Human erythrocyte transglutaminase. Purification and properties

Transglutaminase has been isolated from human erythrocytes, and some of its molecular and catalytic properties have been determined. An enzyme preparation of about 15% purity is readily obtained in about 25% yield after DEAE-cellulose fractionation and gel filtration. In order to achieve this yield of enzyme it is essential to add to the buffers a dialyzable stabilizing factor which is present in the early enzyme fractions. This natural factor can be partly replaced by chelating compounds and totally replaced by ATP, and in practice, the purification of the enzyme is best carried out with ATP present in the buffers. The role of ATP in stabilizing the enzyme is unknown. Complete purification of the erythrocyte transglutaminase can be accomplished by preparative acrylamide gel electrophoresis. The pure enzyme has a molecular weight of 82 000 ± 5000, as established by gel filtration and SDS gel electrophoresis, and its catalytic properties are essentially identical to those of guinea pig liver transglutaminase. The guinea pig liver and the erythrocyte enzymes have also been compared as catalysts for protein modification reactions, and have been found to have quite similar specificity requirements for protein substrates. Both enzymes catalyzed significant incorporation of amines into 4 of 20 soluble proteins tested and into proteins 1, 2 and 3 of the red cell membrane. The partially purified erythrocyte enzyme has been found to be completely satisfactory for protein modification experiments, and the ready availability of outdated human blood and the simple purification procedure should make this enzyme a convenient protein-modifying or crosslinking reagent.     

Endogenous substrates for epidermal transglutaminase.

Potential in vivo substrates for epidermal transglutaminase have been isolated and partially characterized in human stratum corneum and new born rat epidermis. [14C]Putrescine and dansylcadaverine were incorporated into epidermal proteins in vitro. Two high molecular weight proteins incorporated the labels in both the rat ahd human homogenates. One of the proteins was too large to enter a 4% sodium dodecyl sulfate-polyacrylamide spacer gel; the other was seen at the interface between the spacer gel and a 10% sodium dodecyl sulphate-polyacrylamide running gel. These proteins were present in a buffer extract, sodium dodecyl sulphate-dithiothreitol extract and NaOH extract. The labels were also incorporated into protein in the insoluble pellet remaining after the afore-mentioned extractions. The incorporation of putrescine and dansylcadaverine was time dependent, and was inhibited by known inhibitors of epidermal transglutaminase. The two high molecular weight proteins had similar amino acid composition, characterized by high glycine, glutamic acid, serine and aspartic acid. The amino acid composition was similar to, although not identical with, the amino acid composition of alpha-keratin proteins. Epidermal homogenates incubated in the presence of transglutaminase showed progressive insolubilization of the protein. This cross-linking was inhibited by putrescine. [14C]Glycine, [14C]histidine and [4C]proline were incorporated into epidermal proteins in newborn rats in vivo. The glycine-labelled protein became progressively more insoluble when incubated in vitro in the presence of transglutaminase. In vitro incubation with transglutaminase had no effect on the histidine-and proline-labelled proteins.     

Human epidermal transglutaminase: stimulation by trypsin, organic solvents, and chaotropic salts.

The activity of pure human epidermal transglutaminase was enhanced 3- to 10-fold by various treatments. Incubation with trypsin caused a time-dependent enhancement in activity, up to 3 times the initial activity, with no apparent change in electrophoretic mobility as detected by disc and sodium dodecyl sulfate electrophoresis, and with no apparent change in immunological properties. This enhancement was specific for trypsin among the several enzymes tested. Preincubation of transglutaminase with 0.1 to 2.0 m potassium thiocyanate or potassium iodide, and with 10 to 50% solutions of alcohols and other organic solvents caused a time-dependent enhancement of activity up to 10-fold over control. The presence of calcium was required for the observed enhancement. Kinetic studies suggest that the K_m values of the substrates putrescine and casein determined for the native enzyme are similar to those for the stimulated forms of the enzyme. These in vitro methods of altering enzyme activity may be indications of potential in vivo controls of transglutaminase activity.     

Retinoic acid-induced transglutaminase in mouse epidermal cells is distinct from epidermal transglutaminase

Elevated transglutaminase activity and formation of cornified envelopes are markers of terminal differentiation in mouse epidermal cells. Epidermal transglutaminase catalyzes cornified envelope formation and in cultured cells is inducible by calcium ion or phorbol ester tumor promoters. Retinoic acid also induces transglutaminase activity but inhibits cross-linked envelope formation. This apparent paradox might be resolved by the observation that the retinoic acid-induced transglutaminase appears to be either a different enzyme or a markedly altered form of the epidermal enzyme. The retinoic acid-induced transglutaminase is soluble in aqueous buffers, is thermolabile at pH 9.0, 37 degrees C, and elutes from an anion exchange column at 0.4 M NaCl. In contrast, the epidermal enzyme is particulate and requires detergent for solubilization, is relatively thermostable, and elutes from the anion exchanger at 0.25 M NaCl. The retinoic acid-induced enzyme is probably identical with the "tissue" transglutaminase present in liver and in other cells. It is proposed that the transglutaminase induced by retinoic acid may play a role in the inhibition by retinoids of calcium and tumor promoter-induced differentiation.     

Endogenous substrates for epidermal transglutaminase

Potential in vivo substrates for epidermal transglutaminase have been isolated and partially characterized in human stratum corneum and new born rat epidermis. [¹⁴C]Putrescine and dansylcadaverine were incorporated into epidermal protens in vitro. Two high molecular weight proteins incorporated the labels in both the rat and human homogenates. One of the proteins was too large to enter a 4% sodium dodecyl sulfate-polyacrylamide spacer gel; the other was seen at the interface between the spacer gel and a 10% sodium dodecyl sulphate-polyacrylamide running gel. These proteins were present in a buffer extract, sodium dodecyl sulphate-dithiothreitol extract and NaOH extract. The labels were also incorporated into protein in the insoluble pellet remaining after the afore-mentioned extractions. The incorporation of putrescine and dansylcadaverine was time dependent, and was inhibited by known inhibitors of epidermal transglutaminase. The two high molecular weight proteins had similar amino acid composition, characterized by high glycine, glutamic acid, serine and aspartic acid. Their amino acid composition was similar to, although not identical with, the amino acid composition of α-keratin proteins.Epidermal homogenates incubated in the presence of transglutaminase showed progressive insolubilization of the protein. This cross-linking was inhibited by putrescine.[¹⁴C]Glycine, [¹⁴C]histidine and [¹⁴C]proline were incorporated into epidermal proteins in newborn rats in vivo. The glycine-labelled protein became progressively more insoluble when incubated in vitro in the presence of transglutaminase. In vitro incubation with transglutaminase had no effect on the histidine- and proline-labelled proteins.     

Isolation, purification and characterization of bovine epidermal transglutaminase.

A crosslinking enzyme, epidermal transglutaminase, was isolated from soluble proteins of glabrous cow snout epidermis. This enzyme stabilized fibrin clots rendering them insoluble in 2% acetic acid. It also catalyzed the incorporation of the fluorescent amine, dansyl cadaverine, into casein. Epidermal transglutaminase was purified by chromatography upon DEAE-Sephadex A-50, zone electrophoresis in Pevikon, and Sephadex G-200 gel permeation chromatography. The highly purified substance, which had a specific activity of 3267 amine-incorporating units/mg per h and a molecular weight of 55000, behaved as a single molecular species in the analytical ultracentrifuge. It had a sedimentation coefficient of 4.4 S and migrated as a gamma-globulin at pH 8.6; it displayed anomalous migration in polyacrylamide gels containing sodium dodecyl sulfate. The enzyme was dependent upon free calcium ions and a reduced sulfhydryl group for activity. The apparent Km for dansyl cadaverine was 1.2 - 10(-4) at pH 7.5. Monospecific antiserum to bovine epidermal transglutaminase precipitated with the enzyme in agar. The antiserum prevented fibrin crosslinking but enhanced incorporation of dansyl cadaverine into casein by the enzyme. The epidermal enzyme differed biochemically and immunochemically from bovine plasma transglutaminase (Factor XIII).     

Isolation of substrates of epidermal transglutaminase from bovine epidermis.

Substrates of the transglutaminase specific to epidermis were identified by fluorescent labeling of bovine epidermal homogenates with dansyl cadaverine. This lysine analog was preferentially incorporated into a soluble protein of 150,000 MW. A highly insoluble protein was also labeled; this protein was solubilized and extracted following chemical cleavage with cyanogen bromide. The soluble and insoluble substrates of epidermal transglutaminase were immunochemically related, as shown by precipitation in agar or by chromatography on antibody affinity columns. They were distinguished from the fibrous, α-helical and sulfur-rich matrix proteins of skin as well as from fibrinogen and cold insoluble globulin of plasma.     

Molecular cloning of human epidermal transglutaminase cDNA from keratinocytes in culture.

We have isolated a cDNA encoding human epidermal transglutaminase, a key enzyme of terminal differentiation of keratinocytes. A cDNA library from cultured human keratinocytes was screened by a PCR-amplified partial cDNA fragment of the enzyme with oligonucleotide primers based on the homology of the transglutaminase family. The cDNA is 2734 bp coding a protein of 817 amino acids. The several regions including the active site cysteine residue are highly conserved among the transglutaminase family. However, the charged N-terminal domain is unique to the epidermal transglutaminse, suggesting that the region is involved in the function of the enzyme in keratinocytes.     

Phorbol ester tumor promoters induce epidermal transglutaminase activity

Epidermal basal cells in culture have low levels of epidermal transglutaminase, the enzyme responsible for the formation of the cross-linked envelope in differentiated cells. The tumor promoter 12-O-tetradecanoylphorbol-13-acetate and other active (but not inactive) phorbol ester skin tumor promoters induce transglutaminase activity. Sloughing of differentiated cells accompanies the rise in transglutaminase activity. Phorbol esters do not affect transglutaminase activity when added directly to cell lysates. Corticosteroids have little influence on transglutaminase induction by phorbol esters. Retinoic acid induces transglutaminase activity, but activity does not further increase when basal cells are treated with both retinoic acid and 12-O-tetradecanoylphorbol-13-acetate.     

Human erythrocyte transglutaminase: purification and preliminary characterisation

Erythrocyte transglutaminase was purified by anion-exchange chromatography, size exclusion and affinity chromatography. Homogeneity was achieved by an additional step of HPLC size-exclusion chromatography. The molecular mass of the purified enzyme was calculated to be 65,000 Da by size-exclusion chromatography and sucrose-gradient centrifugation, and 92,000 Da by SDS-PAGE, thus suggesting a high degree of asymmetry. The amino-acid composition of erythrocyte transglutaminase differed substantially from that of the guinea-pig liver enzyme, notably with respect to the number of histidine, cysteine and acidic amino-acid residues. The enzyme has an absolute requirement for divalent cations for activity: calcium, manganese, and the lanthanides terbium and gadolinium activate the enzyme in decreasing order of efficacy, while no activity is displayed in the presence of magnesium. In the presence but not in the absence of calcium ions, the enzyme is rapidly inactivated by N-ethylmaleimide and by diethylpyrocarbonate suggesting that the cation influences the reactivity of amino acids essential for catalysis. When erythrocyte proteins are employed as amine acceptors in the presence of calcium, the erythrocyte transglutaminase appears to preferentially modify membrane-associated proteins, although, in the absence of calcium ions and exogenous amines, it displays a pH-dependent interaction with soluble proteins.     

Rabbit liver transglutaminase: physical, chemical, and catalytic properties.

Transglutaminase (R-glutaminyl-peptide:amine alpha-glutamyl-yltransferase [EC 2.3.2.13]) has been purified to apparent homogeneity from extracts of rabbit liver. The enzyme is a single polypeptide chain of approximately 80 000 molecular weight containing one catalytic site per molecule. That the isolated enzyme is the rabbit counterpart of the well-characterized guinea pig liver transglutaminase is evidenced by the similarities in their amino acid compositions and in their enzymic activities toward several substrates, together with the fact that the isolated rabbit enzyme is immunologically distinct from both rabbit plasma and rabbit platelet blood coagulation factor XIII. A striking difference between the catalytic activities of the rabbit and guinea pig enzymes is the low activity of rabbit transglutaminase for hydroxylamine incorporation into benzyloxycarbonyl-L-glutaminylglycine, a reaction for which the guinea pig enzyme shows a high reactivity. This finding reveals the cause of error in an earlier report (Tyler, H.M., and Laki, K. (1967) Biochemistry 6, 3259) that rabbit liver contains little, if any, of the enzyme. Preparation of, and analytical data on, several glutamine-containing peptide derivatives used in this study are reported here.     

Retinoic acid controls expression of epidermal transglutaminase at the pre-translational level

Human epidermal keratinocytes were cultured until sub-confluence in low Ca2+ (0.15 mM) serum-free synthetic MCDB 153 medium. Raising the Ca2+ concentration to 1.15 mM caused an increase in envelope competence as well as plasma membrane associated transglutaminase (TGm) activity. This increase was not observed when the high Ca2+ medium contained retinoic acid. Immunofluorescence studies as well as immunoblotting with the TGm-specific monoclonal antibody B.C1 revealed that retinoic acid inhibits expression of TGm. Isolation and in vitro translation of mRNA with subsequent immunoprecipitation showed that retinoic acid inhibits TGm expression at the pretranslational level.     

Preparation and mechanical properties of edible pectin-soy flour films obtained in the absence or presence of transglutaminase

Whole soy flour and apple pectin were used as raw materials for producing hydrocolloid edible films. The best ratio between the two components (2:1 mg cm(-2), pectin-soy flour) was determined in order to obtain films which could be perfectly handled for their consistence. Films were also prepared in the presence of transglutaminase, an enzyme able to produce isopeptide bonds among the soy polypeptide chains. The latter films showed a smoother surface and higher homogeneity, as demonstrated by microstructural analyses, whereas studies on the mechanical properties indicated that transglutaminase increased their strength and reduced their flexibility. Our results suggest a possible use of the transglutaminase polymerized pectin-soy protein films as edible food or drug coatings.     

Human alpha-fucosidase. Purification and properties.

Human placental alpha-fucosidase (EC 3.2.1.51) has been extensively purified and partially characterized with respect to kinetic and structured properties. Although the enzyme seems to be separated by DEAE-cellulose chromatography in two forms which differ in their molecular weight and thermostability, an interconversion between the two forms takes place during storage and/or electrofocusing so that the same peaks of activity, revealed by the latter technique, are found before and after DEAE-cellulose chrome. The heterogeneous peaks of activity revealed by isoelectrofocusing show a reproducible pattern in the different tissues examined, except in serum where their pI values are consistently more acidic.