MATERIALS AND METHODS Freshly frozen rat, rabbit, guinea pig, and bovine lenses were purchased from Pel-Freez. Human cataract specimens were obtained through the courtesy of Drs. Margaret Gerber and Paul Hauser, Northwestern University. Materials were thawed out just prior to use by immersing in a solution of 50 mM Tris-HCl, pH 7.4/1 mM EDTA at room temperature and, if required, the capsule was removed to allow separation of cortex from nucleus. A hand-operated glass homogenizer was used to break up the cells. Transglutaminase-specific fluorescent staining after electrophoresis of the homogenates on agarose, including photography under UV light, and assays for incorporating [14C]putrescine (Amersham/Searle) into N,N'-dimethylcasein were performed as described (13, 14). Purification of lens transglutaminase was carried out at 40C after dissecting and homogenizing 15 rabbit cortex preparations (ca. 2.7 g wet weight) in 8 ml of 50 mM Tris-HCl, pH 7.4/50 mM NaCl/1 mM EDTA. After centrifugation at 30,000 X g for 30 min, the supernatant was applied to a Sepharose 6B column (2.5 x 88 cm) that had been equilibrated with the same solution. By using this buffer, enzyme activity (monitored by the incorporation of ['4C]putrescine into N,N'-dimethylcasein; ref. 14) emerged between 300 ml and 400 ml of effluent. Glycerol and solid NaCl were added to the collected enzyme fluid to final concentrations of 50% (vol/vol) and 100 mM, respectively. The material was then applied to a column of DEAE-cellulose (1.6 x 7 cm) equilibrated with 50 mM Tris-HCl, pH 7.4/100 mM NaCl/30% glycerol/i mM EDTA. The same solution was used to remove unretained proteins. From measurements of absorbance at 280 nm, it was estimated that less than 5% of the applied proteins were retained by the column. Enzyme was then eluted with a linear gradient of 100-400 mM NaCl in the Tris/NaCVglyceroVEDTA solution. The activity emerged in 1356 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. Proc. Natl. Acad. Sci. USA 78 (1981) 1357 |
| File Format | PDF HTM / HTML |
| Alternate Webpage(s) | http://www.pnas.org/content/78/3/1356.full.pdf |
| Language | English |
| Access Restriction | Open |
| Content Type | Text |
| Resource Type | Article |
